DPP—Ⅳ抑制剂对糖尿病大鼠左室心肌重塑与功能的影响及作用机制
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摘要
研究背景
糖尿病(Diabetes Mellitus, DM)病程缓慢,并发症多且常累及心、脑、肾等全身重要脏器,糖尿病心肌病(diabetic cardiomyopathy, DCM)为常见、严重的并发症之一。DM糖脂代谢紊乱可导致心肌组织病理改变为心肌细胞肥大、凋亡、坏死,间质纤维化,炎症等,表现为进行性左心室结构改变、功能异常,死亡率极高。DM治疗现状目前并不乐观,仍存在诸多间题,单纯降低血糖治疗并不能完全降低心血管病事件。因此,新型降糖药物除具有保护胰岛β细胞功能、降糖作用外,还应具有保护靶器官、降低死亡率的作用,成为当前基础研究与临床治疗策略的重点。
胰高血糖素样肽-1(GLP-1)是主要源于肠道末端L细胞分泌的肠促激素,受体在胰岛、心肌、脑等组织内广泛表达,作用在机体摄入食物后,具有葡萄糖依赖性释放、促进餐后胰岛素分泌等作用。2型DM患者体内,GLP-1分泌水平或活性显著F降,且内源性GLP-1可被二肽基肽酶(DPP-Ⅳ)快速分解。因此,DPP-Ⅳ抑制剂-西格列汀可使内源GLP-1短期不被降解,具有升高GLP-1水平,增强并延迟胰岛素释放来降低血糖。晚近报道证实GLP-1可抑制缺血／再灌注损伤后的细胞凋亡
报道DPP-Ⅳ抑制剂可上调GLUT-4的表达,GLP-1受体兴奋剂可降低2型DM患者高甘油三酯血症、逆转脂肪肝。DM心肌病变中Ⅰ、Ⅲ型胶原沉积、排列紊乱,胶原降解酶(主要基质金属蛋白酶系统MMPs/TIMPs)表达异常,晚近报道,GLP-1受体拮抗剂可抑制脂多糖诱导心肌细胞表达MMP-9,提示可能影响胶原酶的表达。心脏舒张功能不全病人外周血中DPP-Ⅳ活性与超声参数E／E'比值呈正相关。GLP-1受体激动剂可抑制TGF-β1表达。尚未见DPP-Ⅳ抑制剂影响DM大鼠糖脂代谢、心肌脂质含量以及心肌胶原网络代谢的相关研究。
心肌细胞凋亡、坏死广泛存在于DM心肌组织中。DM心肌组织中TNF-α、IL-6表达增加,TNF-α与细胞膜TNFR1结合形成信号转导复合物,可引发细胞内Bax.Bcl-2等基因异常表达,致细胞凋亡。受体相互作用蛋白家族(RIPs)是一类重要调节细胞死亡、存活的信号分子,其中RIP3具有自磷酸化、诱导细胞凋亡与激活NF-kB的作用,是参与TNF-α介导的死亡受体凋亡通路的重要信号分子,且RIP3与线粒体能量代谢有关。DM心肌细胞凋亡中,,RIP3是否参与其中，及DPP-Ⅳ抑制剂对DM心肌细胞凋亡、RIP3表达的影响以及两者的相关性,目前尚未见相关报道。
DPP-Ⅳ抑制剂(西格列汀)通过抑制DPP-Ⅳ活性,提高GLP-1水平、发挥其生物学作用。因此,在既往研究基础上,本部分从动物水平研究西格列汀对DM大鼠心肌组织的炎症、胶原网络代谢、脂质含量及心肌细胞凋亡等病理改变的影响;对DM心肌组织中RIP3的表达及与凋亡的关系：并探讨该药对DM大鼠左心室功能的影响。通过本研究为DM的防治提供更多的方法。
研究目的
1.探讨DPP-Ⅳ抑制剂对DM心肌组织的炎症反应、胶原网络代谢、脂质含量及心肌细胞凋亡等心肌重塑的影响；
2.初步探讨RIP3在DM心肌中的表达及DPP-Ⅳ抑制剂的干预作用;
3.评价DPP-Ⅳ抑制剂对DM大鼠左心功能的影响。
研究方法
1.DM大鼠模型的构建、分组及药物干预
8周龄雄性Wistar大鼠70只,随机分为四组：对照组(n=10只)[Control]；糖尿病组(n=20只)[DM];糖尿病+DPP-Ⅳ抑制剂西格列汀组[(低剂量组,30mg/kg/d)(n=20只)[DPP-IVi(Low)]；糖尿病+DPP-Ⅳ抑制剂西格列汀组(高剂量组,50mg/kg/d)(n=20只)[DPP-IVi(High)]。糖尿病各组高脂饲养4周(w)后,糖尿病各组大鼠腹腔注射小剂量STZ30mg/kg.2型糖尿病大鼠成模的诊断标准为：STZ注射后1周内,连续2次空腹血糖≥11.1mmol/L,为糖尿病模型。模型稳定后持续4w,给予对照组、DM组予以生理盐水灌胃,DM药物干预两组分别给予不同剂量DPP-Ⅳ抑制剂西格列汀研钵碾碎生理盐水稀释后灌胃,均再继续喂养到实验结束,处死称取心脏重量,计算心脏重量／体重[HW/BW(mg/g)]比值后,留取标本。
2.各组大鼠基本指标、糖脂代谢系列指标及GLP-1水平的监测
实验过程中除常规每2周测1次心率、体重、血压与血糖外,期间,根据研究需要,各时间点为0、4、5、9、13、17、21w,抽取颈静脉窦血1.5ml左右,分离血清,全自动生化分析仪测定血总胆固醇、总甘油三酯、低密度脂蛋白胆固醇以及高密度脂蛋白胆固醇水平,以及免疫发光法测定糖化血红蛋白,酶联免疫法法测血清FFA、GLP-1水平,以及空腹血清胰岛素水平,计算胰岛素敏感指数。
3.心电图
根据实验不同时间点,描记肢体导联(Ⅰ、Ⅱ、Ⅲ、aVR、aVL、aVF)心电图,观察各组大鼠心脏电活动情况并记录心电图。
4.超声心动图
分别在0、13w、7w、21w不同时间点进行超声心动图检查,测定指标如下：左室收缩末内径(LVIDs)、左室舒张末内径(LVIDd)、射血分数(LVEF)、左室短轴缩短率(FS)；二尖瓣口E波最大速度、A波最大速度、E／A；组织多普勒(TDI)二尖瓣环左室侧壁测E’波最大速度、A’波最大速度,E'／A',并计算E／E'。
5. Millar导管测左心功能
暴露左室心尖部后,利用10ml注射器针头,内有Milllar导管电极探头直接行心尖穿刺精确控制插入左心室,软件分析心血管参数,左室收缩压(LVSP),左室舒张末压(LVEDP)、左室内压最大上升速率(+dp／dt)、左室内压最大下降速率(-dp/dt)。记录、导出有关数据、图像资料,并计算左室松弛时间常数Tau=P/(-dp/dt)。6
6.心肌组织病理学检测
实验结束处死动物后,进行取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规HE染色、Masson染色及天狼星红染色,观察心肌组织结构、细胞形态、胶原分布情况等。油红O染色用冻存心肌组织标本。以3%戊二醛固定心肌组织后行透射电镜观察。并测心肌羟脯氨酸含量计算胶原含量。
7. TUNEL染色
石蜡切片行免疫组化法检测凋亡细胞,显微镜下观察、计数阳性染色细胞即凋亡细胞百分比数,并进行统计分析。
8.免疫组织化学染色
取心肌组织石蜡切片,分别免疫组化检测左心肌组织炎性因子TNF-α、IL-6,胶原Ⅰ、Ⅲ与胶原降解酶MMP-1、9,以及RIP3的表达,并进行综合分析。
9.实时定量RT-PCR基因水平检测
取-80℃冻存的心肌组织,Trizol法提取心肌组织总RNA,实时定量RT-PCR技术检测RIP3mRNA的表达水平。
10. Western blot检测
取-80℃保存的心肌组织,提取总蛋白,Western-Blot (?)检测左室心肌组织内GLP-1含量及其受体表达,炎性因子TNF-α、IL-6,胶原Ⅰ、Ⅲ,部分胶原降解酶MMP-1、9,以及RIP3的蛋白表达。
11.统计学分析
所用数据用SPSS17.0统计软件进行分析。数值变量资料数据以均数±标准差表示,两组间均数比较采用小样本t检验,多组间均数比较采用One-Way ANOVA方差分析,以P<0.05为差异有统计学意义。
研究结果
1.大鼠的一般情况
糖尿病大鼠造模中,共4只未达标准予以剔除,整个实验中共死亡8只,均为糖尿病各组,最终58只大鼠最终完成实验。其中,Control组10只；DM组15只;DM+DPP-IVi (Low)组16只;DM+DPP-IVi (High)组17只。对照组精神状态好,体重增加,反应敏捷,毛色白而光泽。DM组出现多饮、多尿和消瘦症状,精神差,体重增加迟缓甚至下降,皮毛脏乱无光泽。药物干预后的两组,与DM组相比,一般情况均较好。实验开始时,各组大鼠心率、体重和血压均无差异(P>0.05)。结束时,DM各组与对照组比较,体重下降(P<0.01-0.05),心率、血压升高(P<0.01-0.05)；药物干预后的两组与DM组比较,体重增加及心率、血压不同程度下降(P<0.01-0.05)。
2.血液指标检测结果
2.1各组糖脂代谢指标的比较
造模前、高脂饲养4周后,糖尿病各组存在胰岛素抵抗状态；造模后整个实验过程中,DM组与对照组比较,FBG水平、上午10：00血糖水平及HbA1c,均显著升高(P<0.01)：药物干预后的两组与DM组比较,均不同程度下降(P<0.01-0.05)；造模成功后1周FINS无显著差异,ISI显著下降(P<0.05)；实验末,DM组与对照组、两药物干预组比较,FINS显著下降(P<0.01-0.05)；ISI在实验第5-21周,DM组与对照组比较,显著下降(P<0.05)。
糖尿病造模成功后整个实验过程中,DM各组较对照组,血TC、TG、LDL-C、FFA水平均明显升高(P<0.01-0.05)；药物干预后的两组与DM组比较,均不同程度的降低(P<0.01-0.05)。
2.2检测空腹、口服葡萄糖30min后GLP-1的血浆水平
糖尿病造模成功并且模型稳定后,于第8周开始给予药物干预,DM与对照组比较,分别空腹、口服葡萄糖30min后GLP-1血浆水平均呈不同程度降低(P<0.01-0.05)；药物干预后的两组与DM组比较,在第13-21周,空腹、口服葡萄糖30min后,GLP-1血浆水平均有不同程度的升高(P<0.01-0.05),尤其DM组给予高剂量DPP-Ⅳ抑制剂干预后,与对照组比较,无显著性差异(P>0.05)。
3.心电图的变化
对照组ECG清晰可见P、QRS、T波,QRS波振幅一致。DM组10只心电图异常表现,占66.67%,且QRS“电交替”现象多见,另可见室上速、室速、心动过缓等各种复杂心律失常；两药物干预组中,心电图异常总计14只,占42.42%,未见复杂心律失常且“电交替”减少,提示DPP-Ⅳ抑制剂可改善心脏电活动。
4.超声测量心脏结构和功能指标的变化
左心结构的改变：实验开始时,各组之间左心腔室大小,LVIDs及LVIDd均无显著性差异(P>0.05)；实验结束时,DM组与对照组比较,LLVIDs及LVIDd (?)(?)显著扩大(P<0.05);药物干预后的两组与DM组比较,LVIDs及LVIDd均明显减小(P<0.05)
左室收缩功能的评价：实验开始时,各组大鼠间FS和EF的变化均无显差异(P>0.05)。实验结束时,DM组与对照组比较,FS、LVEF均不同程度降低(P<0.0l-0.05)；药物干预后的两组与DM组比较,FS、LVEF均不同程度升高(P<0.01-0.05)。
左室舒张功能的评价：实验开始时,各项指标均无显著差异(P>0.05);DM组与对照组比较,第13w开始至21w实验结束时,E／A、E'／A’比值不同程度降低(P<0.01-0.05)；E／E’比值不同程度升高(P<0.01-0.05);药物干预后的两组志DM组比较,E／A、E'／A’比值不同程度升高(P<0.01-0.05),E／E’不同程度降低(P<0.01-0.05)。
5. Millar导管测量各组左室功能指标的比较
与对照组比较,DM组LVSP明显下降(P<0.01)、LVEDP升高(P<0.01)、±dp/dt max绝对值降低(P<0.01-0.05)及Tau绝对值延长(P<0.01)。与DM组比较：LVSP在大剂量DPP-Ⅳ抑制剂干预后明显升高(P<0.05),在两组DPP-Ⅳ抑制剂干预后,+dp/dt max (mmHg/s)(?)匀升高(P<0.05),ILVEDP均显著下降(P<0.05),-dp/dt max (mmHg/s)绝对值均升高(P<0.05),左室松弛时间常数(Tau)绝对值不同程度缩短(P<0.01-0.05),大剂量药物干预组Tau缩短更显著。
6.DM大鼠左室心肌重塑及DPP-Ⅳ抑制剂的影响
6.1大体标本病理改变及HE染色
DM组与对照组比较,心脏标本大体形态表现体积较大,HW/BW (mg/g)较大(P<0.05)；HE染色可见左室心腔扩大、室壁肥厚；细胞横截面积明显增加(498.37±14.31vs347.84±12.27μm3, P<0.01);HE染色可见,心肌细胞肥大,可见肌纤维排列紊乱、断裂现象,形态不规则,核大小不均。药物干预后的两组与DM组比较,心脏体积、HW/BW (mg/g)比值变小(P<0.05)；左室心腔缩小,肥厚减轻；心肌细胞横截面积不同程度减少(P<0.01-0.05); HE染色可见心肌细胞体积不同程度减小,细胞排列较规则、有序,核大小尚均一,高剂量药物组则改善更明显。
6.2DPP-Ⅳ抑制剂对DM心肌组织GLP-1含量、GLP-1R表达的影响
实验结束时,Western-Blot提示：与对照组比较,DM组心肌组织GLP-1含量显著降低(P<0.0),GLP-1R表达也减少(P<0.01)；药物干预后的两组与DM组比较,心肌组织GLP-1含量、GLP-1R表达均不同程度升高(P<0.01-0.05),提示DPP-Ⅳ抑制剂干预后,心肌组织内GLP-1含量升高,且可提高GLP-1受体表达的密度。
6.3DPP-Ⅳ抑制剂减少DM心肌组织胶原的含量
Masson染色所见：DM组与对照组大鼠比较,胶原纤维粗大,排列紊乱,分布不均,沉积增多；药物干预后的两组与DM组比较,沉积的心肌胶原减少,排列尚有序、分布也尚均匀。
天狼猩红染色所见：DM组与对照组比较,心肌组织内胶原纤维较明显增加,排列紊乱,且可见不规则网状胶原纤维围绕在小动脉周围;药物干预后的两组与DM组比较,可见心肌组织胶原沉积减少。
胶原含量比较：与对照组比较,DM组左室心肌组织胶原含量(15.86±0.43vs7.06±0.31μg/mg, P<0.01)、胶原容积分数(CVF%)(4.8±0.49vs0.47±0.06,P<0.01-0.05)以及血管周围胶原面积／管腔面积比(PVCA/LA)(2.74±0.37vs0.5±0.02,P<0.01-0.05)均不同程度升高。药物干预后的两组与DM组比较,左室心肌组织胶原含量均不同程度减少。
免疫组化染色：DM组心肌组织与对照组比较,Ⅰ、Ⅲ型胶原阳性表达显著增加；不同剂量DPP-IV抑制剂干预后,可见心肌组织棕色颗粒不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织Ⅰ、Ⅲ型胶原蛋白表达显著升高(P<0.01),药物干预后的两组与DM组比较,均可见心肌组织Ⅰ、Ⅲ型胶原蛋白表达不同程度降低(P<0.01-0.05)。
6.4DPP-Ⅳ抑制剂对糖尿病大鼠心肌胶原酶MMP-1、9表达的影响
免疫组化染色：DM组与对照组比较,MMP-1、9阳性表达显著增加。不同剂量DPP-Ⅳ抑制剂干预后,心肌组织棕色颗粒不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织MMP-1、9蛋白表达显著升高(P<0.05),药物干预后的两组与DM组比较,心肌组织MMP-1、9表达均不同程度降低(P<0.01-0.05)。
6.5DPP-Ⅳ抑制剂对糖尿病大鼠心肌组织炎性因子TNF-α、IL-6表达的影响
免疫组化染色：对照组心肌细胞浆内见少量、稀疏的浅棕色颗粒；DM组心肌细胞胞浆内可见较多浓密深棕色颗粒。与DM组比较,不同剂量DPP-Ⅳ抑制剂干预后,可见心肌组织棕色颗粒分布散在、稀疏，有不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织TNF-α、IL-6蛋白表达显著升高(P＜0.01),药物干预后的两组与DM组比较,心肌组织TNF-α、IL-6蛋白表达均下降(P<0.05)。
6.6DPP-Ⅳ抑制剂减少DM心脏脂质的沉积
油红O染色结果,与对照组比较,DM组心肌脂肪含量(7.03±0.31vs0.32±0.01,P<0.01)、心外膜脂肪组织含量(12.76±0.48vs0.47±0.03,P<0.01)均显著升高；药物干预后的两组与DM组比较,可见心肌组织脂肪含量、心外膜脂肪组织含量均呈不同程度减少(P<0.01-0.05)。
6.7糖尿病心肌组织RIP3的表达及IDPP-Ⅳ抑制剂的干预
免疫组化染色：DM组与对照组比较,RIP3棕黄色阳性颗粒表达增加,且分布在细胞核周围较多,不同剂量DPP-Ⅳ抑制剂干预后,细胞内棕黄色阳性颗粒表达不同程度减少。
实时定量RT-PCR、Western-Blot检测：与对照组比较,DM组大鼠心肌组织RIP3mRNA水平、蛋白水平表达均显著增高(P<0.01);药物干预后的两组与DM组比较,RIP3mRNA水平、蛋白水平表达均不同程度降低(P<0.01-0.05)。
6.8透射电镜观察DPP-Ⅳ抑制剂对糖尿病心肌细胞超微结构的影响
DM心肌细胞核形状不规则,核固缩,染色质聚集,呈现凋亡细胞的早期表现；对照组心肌细胞核仁清晰可见,核膜光滑、完整。药物干预后的两组,细胞核未见明显染色质聚集,无核固缩表现,核膜尚完整、光滑。
6.9TUNEL染色观察DPP-Ⅳ抑制剂对糖尿病心肌组织细胞凋亡发生率的影响
与对照组比较,DM组左室心肌组织凋亡细胞阳性率显著增加(P<0.01),药物干预后的两组与DM组比较,细胞凋亡发生率显著减少(P<0.01)。
6.10初步分析糖尿病心肌组织RIP3mRNA表达与细胞凋亡率的相关性
结合糖尿病心肌组织RIP3mRNA表达与糖尿病心肌组织细胞凋亡百分率,初步分析糖尿病大鼠心肌组织RIP3mRNA表达与细胞凋亡百分率的相关性,得知Pearson相关系数r=0.795(P<0.00]),可以初步判断糖尿病大鼠心肌组织细胞凋亡百分率与RIP3mRNA的表达呈显著正相关。
研究结论
1.DPP-Ⅳ抑制剂影响DM心肌组织炎症反应、胶原代谢、脂质含量及心肌细胞凋亡的改变,逆转DM心肌病理性重塑；
2.DM心肌组织中,RIP3表达上调,且与心肌组织细胞凋亡率呈显著正相关；DPP-Ⅳ抑制剂可干预DM心肌组织中RIP3过表达,抑制心肌细胞凋亡;
3.DPP-Ⅳ抑制剂可显著降低DM大鼠左室E／E’,降低左室舒张末压(LVEDP)和-dp/dt max,增加LVEF和+dp/dt max,缩短Tau。显示DPP-Ⅳ抑制剂明显改善DM大鼠左心室收缩和舒张功能。
研究背景
细胞凋亡系基因调控、主动、程序性死亡的病理生理过程,对维持机体内环境稳态极其重要,糖尿病(DM)心肌病变的主要病理改变之一是心肌细胞凋亡,且随凋亡细胞的增多,最终导致心肌组织重构与心脏功能障碍。
新近研究证实,RIPs (receptor-interacting protein)家族作为一类重要的调节细胞生存、凋亡、坏死的信号分子备受重视,其中RIP3具有自磷酸化、诱导细胞凋亡和激活NF-kB的作用。多种细胞株中过表达RIP3,则会发生细胞凋亡。报道RIP3是TNF-a诱导的细胞凋亡-坏死转换的“分子开关”,且RIP3与线粒体能量代谢、ROS产生等密切相关。RIP3调节能量代谢相关酶相互作用,并产生过量ROS诱发细胞程序性坏死。因此,RIP3参与细胞内多种信号通路,并发挥重要的生理功能。结合课题第一部分的研究,发现DM心肌组织中存在RIP3表达上调,且与心肌组织细胞凋亡率呈显著正相关。但是,高糖刺激下,线粒体功能异常,细胞能量供给障碍,RIP3参与能量代谢相关酶的作用、活性异常等,影响线粒体功能及膜电位改变介导心肌细胞调亡的研究目前国内外尚未见相关报道。
晚近报道,胰高血糖素样肽-1(GLP-1)除降糖作用外,尚具有抑制缺血／再灌注后心肌细胞凋亡、改善心肌梗死后心力衰竭的作用。且GLP-1可能通过激活PI3K、 MAPK信号通路抑制胰岛细胞发生凋亡。课题第一部分动物水平研究发现,DPP-Ⅳ抑制剂通过提高GLP-1水平可抑制DM心肌组织中RIP3过表达、并降低心肌细胞凋亡率。既往研究表明,高糖可直接刺激,或引发Bcl-2表达改变,启动线粒体损伤信号通路,导致线粒体膜通透性增加,线粒体内的细胞色素C释放入胞质,启动caspase级联反应,细胞调亡。但是,国内外尚未见高糖环境中GLP-1对RIP3表达的调节及是否参与以上凋亡通路的研究。
因此,基于既往报道与课题第一部分动物水平的研究,本部分从细胞水平研究提出可能存在的假设：RIP3高表达参与糖尿病心肌细胞TNF-α介导的死亡受体活化的凋亡通路；也参与线粒体损伤的凋亡通路,RIP3直接或间接(通过调节能量代谢过程中产生过量ROS)使线粒体膜通透性改变,线粒体内细胞色素C释放入胞浆,激活型Caspase-3表达增加,发生细胞调亡;而沉默RIP3可改善此病理改变；GLP-1可能通过多个信号通路干预RIP3过表达,从而抑制细胞凋亡。为进一步验证此过程,本部分采用高糖刺激心肌细胞发生凋亡过程中,探讨RIP3在此过程中表达的改变及GLP-1对其影响以及可能存在的机制,为阐明GLP-1、RIP3参与高糖诱导的心室肌细胞凋亡及可能的相关信号通路提供理论依据。
研究目的
1.验证高糖刺激可诱导心室肌细胞调亡,凋亡与RIP3表达相关；
2.探讨高糖诱导心室肌细胞调亡过程中, RIP3在线粒体损伤凋亡途径中的可能作用;
3.探讨高糖刺激下,GLP-1对RIP3表达的影响及可能的信号通路。
研究方法
1.原代心肌细胞培养与鉴定
取1～3日新(?)Wistar(?)鼠心脏,分离出左心室后剪碎、胰酶消化、差速贴壁法获得心肌细胞,可见心肌细胞搏动,并进行原代心肌细胞培养。采用肌钙蛋T单克隆抗体进行免疫荧光染色、荧光显微镜下观察并鉴定为原代培养的心肌细胞。
2.实验分组与干预
体外模拟生理状态、糖尿病状态,加入不同的刺激因素,研究心肌细胞干预后的变化。细胞分别进行处理：心肌细胞分别用不同浓度葡萄糖(5.5、25、33.3、50mmol/L)处理：找出适合实验所需浓度、时间刺激(33.3mmol/L,72h)为高糖(HG)研究对象；分别HG组加入Control siRNA、RIP3siRNA及不同浓度GLP-1干预后孵育细胞,并设计相应对照组。
siRNA技术：采用siRNA技术抑制RIP3表达。将心肌细胞用RIP3siRNA预处理24h,再高糖刺激72h,收取细胞经流式细胞技术和Western-Blot等技术检测细胞调亡及靶蛋白的表达。
3.流式细胞仪检测心肌细胞调亡
采用Annexin V/PI染色流式细胞技术方法,检测心肌细胞的调亡发生情况,得出心肌细胞凋亡的发生率。
4.线粒体膜电位的检测
利用JC-1的方法,经激光共聚焦显微镜观察,检测线粒体膜电位荧光值变化,检测线粒体膜电位(△Ψm)的变化。
5.实时定量RT-PCR检测
收集细胞,提取总RNA,经逆转录反应获得cDNA,以β-actin作参照,通过实时定量RT-PCR技术检测RIP3mRNA水平的表达。
6. Western-Blot检测
收集细胞,提取总蛋白,分别经过SDS-聚丙烯酰胺凝胶电泳分离、转膜、蛋白印记、显色等步骤,检测RIP3、细胞色素C、cleaved caspase3的表达。
研究结果
1.葡萄糖刺激心肌细胞凋亡的时间、浓度的选择
5.5mmol/L[NG]、25%[HG25]、33.3%[HG33.3]、50%[HG50]不同浓度葡萄糖及甘露醇对照组[甘露醇]的培养基孵育细胞0、24、48、72h后,流式细胞仪检测凋亡示,较NG与甘露醇对照组比,HG50刺激48h(P<0.05)、72h(P<0.01)均可显著增加细胞凋亡,HG25刺激72h(P<0.05)可增加细胞凋亡。因此,高浓度葡萄糖可诱导心肌细胞凋亡,结合心肌细胞活力的结果,选用33.3%葡萄刺激72小时为研究对象。
2.RIP3表达与高糖诱导心肌细胞凋亡发生的关系
2.1高糖诱导心肌细胞调亡中RIP3表达升高
(1)利用高浓度葡萄糖(33.3%)[HG]刺激心肌细胞72h后,收取细胞,实时定量RT-PCR及Western-Blot法检测,结果显示：与5.5%浓度葡萄糖[NG]比较,HG组RIP3在mRNA (P<0.05)与蛋白水平(P<0.05)均显著升高;
(2)将原代培养的心肌细胞先应用RIP3siRNA、Control siRNA转染心肌细胞24h后,再予以高糖刺激72h,收取细胞利用实时定量RT-PCR和Western-Blot验证siRNA转染效力,结果RIP3siRNA转染组与HG组、Control siRNA转染组比较,RIP3在mRNA(P<0.05)及蛋白(P<0.05)表达水平显著下调,提示RIP3siRNA能有效抑制RIP3表达。
2.2RIP3siRNA可降低高糖诱导的心肌细胞调亡发生率
采用流式细胞技术分别对5.5mmol/L浓度葡萄糖组(NG)、高糖33.3%浓度葡萄糖组(HG)、HG+Control siRNA转染组、HG+RIP3siRNA转染组的心肌细胞调亡率进行检测,结果显示：NG组比较,高糖可诱导心肌细胞凋亡发生(P<0.01-0.05);与HG组、HG+Control siRNA组比较,RIP3siRNA转染后,心肌细胞凋亡率显著下降(P<0.05)。提示RIP3在细胞调亡过程中发挥重要喽作用,其高表达可诱导高糖刺激的心肌细胞发生凋亡。
3. RIP3高表达通过线粒体凋亡通路诱导高糖刺激的心肌细胞发生凋亡
3.1RIP3siRNA对高糖环境心肌细胞线粒体膜电位(△Ψm)的影响
使用激光共聚焦显微镜观察JC-1单体／聚合物(JC-1monomer/polymer)的荧光值比值,绿色荧光(单体)提示△Ψm下降,细胞凋亡前期；红色荧光(聚合体)提示△ψm较正常,细胞状态正常。
与NG组比较,绿色荧光(单体)／红色荧光(聚合体)比值在HG组(P<0.05)、HG+Contro] siRNA组(P<0.05)显著升高；HG组细胞经RIP3siRNA转染后,与HG、HG+Control siRNA组比较,其比值显著下降(P<0.05)。提示高糖刺激心肌细胞凋亡中,R1P3可能影响线粒体功能,致线粒体△Ψ／m下降。
3.2RIP3siRNA对高糖环境心肌细胞浆细胞色素C(Cyt C)含量、激活型Caspase-3表达的影响
经Western-Blot分析,与NG组比较,HG组、HG+Control siRNA组心肌细胞浆Cyt C含量(P<0.05)、激活型Caspase-3表达(P<0.05)均显著升高；RIP3siRNA转染后,HG+RIP3siRNA组与HG、HG+Control siRNA组比较,心肌细胞浆Cyt C含量(P<0.05)、激活型Caspase-3(P<0.05)表达均显著降低。提示RIP3在高糖环境心肌细胞凋亡中参与线粒体凋亡通路。
4. GLP-1对RIP3表达的调控及机制
4.1GLP-1对高糖诱导心肌细胞RIP3表达的影响及信号通路
Western-Blot结果显示：与单纯HG刺激组比较,HG+GLP-1(1nmol/L)组无差异(P>0.05),HG+GLP-1(10nmol/L)组RIP3蛋白表达明显下调(P<0.01)；加HG+GLP-1(10nmol/L)之前分别加MAPK阻断剂(LY294002)、PI3K阻断剂(U0126)干预的2组与HG+GLP-1(lOnmol/L)组比较,RIP3蛋白表达升高亦有明显差异(P<0.05)。提示GLP-1呈浓度依赖抑制高糖诱导的RIP3过表达,且可能部分通过MAPK、PI3K信号通路调控此过程。
4.2GLP-1通过抑制RIP3表达降低高糖刺激的心肌细胞凋亡率
本实验中,采用GLP-1(10nmol/L)干预、沉默RIP3分别交换先后顺序干预细胞,共5组,利用流式细胞仪结果显示,与单纯高糖刺激组(HG)比较,GLP-1干预、沉默RIP3均可降低细胞凋亡率(P<0.01-0.05)；与HG+GLP-1(10nmol/L)比较,沉默RIP3组[HG+GLP-1(10nmol/L)+RIP3siRNA、HG+RIP3siRNA、HG+RIP3siRNA+GLP-1(10nmol/L)]细胞凋亡率也显著降低(P〈0.05)；GLP-干预后沉默RIP3组与单纯GLP-1于预组比较,细胞凋亡率降低(P<0.05)；单纯沉默RIP3组与沉默RIP3后加GLP-1干预组比较,组间无统计学差异(P>0.05)。
4.3GLP-1抑制RIP3表达降低高糖环境细胞浆Cyt C含量、激活型Caspase-3表达
Western-Blot分析,与HG组比较,lHG+GLP-1(10nmol/L)(?)且心肌细胞浆Cyt C含量、激活型Caspase-3表达均显著减少(P<0.05)；HG分别经RIP3siRNA转染干预、GLP-1(lOnmol/L)干预后,心肌细胞浆Cyt C含量、激活型Caspase-3表达与HG组(P<0.01)、HG+GLP-1(10nmol/L)组(P<0.05)均显著减少。
此实验提示GLP-1可能部分通过MAPK、PI3K(?)信号抑制RIP3表达,从而降低高糖刺激的心肌细胞凋亡率。
研究结论
1.高糖刺激可诱导心肌细胞调亡,同时RIP3表达增高：沉默RIP3后细胞凋亡率降低;
2.高糖刺激心肌细胞凋亡中,RIP3可能通过影响线粒体能量代谢,膜电位降低、通透性增加,线粒体内细胞色素C释放入胞浆,启动Caspase级联反应,诱导细胞凋亡;
3.GLP-1可能部分通过MAPK、P13K信号通路抑制RIP3表达,进而抑制高糖诱导的心肌细胞凋亡。
Background
The duration of diabetes is very slow. During the course if diabetes, many complications could damage the main organs such as the heart, the brain and the kidney. Diabetic cardiomyopathy is one of the relatively common and serious complications. The main pathological changes of diabetic cardiomyopathy are myocardial hypertrophy, apoptosis or necrosis of myocardiocytes, myocardial fibrosis and inflammation. The pathogenesis of diabetic cardiomyopathy is very complex, possibly related to the disorders of myocardial glucose metabolism and energy metabolism, calcium overload, myocardial RAS activity, oxidative stress, insulin resistance, cardiac autonomic neuropathy and TGF-/β or TNF-α abnormal expression. The clinical findings of diabetic cardiomyopathy are usually progressive abnormal changes of left ventricular structure and function, or even high mortality rate. However, the current treatment of diabetes is not so satisfactory that there are still many deficiencies and the incidence of diabetic complications is still high. Therefore, the novel anti diabetic drugs should not only lower the blood glucose and protect the function of islet beta cells, but also protect the main target organs and delay the progressive lesions of diabetic cardiomyopathy in order to reduce the mortality. Hence, the treatment of diabetes becomes the focus of the current strategies of the basic research and clinical treatment.
Glucagon-like peptide-1(GLP-I) is one of the incretin hormones secreted from the distal intestine L cells and GLP-1receptors (GLP-1R) are widely expressed in the tissues islated from the islets, myocardium and the brain. GLP-I begins to work after the food intake in the body, which has a glucose-dependent release and promotes the secretion function of postprandial insulin. The serum levels or activities of GLP-1usually decrease in diabetic patients and the endogenous GLP-1could be dissolved rapidly by dipeptidyl peptidase (DPP-IV). Therefore, sitagliptin as one of the DPP-IV inhibitors could make the endogenous GLP-1not be degraded in the short term, elevate the serum levels of GLP-1and enhance or delay the release of insulin in order to lower the blood glucose. Recent reports demonstrated that GLP-1could play a key role in the progress of inhibiting apoptosis during the course of ischemia/reperfusion injury and improve the cardiac function.
DPP-IV inhibitor was reported to function as the up-regulation of the expression of GLUT-4. GLP-1receptor agonist can reduce the hypertriglyceridemia and reverse fatty liver in diabetic patients. However, little research is reported about the effects of DPP-IV inhibitors on the glucose and lipid metabolism in myocardium.
The characteristic pathological changes of myocardial lesions in diabetic patients are the imbalances of extracellular matrix, the deposition or disorders arranged of type I and Ⅲ collagen and the abnormal expression of degrading enzyme (main matrix metalloproteinase system MMPs/TIMPs). Recently, GLP-1receptor antagonists can inhibit the expression of MMP-9induced by lipopolysaccharide in the myocardial cells, which implied GLP-1could inhibit the expression of collagenase. The DPP-IV activity in peripheral blood of the patients with cardiac diastolic dysfunction was positively associated with the E/E'ratio measured by echocardiology and the GLP-1receptor agonist can inhibit the expression of TGF-β1. However, no research is reported about the effects of DPP-IV inhibitor on the network of the cardiac collagen metabolism in diabetic rats and cardiac diastolic dysfunction.
Apoptosis and necrosis of myocardial cells exist widely and the expression of TNF-a or IL-6increase in the myocardial tissue with diabetic rats. The formation of signal transduction complexes results from the combination of TNF-a and TNFR1in cell membrane, can lead to the abnormal expression of Bax, Bcl-2. Receptor interacting protein family (RIPs) is an important signaling moleculear regulating death and survival of the cells. RIP3has autophosphoryl ation, induction of apoptosis and activation of NF-κB effect, is an important signal molecules involved in TNF-a mediated by death receptor pathway of apoptosis, and RIP3and mitochondrial energy metabolism. However, no related research are reported about whether RIP3is involved in apoptosis of myocardial cells in diabetic rats and the effect of DPP-Ⅳ inhibitor on the expression of RIP3in apoptotic myocardial cell, and even the correlation between the two factors.
Sitagliptin as one of the DPP-Ⅳ inhibitors (inhibiting enzyme DPP-IV activity) plays its biological role via improving the levels of GLP-1. Hence, the effects of sitagliptin on inflammation, network of collagen metabolism, lipid content and myocardial apoptosis in diabetic rats would be studied in this part based on the previous studies. This work would detect the effect of sitagliptin on the expression of RIP3in myocardial tissues in diabetic rats and the relationship with apoptosis and explore the effect of the drug on left ventricular function in diabetic rats. More additional treatments would be provided through the research for the prevention and treatment of diabetes.
Objectives
1. To investigate the effects of DPP-IV inhibitors on the pathological remodeling including inflammation, network of collagen metabolism, lipids contents and apoptosis of myocardial cells in the myocardial tissues of diabetic rats.
2. Preliminary study on the relationships between the expressions of RIP3in the diabetic myocardial tissues and apoptosis of cells, and interventional effects of DPP-IV inhibitors on the above pathological changes.
3. To evaluate the effects of the DPP-Ⅳ inhibitors on cardiac function of left ventricle in diabetic rats.
Methods
1. Establishment and groupings of the Type2diabetic rats
Seventy Wistar rats of eight weeks old and male were randomly divided into four groupsrcontrol group (n=10)[Control]; diabetes mellitus group (n=20)[DM]; diabeties with DPP-IV inhibitors (sitagliptin low dose group,30mg/kg/d)(n=20)[DPP-IVi (Low)]; diabetic treated with DPP-IV inhibitors(sitagliptin high dose group50mg/kg/d)(n=20),[DPP-IVi (High)]. All the subgroups of diabetic rats were injectioned with small dose of STZ30mg/kg by intraperitoneal style after high fat diet for4weeks, and the control groups were injected with the same dose of citrate buffer (pH4.5). Diagnostic criteria of type2diabetic rats:two consecutive fasting glucose≥11.1mmol/L after STZ injection in one week were regarded as type2diabetic models. The control groups and DM groups were given normal saline, and another DM groups interventioned were given different doses of DPP-Ⅳ inhibitor-sitagliptin diluted gastric lavage were to continue feeding until the end of the experiment, put to death, heart weight (HW), heart weight/body weight calculation [HW/BW(mg/g)]ratio, the specimen were reserved.
2. Monitoring of the basic indicators of the metabolism index of blood glucose or lipids, and the levels of GLP-1in each groups
The experimental process in addition to the conventional measured every2weeks for1times the heart rate, weight, blood pressure and blood glucose, during the period of this schedule, according to the need of research, the time point of0,4,5,9,13,17,21W, jugular venous sinus blood about1.5ml were extractioned, separated with serum, then serum total cholesterol, full automatic biochemical analyzer total triglyceride, low density lipoprotein cholesterol and high density lipoprotein cholesterol levels, glycosylated hemoglobin were measured as well as the immunoluminometric assay. The serum FFA, levels of GLP-1were measured with enzyme linked immunosorbent assay (ELISA) method, and fasting serum insulin level, insulin sensitivity index were calculated.
3. ECG
At different time points of this study, the cardiac electrical activity by ECG was recorded with the limb leads (Ⅰ, Ⅱ, Ⅲ, aVR, aVL, aVF) of rats were evaluated.
4. Echocardiology
Echocardiology were examinated at different time points of0,13W,17w,21W and measured as these follows:left ventricular end systolic diameter (LVIDs), left ventricular end diastolic diameter (LVIDd), ejection fraction (LVEF), left ventricular fractional shortening (FS); mitral E wave maximum speed, the maximum A wave speed, E/A; tissue Doppler image (TDI) of mitral annulus left ventricular lateral wall measured E'wave maximum velocity, maximum velocity of E wave A'. Then EVA' and E/E' were calculated.
5. Left ventricular function measured by Millar catheter
Direct apical puncture:exposure to the apex of the left ventricle, using10ml syringe needle, a Milllar catheter electrode probe directly for precise control of apical puncture into the left ventricle, Lab Chart Pro software to calculate cardiovascular parameters, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular pressure maximum rate of rise (+dp/dt), left ventricular pressure maximum falling rate (-dp/dt) and calculate Tau. Record and export the data, image data analysis.
6. Myocardial pathology
At the end, the rats were killed. Then the hearts were collected, fixed, dehydrated, transparent, paraffin, paraffin embedding, slicing, HE staining, Masson staining, and Sirius red staining in order to observe the myocardial tissue structure, cell morphology, collagen distribution et al. The frozen myocardial tissue specimens were stained with oil red O. The myocardial tissues were fixed with3%glutaraldehyde and transmission electron microscope and myocardial collagen contents were calculated and determinated with hydroxyproline content.
7. TUNEL Staining
The detection of apoptotic cells in paraffin sections by immunohisto chemistry. The percentage of the numbers of apoptotic cells were observed by microscope, and then the positive stained cells were counted by statistical analysis. The specific operation methods were dealed with the reference TUNEL apoptosis Kit (Calbiochem) steps.
8. Immunohistochemical Staining
The inflammatory factor TNF-a and IL-6, type Ⅰ and Ⅲ collagens and collagen degrading enzyme MMP-1and9, and RIP3expressions in the paraffin sections of left ventricular myocardial tissues by immunohistochemical detection were observed and then comprehensive analysis were done well.
9. Real-time quantitative RT-PCR
The total RNAs of myocardial Trizol were extracted from the myocardial tissues reserved at-80℃. The expression levels of RIP3mRNA were detected with real-time quantitative RT-PCR.
10. Western-blot
The total protein was extracted from the myocardial tissues cryopre served at-80℃. The GLP-1content and expression of GLP-1receptors, the inflammatory factor TNF-α and IL-6, type Ⅰ and Ⅲ collagens and collagen degrading enzyme MMP-1and9, and RIP3expressions were detected with Western-Blot method.
11. Statistical analysis
The data used by SPSS17.0statistical software for analysis. Numerical variable data were expressed by mean±standard deviation. The Comparison between the two groups was calculated with the small sample t test. The Comparison in different groups was calculated by One-Way ANOVA analysis of variance and P<0.05is regarded as significant difference.
Results
1. General situation of all the rats
During the course of the establishment of the type2diabetic rats models, a total of four is eliminated because of not up to standard and five rats died in the whole experiment. Finally, fifty-eight rats were involved in this experiment, and were divided into four groups:the Control group including10rats; the Diabetic group including15rats; the DM+DPP-Ⅳi (Low) group including16rats; and the DM+DPP-Ⅳi (High) group including17rats. The Control group had a good mental state, weight increased significantly, quick reaction, hair color white and shiny. However, polydipsia, polyuria and weight loss of symptoms occurred in DM groups, and poor spirit, slow weight gain or even declining, fur dirty and dull. The results compared with the DM group were slightly better in the treatment groups with different doses of DPP-Ⅳ inhibitors in DM rats. At the beginning of the experiment, no differences exist in the heart rate, blood pressure and body weight of rats in each group (P>0.05). But at the end, compared with the control group, the body weight decreased (P<0.01-0.05), the heart rate, blood pressure increased (P<0.01-0.05), and after different doses of DPP-Ⅳ inhibitors treatment, body weight increased, blood pressure and heart rate decreased in different degrees(P<0.01-0.05).
2. Results of the blood examination
2.1Comparison of metabolism of glucose and lipids in different groups
The phenomena of insulin resistance exist in all diabetic subgroups after four weeks with high-fat feeding before establishment of the models. During the whole process of the experiment, compared with the control group, the serum levels of FBG, blood glucose at10:00am and HbA1c, increased significantly (P<0.01) in DM group. Compared with the DM groups, all the indexes above in the DM groups with different doses of DPP-Ⅳ inhibitors decreased (P<0.01-0.05). No significant difference exists in FINS, but ISI decreased significantly (P<0.05) after one week establishing the models. However, at the end, compared with the control group and different doses of drug intervention groups, FINS decreased significantly (P<0.01-0.05) in DM group and from NO.5to21week, ISI decreased significantly (P<0.05) in DM group compared with the control group. The serum levels of TC, TG, LDL-C, and FFA increased significantly (P<0.01-0.05) in all the DM subgroups during the whole process of diabetes, and all these issues decreased in different degree (P<0.01-0.05) after oral different doses of DPP-Ⅳ inhibitors compared with the DM group.
2.2Serum levels of GLP-1examinated in fasting and oral glucose after30min
Diabetic model was successful and stable model, was treated for eighth weeks, DM compared with the control group, respectively, after oral glucose30min fasting plasma levels of GLP-1were decreased in different degree (P<0.01-0.05); different doses of DPP-Ⅳ inhibitors treatment, compared with the DM group, in the13-21weeks, fasting, oral glucose30min, GLP-1elevated plasma levels in varying degrees (P<0.01-0.05), especially the DM group were given high doses of DPP-Ⅳ inhibitor treatment, compared with the control group, no significant difference (P>0.05).
3. Results of ECG
ECG of the control group demonstrated P wave, QRS wave, T wave were visible and amplitude consistency of QRS waves. Abnormal ECG were found in ten diabetic rats, accounted for66.67%, and QRS "electrical alternans" phenomena were seen more and also including super ventricular tachycardia, ventricular tachycardia, slow heartbeat and other complex arrhythmia in the DM group. However, abnormal ECG were found in only fourteen diabetic rats with DPP-Ⅳ inhibitors, accounted for42.42%, in addition, no complicated arrhythmia occurred and "QRS alternans" reduced in the DM groups treated with DPP-IV inhibitors, suggesting that DPP-IV inhibitors improved the electrical activity of the heart.
4. Cardiac structure and function measured by echocardiology
Structure changes of left ventricule:at the beginning, no significant difference was found in the size of left heart chambers, LVIDs and LVIDd (P>0.05). However, at the end, compared with the control group, LVIDs and LVIDd of the DM group increased significantly (P<0.05). Compared with the DM group, LVIDs and LVIDd of the group treated with different doses of DPP-Ⅳ inhibitors decreased significantly (P<0.05).
Systolic function of the left ventricule:at the beginning, no significant differences of FS and EF existed in each group (P>0.05). However, at the end, compared with the control group, FS and LVEF decreased significantly (P<0.01-0.05) and compared with the DM group, FS and LVEF elevated significantly (P<0.01-0.05).
Diastolic function of the left ventricule:at the beginning, all indexes were showed no significant difference (P>0.05). Compared with the control group, during the period of13w-terminal21w, E/A, E'/A' ratio decreased (P<0.01-0.05) and E/E'ratio increased (P<0.01-0.05). Compared to the DM group, E/A and E'/A' ratio increased (P<0.01-0.05) and E/E 'decreased (P<0.01-0.05) were found in the DM group treated with DPP-Ⅳ inhibitors.
5. Parameters of left ventricular function measured by Millar catheter
Compared with the control group, LVSP decreased significantly (P<0.01), LVEDP increased (P<0.01),+dp/dt Max and-dp/dt Max absolute value decreased (P<0.01-0.05). Compared with the DM group, LVSP increased significantly in the DM treated with high doses of DPP-Ⅳ inhibitors (P<0.05),+dp/dt max (mmHg/s) increased significantly in different doses of DPP-Ⅳ inhibitors (P<0.05). LVEDP decreased significantly (P<0.01-0.05),-dp/dt max (mmHg/s) increased (P<0.05) and Tau decreased (P<0.05) in the DM group treated with different doses of DPP-Ⅳ inhibitors.
6. Effects of DPP-Ⅳ inhibitors on myocardial remodeling of left ventricle in diabetic rats
6.1Gross pathology and HE staining
Comparison the control group, cardiac gross morphological features demonstrated large sizes, high HW/BW (mg/g)(P<0.05) and Left ventricular cavity enlargement and ventricular wall hypertrophy occurred and cellular cross-sectional area increased significantly (498.37±14.31vs347.84±12.27μM3, P<0.01). HE staining:myocardial cell hypertrophy, muscle fibers derange ment or fracture phenomenon, irregular shape, uneven size of nuclei occurred in the DM group. Comparison the DM group, cardiac sizes, HW/BW (mg/g) ratio became smaller (P<0.05), left ventricular hypertrophy alleviated, cardio myocyte cross-sectional area decreased significantly (p<0.01-0.05) and HE staining:myo cardial cellular sizes reduced, cells in a regular arrangement, nuclear uniform size in the DM group treated with DPP-IV inhibitors.
6.2Effect of DPP-IV inhibitors on the content of GLP-1and the expression of GLP-1Receptor in diabetic myocardial tissue
Western-Blot:at the end, compared with the control group, the content of GLP-1(P<0.01) and the expression of GLP-1R (p<0.01) decreased significant ly in diabetic myocardial tissues. Compared with the DM group, the content of GLP-1and the expression of GLP-IR increased in the DM group treated with DPP-IV inhibitors.6.3Effects of DPP-IV inhibitors on myocardial collagen metabolism
Masson staining:compared with the control, the collagen fibers arranged in disorder, rough, uneven distribution, sedimentary increased in the DM group, and compared with DM group, the myocardial collagen deposition reduction, arrayed orderly, distribution was uniform in the DM group treated with different doses of DPP-Ⅳ inhibitors.
Sirius red staining:compared with the control group, collagen fibers signi ficantly increased, disorganized, and irregular reticular collagen fibers around small arteries in the DM group and the myocardial collagen deposition decreased in the group treated with different doses of DPP-Ⅳ inhibitors.
Compared with the control group, the collagen content in diabetic myocar dial tissue (15.86±0.43vs7.06±0.31μg/mg, P<0.01), CVF%(4.8±0.49vs0.47±0.06, P<0.01-0.05) and perivascular collagen area/lumen area ratio increased (PVCA/LA)(2.74±0.37vs0.5±0.02,P<0.01-0.05) in the DM group. Compared with the DM group, the collagen content of left ventricular tissue decreased in in the DM group treated with DPP-Ⅳ inhibitors.
Immunohistochemical staining:compared with the control group, the positive expression of type Ⅰ and Ⅲ collagen increased significantly in diabetic myocar dial tissues, and brown granules decreased after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of type Ⅰ and Ⅲ collagen in diabetic myocardial tissue increased significantly (P<0.01) and expression decreased significantly (P<0.01-0.05) after treatment with DPP-IV inhibitors.
6.4MMP-1,9reduced in DM myocardial tissues treated with DPP-IV inhibitors
Immunohistochemical staining:compared with the control group, the positive expression of MMP-I, MMP-9increased significantly in diabetic myocardial tissues and brown granules decreased after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of MMP-1, MMP-9in diabetic myocardial tissues increased significantly (P<0.05) and expression decreased significantly (P<0.01-0.05) after treatment with DPP-Ⅳ inhibitors.
6.5Inflammatory factors TNF-a and IL-6reduced in DM myocardial tissues treated with DPP-Ⅳ inhibitors
Immunohistochemical staining:a few sparse pale or brown granules in the control group and with more densely dark or brown granules in myocardial cellular cytoplasm. Brown particles, scattered distribution in sparse reduced after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of TNF-a and IL-6in diabetic myocardial tissue increased significantly (P<0.01) and expression decreased significantly (P<0.05) after treatment with DPP-Ⅳ inhibitors.
6.6Fatty deposition reduced in diabetic myocardial and epicardial tissues treated with DPP-IV inhibitors by Oil red O staining
Compared with the control group, lipids content of diabetic myocardium (7.03±0.31vs0.32±0.01, P<0.01) and that of epicardial adipose tissue (12.76±0.48vs0.32±0.01, P<0.01) increased significantly. Compared with the DM group, lipid content, epicardial adipose tissue reduced after treatment of different doses of DPP-IV inhibitors (P<0.01-0.05).
6.7The expression of RIP3in diabetic myocardial tissues and DM group treated with DPP-Ⅳ inhibitors
Immunohistochemical staining:compared with the control group, RIP3brown positive particles increased and distributed in the nucleus around more in diabetic myocardial tissues, and brown positive particles in the cell decreased after treatment with different doses of DPP-Ⅳ inhibitors.
Quantitative real-time RT-PCR and Western-Blot:compared with the control group, mRNA level and protein expression of RIP3increased significantly (P<0.01-0.05) in diabetic myocardial tissues, and compared with the DM group, mRNA level and protein expression of RIP3decreased (P<0.01-0.05) after treatment with DPP-Ⅳ inhibitors.
6.8Effects of DPP-Ⅳ inhibitors on DM myocardial ultrastructure by TEM observation
Myocardial irregular nuclei, nuclear pyknosis, chromatin aggregation, early presentation of apoptotic cells in the DM group. Myocardial cellular nucleolus more clear, nuclear membrane smooth and complete were seen in the control group. No obvious aggregation of chromatin, no nuclear pyknosis and nuclear membrane still intact, smooth were seen in the DM rats after treatment with DPP-Ⅳ inhibitors.
6.9Effects of DPP-Ⅳ inhibitors on apoptosis of diabetic myocardial tissues by TUNEL staining
Compared with the control group, the percentage of apoptotic cells increased significantly (P<0.01) and compared with DM group, the apoptosis rate reduced significantly (P<0.01) after after treatment with DPP-IV inhibitors.
6.10Analysis of the relationships between the expression RIP3mRNA and cellular apoptotic rate in diabetic myocardial tissues
Combined with the relationships between the expression RIP3mRNA and cellular apoptotic rate in diabetic myocardial tissues, Pearson correlation coefficient r=0.795(P0.001), the percentage of apoptosis positive related to RIP3mRNA in diabetic myocardial tissues.
Conclusions
1. DPP-IV inhibitors could exactly impove the pathological remodeling including inflammation, metabolism of collagen network, lipids content and apoptosis of myocardial cells in type2diabetic rats.
2. The expressions of RIP3were up-regulated and positively correlated with apoptosis of the cells in diabetic myocardial tissues and DPP-IV inhibitors could down-regulate the expression of RIP3and inhibit apoptotic cells of the myocardial tissues.
3. DPP-IV inhibitors could significantly reduce the E/E'ratio in diabetic rats, improve systolic blood pressure (LVSP), reduced left ventricular end diastolic pressure (LVEDP),±dp/dt max and Tau. DPP-IV inhibitors could significantly improve both systolic and diastolic functions of the left ventricle in type2diabetic rats.
Background
Apoptosis is a pathophysiological process regulated by gene control, active, and programmed cell death, and is extremely important for maintaining homeostasis. Apoptosis of myocardial cells is one of the main pathologic changes in diabetic myocardial lesions and increased with the increase of apoptotic cells, eventually myocardial tissue remodeling and cardiac dysfunc tion occurred.
Recent studies demonstrated that RIPs (receptor-interacting protein) family as a kind of regulation of cell survival, apoptosis, necrosis of important signal molecules has attracted increasing attention, in which RIP3has autophosphoryla tion, induction of apoptosis and activation of NF-κB. Overexpression of RIP3in various cell lines, would undergo apoptosis. Zhang DW et al. reported that RIP3was TNF-a induced apoptosis and necrosis of mutual conversion "molecular switch", and RIP3was closely related to mitochondrial energy metabolism of cells and ROS released. RIP3regulated enzyme interactions related to energy metabolism, and overproduction of ROS induced programmed cell necrosis. Therefore, RIP3was involved in many intracellular signal pathways, and play an important physiological function. Combined with the research of the first part of this study, up-regulated expression of RIP3was found in diabetic myocardial tissues, and significantly positive correlated with myocardial apoptosis rate. However, in high glucose conditions, mitochondrial dysfunction, cellular energy supply disorder, RIP3was involved in energic metabolism related enzyme action, activity, effect of mitochondrial function and membrane potential changes of myocardial cells mediated apoptosis has not been reported.
Recent reports, glucagon-like peptide-1(GLP-1) in addition to hypoglycemic effect, still could inhibit myocardial apoptosis after ischemia/reperfusion or myocardial infarct tion, improve heart failure. GLP-1might inhibit pancreatic islet cells apoptosis via activating P13K. MAPK signaling. First part of this study demonstrated that DPP-1V inhibitors inhibited RIP3expression in diabetic myocardium and decreased myocardial cellular apoptosis by increasing the level of GLP-1. Previous study showed that, high glucose might directly stimulate or cause changes of Bax, Bcl-2expression, start the mitochondrial damage pathway, leading to increased mitochondrial membrane permeability, cytochrome C released from mitochondrial into the cytoplasm, start the caspase cascade reaction, cell apoptosis. However, in high glucose environment, research on the regulation of RIP3expression is involved in the apoptosis pathway via GLP-1is no reported till now.
Hence, study on the previous reports and first task based on the animal levels, this part will study and put forward possible hypotheses:over-expression of RIP3participates in the apoptotic pathway of diabetic myocardial cells via TNF-a mediated death receptor, also involved in mitochondrial injury. RIP3directly or indirectly (through the process of adjusting the energy to produce excess of ROS) cause the mitochondrial membrane permeability changes, mitochondrial cytochrome C releasing into the cytosol. activated Caspase-3expression, cell apoptosis, while RIP3silence could improve the pathological change. GLP-1might inhibit overexpression of RIP3via multiple signaling pathways. To further verify this process, this part adopts high glucose-induced apoptosis process, to explore changes of RIP3expression in this process and effect of GLP-1and the possible mechanisms, and provide a theoretical basis for the description of GLP-1, RIP3in high glucose induced apoptosis in ventricular myocytes and the possible signal pathway.
Objectives
1. To verify stimulation with high glucose can induce ventricular myocardial apopto sis, and apoptosis associated with expression of RIP3.
2. To investigate the apoptotic process of ventricular myocardiocytes induced by high glucose, RIP3may play a key role in apoptosis via the mitochondrial damage.
3. To explore the possible signal pathway of effects of GLP-1on the expression of RIP3in high glucose conditions.
Methods
1. Culture and identification of primary myocardial cells
1-3days of newborn Wistar rats isolated left ventricular posterior shear, trypsin digestion, differential centrifugation to obtain myocardial cells, the pulsation of myocardial cells, and the primary myocardial cells cultured. The troponin T monoclonal antibodies by immunofluorescence staining, fluorescence microscope and identified as the primary cultured myocardial cells.
2. Experimental groups and intervention
In vitro physiological state, diabetes status, adding stimulus to different factors, prognosis of myocardial cells. Cells were treated separately:myocardial cells were treated with different concentrations of glucose (5.5,25,33.3,50mmol/L). To identify suitable for the required concentration, time stimulation (33.3mmol/L,72h) for high (HG) the research object. HG group with Control siRNA, RIP3siRNA and GLP-1of different concentrations after incubation of cells, and design corresponding control group.
siRNA Technology:inhibition of RIP3expression by siRNA technology. RIP3siRNA pretreatment of24h with myocardial cells induced by high glucose, and72h, expression of charged cells by flow cytometry and Western-Blot technique for the detection of cell apoptosis and the target protein.
3. Myocardial apoptosis was detected by flow cytometry
Using Annexin V/PI flow cytometry staining method, detection of myocardial cellular apoptosis occurrence, incidence rate of the myocardial cell apoptosis.
4. Detection of mitochondrial membrane potential
Using the JC-1method, by laser confocal microscopy, fluorescence detection of mitochondrial membrane potential change in value, detection of mitochondrial membrane potential (AΨm) changes.
5. real-time quantitative RT-PCR detection
Cells were collected, total RNA was extracted by reverse transcription reaction, cDNA, β-actin as a reference, through the detection of RIP3expression of mRNA by RT-PCR.
6. Western-Blot detection
All the cells were collected, extracted total protein, respectively by SDS-poly acrylamide gel electrophoresis separation, membrane protein, mark, color and other steps, to detect the expression of RIP3, cytochrome C, Caspase-3, cleaved caspase3.
Results
1. Myocardial apoptosis induced by Selection of the glucose concentrations and choices of time
5.5mmol/L[NG],25%[HG25],33.3%[HG33.3],50%[HG50] of different concentra tions of glucose and mannitol in control medium with mannitol Group was0,24,48cells,72h, flow cytomctry apoptosis is shown, NG and mannitol than in control group, HG33.3, HG50stimulation of48h (P<0.05),72h (P<0.01) could significantly increase cellular apoptosis, HG25stimulation of72h (P<0.05) can increase cellular apoptosis. Therefore, the high concentration of glucose can induce the apoptosis of myocardial cells, combined with myocardial cellular viability results, with33.3%glucose for72hours as the object of study.
2. Relationships between the expression of RIP3and apoptosis of myocardial cells induced by high glucose
2.1Overexpression of RIP3during the course of apoptosis induced by high glucose in myocardial cells
(1) Myocardial cells after stimulation of72h with a high concentration of glucose (33.3%)[HG], collected. The results were displayed by real-time quantitative RT-PCR and Western-Blot assay:compared with5.5%concentrations of glucose[NG], both mRNA (P<0.05) and protein (P<0.05) levels of the expressions of RIP;, elevated significantly in HG group.
(2) RIP3siRNA, Control siRNA were transfected into primary cultured cardiomyo cytes after24h, then the cells stimulated with high glucose for72h. siRNA transfection efficiency was verified by RT-PCR and Western-Blot. Compared with the HG group and control siRNA transfection group, RIP3siRNA transfection group demonstrated that the expressions of both mRNAs (P<0.05) and protein (P<0.05) decreased significantly, suggesting that RIP3siRNA inhibited the expression of RIP3effe
糖尿病(Diabetes Mellitus, DM)病程缓慢,并发症多且常累及心、脑、肾等全身重要脏器,糖尿病心肌病(diabetic cardiomyopathy, DCM)为常见、严重的并发症之一。DM糖脂代谢紊乱可导致心肌组织病理改变为心肌细胞肥大、凋亡、坏死,间质纤维化,炎症等,表现为进行性左心室结构改变、功能异常,死亡率极高。DM治疗现状目前并不乐观,仍存在诸多间题,单纯降低血糖治疗并不能完全降低心血管病事件。因此,新型降糖药物除具有保护胰岛β细胞功能、降糖作用外,还应具有保护靶器官、降低死亡率的作用,成为当前基础研究与临床治疗策略的重点。
胰高血糖素样肽-1(GLP-1)是主要源于肠道末端L细胞分泌的肠促激素,受体在胰岛、心肌、脑等组织内广泛表达,作用在机体摄入食物后,具有葡萄糖依赖性释放、促进餐后胰岛素分泌等作用。2型DM患者体内,GLP-1分泌水平或活性显著F降,且内源性GLP-1可被二肽基肽酶(DPP-Ⅳ)快速分解。因此,DPP-Ⅳ抑制剂-西格列汀可使内源GLP-1短期不被降解,具有升高GLP-1水平,增强并延迟胰岛素释放来降低血糖。晚近报道证实GLP-1可抑制缺血／再灌注损伤后的细胞凋亡
报道DPP-Ⅳ抑制剂可上调GLUT-4的表达,GLP-1受体兴奋剂可降低2型DM患者高甘油三酯血症、逆转脂肪肝。DM心肌病变中Ⅰ、Ⅲ型胶原沉积、排列紊乱,胶原降解酶(主要基质金属蛋白酶系统MMPs/TIMPs)表达异常,晚近报道,GLP-1受体拮抗剂可抑制脂多糖诱导心肌细胞表达MMP-9,提示可能影响胶原酶的表达。心脏舒张功能不全病人外周血中DPP-Ⅳ活性与超声参数E／E'比值呈正相关。GLP-1受体激动剂可抑制TGF-β1表达。尚未见DPP-Ⅳ抑制剂影响DM大鼠糖脂代谢、心肌脂质含量以及心肌胶原网络代谢的相关研究。
心肌细胞凋亡、坏死广泛存在于DM心肌组织中。DM心肌组织中TNF-α、IL-6表达增加,TNF-α与细胞膜TNFR1结合形成信号转导复合物,可引发细胞内Bax.Bcl-2等基因异常表达,致细胞凋亡。受体相互作用蛋白家族(RIPs)是一类重要调节细胞死亡、存活的信号分子,其中RIP3具有自磷酸化、诱导细胞凋亡与激活NF-kB的作用,是参与TNF-α介导的死亡受体凋亡通路的重要信号分子,且RIP3与线粒体能量代谢有关。DM心肌细胞凋亡中,,RIP3是否参与其中，及DPP-Ⅳ抑制剂对DM心肌细胞凋亡、RIP3表达的影响以及两者的相关性,目前尚未见相关报道。
DPP-Ⅳ抑制剂(西格列汀)通过抑制DPP-Ⅳ活性,提高GLP-1水平、发挥其生物学作用。因此,在既往研究基础上,本部分从动物水平研究西格列汀对DM大鼠心肌组织的炎症、胶原网络代谢、脂质含量及心肌细胞凋亡等病理改变的影响;对DM心肌组织中RIP3的表达及与凋亡的关系：并探讨该药对DM大鼠左心室功能的影响。通过本研究为DM的防治提供更多的方法。
研究目的
1.探讨DPP-Ⅳ抑制剂对DM心肌组织的炎症反应、胶原网络代谢、脂质含量及心肌细胞凋亡等心肌重塑的影响；
2.初步探讨RIP3在DM心肌中的表达及DPP-Ⅳ抑制剂的干预作用;
3.评价DPP-Ⅳ抑制剂对DM大鼠左心功能的影响。
研究方法
1.DM大鼠模型的构建、分组及药物干预
8周龄雄性Wistar大鼠70只,随机分为四组：对照组(n=10只)[Control]；糖尿病组(n=20只)[DM];糖尿病+DPP-Ⅳ抑制剂西格列汀组[(低剂量组,30mg/kg/d)(n=20只)[DPP-IVi(Low)]；糖尿病+DPP-Ⅳ抑制剂西格列汀组(高剂量组,50mg/kg/d)(n=20只)[DPP-IVi(High)]。糖尿病各组高脂饲养4周(w)后,糖尿病各组大鼠腹腔注射小剂量STZ30mg/kg.2型糖尿病大鼠成模的诊断标准为：STZ注射后1周内,连续2次空腹血糖≥11.1mmol/L,为糖尿病模型。模型稳定后持续4w,给予对照组、DM组予以生理盐水灌胃,DM药物干预两组分别给予不同剂量DPP-Ⅳ抑制剂西格列汀研钵碾碎生理盐水稀释后灌胃,均再继续喂养到实验结束,处死称取心脏重量,计算心脏重量／体重[HW/BW(mg/g)]比值后,留取标本。
2.各组大鼠基本指标、糖脂代谢系列指标及GLP-1水平的监测
实验过程中除常规每2周测1次心率、体重、血压与血糖外,期间,根据研究需要,各时间点为0、4、5、9、13、17、21w,抽取颈静脉窦血1.5ml左右,分离血清,全自动生化分析仪测定血总胆固醇、总甘油三酯、低密度脂蛋白胆固醇以及高密度脂蛋白胆固醇水平,以及免疫发光法测定糖化血红蛋白,酶联免疫法法测血清FFA、GLP-1水平,以及空腹血清胰岛素水平,计算胰岛素敏感指数。
3.心电图
根据实验不同时间点,描记肢体导联(Ⅰ、Ⅱ、Ⅲ、aVR、aVL、aVF)心电图,观察各组大鼠心脏电活动情况并记录心电图。
4.超声心动图
分别在0、13w、7w、21w不同时间点进行超声心动图检查,测定指标如下：左室收缩末内径(LVIDs)、左室舒张末内径(LVIDd)、射血分数(LVEF)、左室短轴缩短率(FS)；二尖瓣口E波最大速度、A波最大速度、E／A；组织多普勒(TDI)二尖瓣环左室侧壁测E’波最大速度、A’波最大速度,E'／A',并计算E／E'。
5. Millar导管测左心功能
暴露左室心尖部后,利用10ml注射器针头,内有Milllar导管电极探头直接行心尖穿刺精确控制插入左心室,软件分析心血管参数,左室收缩压(LVSP),左室舒张末压(LVEDP)、左室内压最大上升速率(+dp／dt)、左室内压最大下降速率(-dp/dt)。记录、导出有关数据、图像资料,并计算左室松弛时间常数Tau=P/(-dp/dt)。6
6.心肌组织病理学检测
实验结束处死动物后,进行取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规HE染色、Masson染色及天狼星红染色,观察心肌组织结构、细胞形态、胶原分布情况等。油红O染色用冻存心肌组织标本。以3%戊二醛固定心肌组织后行透射电镜观察。并测心肌羟脯氨酸含量计算胶原含量。
7. TUNEL染色
石蜡切片行免疫组化法检测凋亡细胞,显微镜下观察、计数阳性染色细胞即凋亡细胞百分比数,并进行统计分析。
8.免疫组织化学染色
取心肌组织石蜡切片,分别免疫组化检测左心肌组织炎性因子TNF-α、IL-6,胶原Ⅰ、Ⅲ与胶原降解酶MMP-1、9,以及RIP3的表达,并进行综合分析。
9.实时定量RT-PCR基因水平检测
取-80℃冻存的心肌组织,Trizol法提取心肌组织总RNA,实时定量RT-PCR技术检测RIP3mRNA的表达水平。
10. Western blot检测
取-80℃保存的心肌组织,提取总蛋白,Western-Blot (?)检测左室心肌组织内GLP-1含量及其受体表达,炎性因子TNF-α、IL-6,胶原Ⅰ、Ⅲ,部分胶原降解酶MMP-1、9,以及RIP3的蛋白表达。
11.统计学分析
所用数据用SPSS17.0统计软件进行分析。数值变量资料数据以均数±标准差表示,两组间均数比较采用小样本t检验,多组间均数比较采用One-Way ANOVA方差分析,以P<0.05为差异有统计学意义。
研究结果
1.大鼠的一般情况
糖尿病大鼠造模中,共4只未达标准予以剔除,整个实验中共死亡8只,均为糖尿病各组,最终58只大鼠最终完成实验。其中,Control组10只；DM组15只;DM+DPP-IVi (Low)组16只;DM+DPP-IVi (High)组17只。对照组精神状态好,体重增加,反应敏捷,毛色白而光泽。DM组出现多饮、多尿和消瘦症状,精神差,体重增加迟缓甚至下降,皮毛脏乱无光泽。药物干预后的两组,与DM组相比,一般情况均较好。实验开始时,各组大鼠心率、体重和血压均无差异(P>0.05)。结束时,DM各组与对照组比较,体重下降(P<0.01-0.05),心率、血压升高(P<0.01-0.05)；药物干预后的两组与DM组比较,体重增加及心率、血压不同程度下降(P<0.01-0.05)。
2.血液指标检测结果
2.1各组糖脂代谢指标的比较
造模前、高脂饲养4周后,糖尿病各组存在胰岛素抵抗状态；造模后整个实验过程中,DM组与对照组比较,FBG水平、上午10：00血糖水平及HbA1c,均显著升高(P<0.01)：药物干预后的两组与DM组比较,均不同程度下降(P<0.01-0.05)；造模成功后1周FINS无显著差异,ISI显著下降(P<0.05)；实验末,DM组与对照组、两药物干预组比较,FINS显著下降(P<0.01-0.05)；ISI在实验第5-21周,DM组与对照组比较,显著下降(P<0.05)。
糖尿病造模成功后整个实验过程中,DM各组较对照组,血TC、TG、LDL-C、FFA水平均明显升高(P<0.01-0.05)；药物干预后的两组与DM组比较,均不同程度的降低(P<0.01-0.05)。
2.2检测空腹、口服葡萄糖30min后GLP-1的血浆水平
糖尿病造模成功并且模型稳定后,于第8周开始给予药物干预,DM与对照组比较,分别空腹、口服葡萄糖30min后GLP-1血浆水平均呈不同程度降低(P<0.01-0.05)；药物干预后的两组与DM组比较,在第13-21周,空腹、口服葡萄糖30min后,GLP-1血浆水平均有不同程度的升高(P<0.01-0.05),尤其DM组给予高剂量DPP-Ⅳ抑制剂干预后,与对照组比较,无显著性差异(P>0.05)。
3.心电图的变化
对照组ECG清晰可见P、QRS、T波,QRS波振幅一致。DM组10只心电图异常表现,占66.67%,且QRS“电交替”现象多见,另可见室上速、室速、心动过缓等各种复杂心律失常；两药物干预组中,心电图异常总计14只,占42.42%,未见复杂心律失常且“电交替”减少,提示DPP-Ⅳ抑制剂可改善心脏电活动。
4.超声测量心脏结构和功能指标的变化
左心结构的改变：实验开始时,各组之间左心腔室大小,LVIDs及LVIDd均无显著性差异(P>0.05)；实验结束时,DM组与对照组比较,LLVIDs及LVIDd (?)(?)显著扩大(P<0.05);药物干预后的两组与DM组比较,LVIDs及LVIDd均明显减小(P<0.05)
左室收缩功能的评价：实验开始时,各组大鼠间FS和EF的变化均无显差异(P>0.05)。实验结束时,DM组与对照组比较,FS、LVEF均不同程度降低(P<0.0l-0.05)；药物干预后的两组与DM组比较,FS、LVEF均不同程度升高(P<0.01-0.05)。
左室舒张功能的评价：实验开始时,各项指标均无显著差异(P>0.05);DM组与对照组比较,第13w开始至21w实验结束时,E／A、E'／A’比值不同程度降低(P<0.01-0.05)；E／E’比值不同程度升高(P<0.01-0.05);药物干预后的两组志DM组比较,E／A、E'／A’比值不同程度升高(P<0.01-0.05),E／E’不同程度降低(P<0.01-0.05)。
5. Millar导管测量各组左室功能指标的比较
与对照组比较,DM组LVSP明显下降(P<0.01)、LVEDP升高(P<0.01)、±dp/dt max绝对值降低(P<0.01-0.05)及Tau绝对值延长(P<0.01)。与DM组比较：LVSP在大剂量DPP-Ⅳ抑制剂干预后明显升高(P<0.05),在两组DPP-Ⅳ抑制剂干预后,+dp/dt max (mmHg/s)(?)匀升高(P<0.05),ILVEDP均显著下降(P<0.05),-dp/dt max (mmHg/s)绝对值均升高(P<0.05),左室松弛时间常数(Tau)绝对值不同程度缩短(P<0.01-0.05),大剂量药物干预组Tau缩短更显著。
6.DM大鼠左室心肌重塑及DPP-Ⅳ抑制剂的影响
6.1大体标本病理改变及HE染色
DM组与对照组比较,心脏标本大体形态表现体积较大,HW/BW (mg/g)较大(P<0.05)；HE染色可见左室心腔扩大、室壁肥厚；细胞横截面积明显增加(498.37±14.31vs347.84±12.27μm3, P<0.01);HE染色可见,心肌细胞肥大,可见肌纤维排列紊乱、断裂现象,形态不规则,核大小不均。药物干预后的两组与DM组比较,心脏体积、HW/BW (mg/g)比值变小(P<0.05)；左室心腔缩小,肥厚减轻；心肌细胞横截面积不同程度减少(P<0.01-0.05); HE染色可见心肌细胞体积不同程度减小,细胞排列较规则、有序,核大小尚均一,高剂量药物组则改善更明显。
6.2DPP-Ⅳ抑制剂对DM心肌组织GLP-1含量、GLP-1R表达的影响
实验结束时,Western-Blot提示：与对照组比较,DM组心肌组织GLP-1含量显著降低(P<0.0),GLP-1R表达也减少(P<0.01)；药物干预后的两组与DM组比较,心肌组织GLP-1含量、GLP-1R表达均不同程度升高(P<0.01-0.05),提示DPP-Ⅳ抑制剂干预后,心肌组织内GLP-1含量升高,且可提高GLP-1受体表达的密度。
6.3DPP-Ⅳ抑制剂减少DM心肌组织胶原的含量
Masson染色所见：DM组与对照组大鼠比较,胶原纤维粗大,排列紊乱,分布不均,沉积增多；药物干预后的两组与DM组比较,沉积的心肌胶原减少,排列尚有序、分布也尚均匀。
天狼猩红染色所见：DM组与对照组比较,心肌组织内胶原纤维较明显增加,排列紊乱,且可见不规则网状胶原纤维围绕在小动脉周围;药物干预后的两组与DM组比较,可见心肌组织胶原沉积减少。
胶原含量比较：与对照组比较,DM组左室心肌组织胶原含量(15.86±0.43vs7.06±0.31μg/mg, P<0.01)、胶原容积分数(CVF%)(4.8±0.49vs0.47±0.06,P<0.01-0.05)以及血管周围胶原面积／管腔面积比(PVCA/LA)(2.74±0.37vs0.5±0.02,P<0.01-0.05)均不同程度升高。药物干预后的两组与DM组比较,左室心肌组织胶原含量均不同程度减少。
免疫组化染色：DM组心肌组织与对照组比较,Ⅰ、Ⅲ型胶原阳性表达显著增加；不同剂量DPP-IV抑制剂干预后,可见心肌组织棕色颗粒不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织Ⅰ、Ⅲ型胶原蛋白表达显著升高(P<0.01),药物干预后的两组与DM组比较,均可见心肌组织Ⅰ、Ⅲ型胶原蛋白表达不同程度降低(P<0.01-0.05)。
6.4DPP-Ⅳ抑制剂对糖尿病大鼠心肌胶原酶MMP-1、9表达的影响
免疫组化染色：DM组与对照组比较,MMP-1、9阳性表达显著增加。不同剂量DPP-Ⅳ抑制剂干预后,心肌组织棕色颗粒不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织MMP-1、9蛋白表达显著升高(P<0.05),药物干预后的两组与DM组比较,心肌组织MMP-1、9表达均不同程度降低(P<0.01-0.05)。
6.5DPP-Ⅳ抑制剂对糖尿病大鼠心肌组织炎性因子TNF-α、IL-6表达的影响
免疫组化染色：对照组心肌细胞浆内见少量、稀疏的浅棕色颗粒；DM组心肌细胞胞浆内可见较多浓密深棕色颗粒。与DM组比较,不同剂量DPP-Ⅳ抑制剂干预后,可见心肌组织棕色颗粒分布散在、稀疏，有不同程度减少。
Western-Blot:DM组与对照组比较,心肌组织TNF-α、IL-6蛋白表达显著升高(P＜0.01),药物干预后的两组与DM组比较,心肌组织TNF-α、IL-6蛋白表达均下降(P<0.05)。
6.6DPP-Ⅳ抑制剂减少DM心脏脂质的沉积
油红O染色结果,与对照组比较,DM组心肌脂肪含量(7.03±0.31vs0.32±0.01,P<0.01)、心外膜脂肪组织含量(12.76±0.48vs0.47±0.03,P<0.01)均显著升高；药物干预后的两组与DM组比较,可见心肌组织脂肪含量、心外膜脂肪组织含量均呈不同程度减少(P<0.01-0.05)。
6.7糖尿病心肌组织RIP3的表达及IDPP-Ⅳ抑制剂的干预
免疫组化染色：DM组与对照组比较,RIP3棕黄色阳性颗粒表达增加,且分布在细胞核周围较多,不同剂量DPP-Ⅳ抑制剂干预后,细胞内棕黄色阳性颗粒表达不同程度减少。
实时定量RT-PCR、Western-Blot检测：与对照组比较,DM组大鼠心肌组织RIP3mRNA水平、蛋白水平表达均显著增高(P<0.01);药物干预后的两组与DM组比较,RIP3mRNA水平、蛋白水平表达均不同程度降低(P<0.01-0.05)。
6.8透射电镜观察DPP-Ⅳ抑制剂对糖尿病心肌细胞超微结构的影响
DM心肌细胞核形状不规则,核固缩,染色质聚集,呈现凋亡细胞的早期表现；对照组心肌细胞核仁清晰可见,核膜光滑、完整。药物干预后的两组,细胞核未见明显染色质聚集,无核固缩表现,核膜尚完整、光滑。
6.9TUNEL染色观察DPP-Ⅳ抑制剂对糖尿病心肌组织细胞凋亡发生率的影响
与对照组比较,DM组左室心肌组织凋亡细胞阳性率显著增加(P<0.01),药物干预后的两组与DM组比较,细胞凋亡发生率显著减少(P<0.01)。
6.10初步分析糖尿病心肌组织RIP3mRNA表达与细胞凋亡率的相关性
结合糖尿病心肌组织RIP3mRNA表达与糖尿病心肌组织细胞凋亡百分率,初步分析糖尿病大鼠心肌组织RIP3mRNA表达与细胞凋亡百分率的相关性,得知Pearson相关系数r=0.795(P<0.00]),可以初步判断糖尿病大鼠心肌组织细胞凋亡百分率与RIP3mRNA的表达呈显著正相关。
研究结论
1.DPP-Ⅳ抑制剂影响DM心肌组织炎症反应、胶原代谢、脂质含量及心肌细胞凋亡的改变,逆转DM心肌病理性重塑；
2.DM心肌组织中,RIP3表达上调,且与心肌组织细胞凋亡率呈显著正相关；DPP-Ⅳ抑制剂可干预DM心肌组织中RIP3过表达,抑制心肌细胞凋亡;
3.DPP-Ⅳ抑制剂可显著降低DM大鼠左室E／E’,降低左室舒张末压(LVEDP)和-dp/dt max,增加LVEF和+dp/dt max,缩短Tau。显示DPP-Ⅳ抑制剂明显改善DM大鼠左心室收缩和舒张功能。
研究背景
细胞凋亡系基因调控、主动、程序性死亡的病理生理过程,对维持机体内环境稳态极其重要,糖尿病(DM)心肌病变的主要病理改变之一是心肌细胞凋亡,且随凋亡细胞的增多,最终导致心肌组织重构与心脏功能障碍。
新近研究证实,RIPs (receptor-interacting protein)家族作为一类重要的调节细胞生存、凋亡、坏死的信号分子备受重视,其中RIP3具有自磷酸化、诱导细胞凋亡和激活NF-kB的作用。多种细胞株中过表达RIP3,则会发生细胞凋亡。报道RIP3是TNF-a诱导的细胞凋亡-坏死转换的“分子开关”,且RIP3与线粒体能量代谢、ROS产生等密切相关。RIP3调节能量代谢相关酶相互作用,并产生过量ROS诱发细胞程序性坏死。因此,RIP3参与细胞内多种信号通路,并发挥重要的生理功能。结合课题第一部分的研究,发现DM心肌组织中存在RIP3表达上调,且与心肌组织细胞凋亡率呈显著正相关。但是,高糖刺激下,线粒体功能异常,细胞能量供给障碍,RIP3参与能量代谢相关酶的作用、活性异常等,影响线粒体功能及膜电位改变介导心肌细胞调亡的研究目前国内外尚未见相关报道。
晚近报道,胰高血糖素样肽-1(GLP-1)除降糖作用外,尚具有抑制缺血／再灌注后心肌细胞凋亡、改善心肌梗死后心力衰竭的作用。且GLP-1可能通过激活PI3K、 MAPK信号通路抑制胰岛细胞发生凋亡。课题第一部分动物水平研究发现,DPP-Ⅳ抑制剂通过提高GLP-1水平可抑制DM心肌组织中RIP3过表达、并降低心肌细胞凋亡率。既往研究表明,高糖可直接刺激,或引发Bcl-2表达改变,启动线粒体损伤信号通路,导致线粒体膜通透性增加,线粒体内的细胞色素C释放入胞质,启动caspase级联反应,细胞调亡。但是,国内外尚未见高糖环境中GLP-1对RIP3表达的调节及是否参与以上凋亡通路的研究。
因此,基于既往报道与课题第一部分动物水平的研究,本部分从细胞水平研究提出可能存在的假设：RIP3高表达参与糖尿病心肌细胞TNF-α介导的死亡受体活化的凋亡通路；也参与线粒体损伤的凋亡通路,RIP3直接或间接(通过调节能量代谢过程中产生过量ROS)使线粒体膜通透性改变,线粒体内细胞色素C释放入胞浆,激活型Caspase-3表达增加,发生细胞调亡;而沉默RIP3可改善此病理改变；GLP-1可能通过多个信号通路干预RIP3过表达,从而抑制细胞凋亡。为进一步验证此过程,本部分采用高糖刺激心肌细胞发生凋亡过程中,探讨RIP3在此过程中表达的改变及GLP-1对其影响以及可能存在的机制,为阐明GLP-1、RIP3参与高糖诱导的心室肌细胞凋亡及可能的相关信号通路提供理论依据。
研究目的
1.验证高糖刺激可诱导心室肌细胞调亡,凋亡与RIP3表达相关；
2.探讨高糖诱导心室肌细胞调亡过程中, RIP3在线粒体损伤凋亡途径中的可能作用;
3.探讨高糖刺激下,GLP-1对RIP3表达的影响及可能的信号通路。
研究方法
1.原代心肌细胞培养与鉴定
取1～3日新(?)Wistar(?)鼠心脏,分离出左心室后剪碎、胰酶消化、差速贴壁法获得心肌细胞,可见心肌细胞搏动,并进行原代心肌细胞培养。采用肌钙蛋T单克隆抗体进行免疫荧光染色、荧光显微镜下观察并鉴定为原代培养的心肌细胞。
2.实验分组与干预
体外模拟生理状态、糖尿病状态,加入不同的刺激因素,研究心肌细胞干预后的变化。细胞分别进行处理：心肌细胞分别用不同浓度葡萄糖(5.5、25、33.3、50mmol/L)处理：找出适合实验所需浓度、时间刺激(33.3mmol/L,72h)为高糖(HG)研究对象；分别HG组加入Control siRNA、RIP3siRNA及不同浓度GLP-1干预后孵育细胞,并设计相应对照组。
siRNA技术：采用siRNA技术抑制RIP3表达。将心肌细胞用RIP3siRNA预处理24h,再高糖刺激72h,收取细胞经流式细胞技术和Western-Blot等技术检测细胞调亡及靶蛋白的表达。
3.流式细胞仪检测心肌细胞调亡
采用Annexin V/PI染色流式细胞技术方法,检测心肌细胞的调亡发生情况,得出心肌细胞凋亡的发生率。
4.线粒体膜电位的检测
利用JC-1的方法,经激光共聚焦显微镜观察,检测线粒体膜电位荧光值变化,检测线粒体膜电位(△Ψm)的变化。
5.实时定量RT-PCR检测
收集细胞,提取总RNA,经逆转录反应获得cDNA,以β-actin作参照,通过实时定量RT-PCR技术检测RIP3mRNA水平的表达。
6. Western-Blot检测
收集细胞,提取总蛋白,分别经过SDS-聚丙烯酰胺凝胶电泳分离、转膜、蛋白印记、显色等步骤,检测RIP3、细胞色素C、cleaved caspase3的表达。
研究结果
1.葡萄糖刺激心肌细胞凋亡的时间、浓度的选择
5.5mmol/L[NG]、25%[HG25]、33.3%[HG33.3]、50%[HG50]不同浓度葡萄糖及甘露醇对照组[甘露醇]的培养基孵育细胞0、24、48、72h后,流式细胞仪检测凋亡示,较NG与甘露醇对照组比,HG50刺激48h(P<0.05)、72h(P<0.01)均可显著增加细胞凋亡,HG25刺激72h(P<0.05)可增加细胞凋亡。因此,高浓度葡萄糖可诱导心肌细胞凋亡,结合心肌细胞活力的结果,选用33.3%葡萄刺激72小时为研究对象。
2.RIP3表达与高糖诱导心肌细胞凋亡发生的关系
2.1高糖诱导心肌细胞调亡中RIP3表达升高
(1)利用高浓度葡萄糖(33.3%)[HG]刺激心肌细胞72h后,收取细胞,实时定量RT-PCR及Western-Blot法检测,结果显示：与5.5%浓度葡萄糖[NG]比较,HG组RIP3在mRNA (P<0.05)与蛋白水平(P<0.05)均显著升高;
(2)将原代培养的心肌细胞先应用RIP3siRNA、Control siRNA转染心肌细胞24h后,再予以高糖刺激72h,收取细胞利用实时定量RT-PCR和Western-Blot验证siRNA转染效力,结果RIP3siRNA转染组与HG组、Control siRNA转染组比较,RIP3在mRNA(P<0.05)及蛋白(P<0.05)表达水平显著下调,提示RIP3siRNA能有效抑制RIP3表达。
2.2RIP3siRNA可降低高糖诱导的心肌细胞调亡发生率
采用流式细胞技术分别对5.5mmol/L浓度葡萄糖组(NG)、高糖33.3%浓度葡萄糖组(HG)、HG+Control siRNA转染组、HG+RIP3siRNA转染组的心肌细胞调亡率进行检测,结果显示：NG组比较,高糖可诱导心肌细胞凋亡发生(P<0.01-0.05);与HG组、HG+Control siRNA组比较,RIP3siRNA转染后,心肌细胞凋亡率显著下降(P<0.05)。提示RIP3在细胞调亡过程中发挥重要喽作用,其高表达可诱导高糖刺激的心肌细胞发生凋亡。
3. RIP3高表达通过线粒体凋亡通路诱导高糖刺激的心肌细胞发生凋亡
3.1RIP3siRNA对高糖环境心肌细胞线粒体膜电位(△Ψm)的影响
使用激光共聚焦显微镜观察JC-1单体／聚合物(JC-1monomer/polymer)的荧光值比值,绿色荧光(单体)提示△Ψm下降,细胞凋亡前期；红色荧光(聚合体)提示△ψm较正常,细胞状态正常。
与NG组比较,绿色荧光(单体)／红色荧光(聚合体)比值在HG组(P<0.05)、HG+Contro] siRNA组(P<0.05)显著升高；HG组细胞经RIP3siRNA转染后,与HG、HG+Control siRNA组比较,其比值显著下降(P<0.05)。提示高糖刺激心肌细胞凋亡中,R1P3可能影响线粒体功能,致线粒体△Ψ／m下降。
3.2RIP3siRNA对高糖环境心肌细胞浆细胞色素C(Cyt C)含量、激活型Caspase-3表达的影响
经Western-Blot分析,与NG组比较,HG组、HG+Control siRNA组心肌细胞浆Cyt C含量(P<0.05)、激活型Caspase-3表达(P<0.05)均显著升高；RIP3siRNA转染后,HG+RIP3siRNA组与HG、HG+Control siRNA组比较,心肌细胞浆Cyt C含量(P<0.05)、激活型Caspase-3(P<0.05)表达均显著降低。提示RIP3在高糖环境心肌细胞凋亡中参与线粒体凋亡通路。
4. GLP-1对RIP3表达的调控及机制
4.1GLP-1对高糖诱导心肌细胞RIP3表达的影响及信号通路
Western-Blot结果显示：与单纯HG刺激组比较,HG+GLP-1(1nmol/L)组无差异(P>0.05),HG+GLP-1(10nmol/L)组RIP3蛋白表达明显下调(P<0.01)；加HG+GLP-1(10nmol/L)之前分别加MAPK阻断剂(LY294002)、PI3K阻断剂(U0126)干预的2组与HG+GLP-1(lOnmol/L)组比较,RIP3蛋白表达升高亦有明显差异(P<0.05)。提示GLP-1呈浓度依赖抑制高糖诱导的RIP3过表达,且可能部分通过MAPK、PI3K信号通路调控此过程。
4.2GLP-1通过抑制RIP3表达降低高糖刺激的心肌细胞凋亡率
本实验中,采用GLP-1(10nmol/L)干预、沉默RIP3分别交换先后顺序干预细胞,共5组,利用流式细胞仪结果显示,与单纯高糖刺激组(HG)比较,GLP-1干预、沉默RIP3均可降低细胞凋亡率(P<0.01-0.05)；与HG+GLP-1(10nmol/L)比较,沉默RIP3组[HG+GLP-1(10nmol/L)+RIP3siRNA、HG+RIP3siRNA、HG+RIP3siRNA+GLP-1(10nmol/L)]细胞凋亡率也显著降低(P〈0.05)；GLP-干预后沉默RIP3组与单纯GLP-1于预组比较,细胞凋亡率降低(P<0.05)；单纯沉默RIP3组与沉默RIP3后加GLP-1干预组比较,组间无统计学差异(P>0.05)。
4.3GLP-1抑制RIP3表达降低高糖环境细胞浆Cyt C含量、激活型Caspase-3表达
Western-Blot分析,与HG组比较,lHG+GLP-1(10nmol/L)(?)且心肌细胞浆Cyt C含量、激活型Caspase-3表达均显著减少(P<0.05)；HG分别经RIP3siRNA转染干预、GLP-1(lOnmol/L)干预后,心肌细胞浆Cyt C含量、激活型Caspase-3表达与HG组(P<0.01)、HG+GLP-1(10nmol/L)组(P<0.05)均显著减少。
此实验提示GLP-1可能部分通过MAPK、PI3K(?)信号抑制RIP3表达,从而降低高糖刺激的心肌细胞凋亡率。
研究结论
1.高糖刺激可诱导心肌细胞调亡,同时RIP3表达增高：沉默RIP3后细胞凋亡率降低;
2.高糖刺激心肌细胞凋亡中,RIP3可能通过影响线粒体能量代谢,膜电位降低、通透性增加,线粒体内细胞色素C释放入胞浆,启动Caspase级联反应,诱导细胞凋亡;
3.GLP-1可能部分通过MAPK、P13K信号通路抑制RIP3表达,进而抑制高糖诱导的心肌细胞凋亡。
Background
The duration of diabetes is very slow. During the course if diabetes, many complications could damage the main organs such as the heart, the brain and the kidney. Diabetic cardiomyopathy is one of the relatively common and serious complications. The main pathological changes of diabetic cardiomyopathy are myocardial hypertrophy, apoptosis or necrosis of myocardiocytes, myocardial fibrosis and inflammation. The pathogenesis of diabetic cardiomyopathy is very complex, possibly related to the disorders of myocardial glucose metabolism and energy metabolism, calcium overload, myocardial RAS activity, oxidative stress, insulin resistance, cardiac autonomic neuropathy and TGF-/β or TNF-α abnormal expression. The clinical findings of diabetic cardiomyopathy are usually progressive abnormal changes of left ventricular structure and function, or even high mortality rate. However, the current treatment of diabetes is not so satisfactory that there are still many deficiencies and the incidence of diabetic complications is still high. Therefore, the novel anti diabetic drugs should not only lower the blood glucose and protect the function of islet beta cells, but also protect the main target organs and delay the progressive lesions of diabetic cardiomyopathy in order to reduce the mortality. Hence, the treatment of diabetes becomes the focus of the current strategies of the basic research and clinical treatment.
Glucagon-like peptide-1(GLP-I) is one of the incretin hormones secreted from the distal intestine L cells and GLP-1receptors (GLP-1R) are widely expressed in the tissues islated from the islets, myocardium and the brain. GLP-I begins to work after the food intake in the body, which has a glucose-dependent release and promotes the secretion function of postprandial insulin. The serum levels or activities of GLP-1usually decrease in diabetic patients and the endogenous GLP-1could be dissolved rapidly by dipeptidyl peptidase (DPP-IV). Therefore, sitagliptin as one of the DPP-IV inhibitors could make the endogenous GLP-1not be degraded in the short term, elevate the serum levels of GLP-1and enhance or delay the release of insulin in order to lower the blood glucose. Recent reports demonstrated that GLP-1could play a key role in the progress of inhibiting apoptosis during the course of ischemia/reperfusion injury and improve the cardiac function.
DPP-IV inhibitor was reported to function as the up-regulation of the expression of GLUT-4. GLP-1receptor agonist can reduce the hypertriglyceridemia and reverse fatty liver in diabetic patients. However, little research is reported about the effects of DPP-IV inhibitors on the glucose and lipid metabolism in myocardium.
The characteristic pathological changes of myocardial lesions in diabetic patients are the imbalances of extracellular matrix, the deposition or disorders arranged of type I and Ⅲ collagen and the abnormal expression of degrading enzyme (main matrix metalloproteinase system MMPs/TIMPs). Recently, GLP-1receptor antagonists can inhibit the expression of MMP-9induced by lipopolysaccharide in the myocardial cells, which implied GLP-1could inhibit the expression of collagenase. The DPP-IV activity in peripheral blood of the patients with cardiac diastolic dysfunction was positively associated with the E/E'ratio measured by echocardiology and the GLP-1receptor agonist can inhibit the expression of TGF-β1. However, no research is reported about the effects of DPP-IV inhibitor on the network of the cardiac collagen metabolism in diabetic rats and cardiac diastolic dysfunction.
Apoptosis and necrosis of myocardial cells exist widely and the expression of TNF-a or IL-6increase in the myocardial tissue with diabetic rats. The formation of signal transduction complexes results from the combination of TNF-a and TNFR1in cell membrane, can lead to the abnormal expression of Bax, Bcl-2. Receptor interacting protein family (RIPs) is an important signaling moleculear regulating death and survival of the cells. RIP3has autophosphoryl ation, induction of apoptosis and activation of NF-κB effect, is an important signal molecules involved in TNF-a mediated by death receptor pathway of apoptosis, and RIP3and mitochondrial energy metabolism. However, no related research are reported about whether RIP3is involved in apoptosis of myocardial cells in diabetic rats and the effect of DPP-Ⅳ inhibitor on the expression of RIP3in apoptotic myocardial cell, and even the correlation between the two factors.
Sitagliptin as one of the DPP-Ⅳ inhibitors (inhibiting enzyme DPP-IV activity) plays its biological role via improving the levels of GLP-1. Hence, the effects of sitagliptin on inflammation, network of collagen metabolism, lipid content and myocardial apoptosis in diabetic rats would be studied in this part based on the previous studies. This work would detect the effect of sitagliptin on the expression of RIP3in myocardial tissues in diabetic rats and the relationship with apoptosis and explore the effect of the drug on left ventricular function in diabetic rats. More additional treatments would be provided through the research for the prevention and treatment of diabetes.
Objectives
1. To investigate the effects of DPP-IV inhibitors on the pathological remodeling including inflammation, network of collagen metabolism, lipids contents and apoptosis of myocardial cells in the myocardial tissues of diabetic rats.
2. Preliminary study on the relationships between the expressions of RIP3in the diabetic myocardial tissues and apoptosis of cells, and interventional effects of DPP-IV inhibitors on the above pathological changes.
3. To evaluate the effects of the DPP-Ⅳ inhibitors on cardiac function of left ventricle in diabetic rats.
Methods
1. Establishment and groupings of the Type2diabetic rats
Seventy Wistar rats of eight weeks old and male were randomly divided into four groupsrcontrol group (n=10)[Control]; diabetes mellitus group (n=20)[DM]; diabeties with DPP-IV inhibitors (sitagliptin low dose group,30mg/kg/d)(n=20)[DPP-IVi (Low)]; diabetic treated with DPP-IV inhibitors(sitagliptin high dose group50mg/kg/d)(n=20),[DPP-IVi (High)]. All the subgroups of diabetic rats were injectioned with small dose of STZ30mg/kg by intraperitoneal style after high fat diet for4weeks, and the control groups were injected with the same dose of citrate buffer (pH4.5). Diagnostic criteria of type2diabetic rats:two consecutive fasting glucose≥11.1mmol/L after STZ injection in one week were regarded as type2diabetic models. The control groups and DM groups were given normal saline, and another DM groups interventioned were given different doses of DPP-Ⅳ inhibitor-sitagliptin diluted gastric lavage were to continue feeding until the end of the experiment, put to death, heart weight (HW), heart weight/body weight calculation [HW/BW(mg/g)]ratio, the specimen were reserved.
2. Monitoring of the basic indicators of the metabolism index of blood glucose or lipids, and the levels of GLP-1in each groups
The experimental process in addition to the conventional measured every2weeks for1times the heart rate, weight, blood pressure and blood glucose, during the period of this schedule, according to the need of research, the time point of0,4,5,9,13,17,21W, jugular venous sinus blood about1.5ml were extractioned, separated with serum, then serum total cholesterol, full automatic biochemical analyzer total triglyceride, low density lipoprotein cholesterol and high density lipoprotein cholesterol levels, glycosylated hemoglobin were measured as well as the immunoluminometric assay. The serum FFA, levels of GLP-1were measured with enzyme linked immunosorbent assay (ELISA) method, and fasting serum insulin level, insulin sensitivity index were calculated.
3. ECG
At different time points of this study, the cardiac electrical activity by ECG was recorded with the limb leads (Ⅰ, Ⅱ, Ⅲ, aVR, aVL, aVF) of rats were evaluated.
4. Echocardiology
Echocardiology were examinated at different time points of0,13W,17w,21W and measured as these follows:left ventricular end systolic diameter (LVIDs), left ventricular end diastolic diameter (LVIDd), ejection fraction (LVEF), left ventricular fractional shortening (FS); mitral E wave maximum speed, the maximum A wave speed, E/A; tissue Doppler image (TDI) of mitral annulus left ventricular lateral wall measured E'wave maximum velocity, maximum velocity of E wave A'. Then EVA' and E/E' were calculated.
5. Left ventricular function measured by Millar catheter
Direct apical puncture:exposure to the apex of the left ventricle, using10ml syringe needle, a Milllar catheter electrode probe directly for precise control of apical puncture into the left ventricle, Lab Chart Pro software to calculate cardiovascular parameters, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular pressure maximum rate of rise (+dp/dt), left ventricular pressure maximum falling rate (-dp/dt) and calculate Tau. Record and export the data, image data analysis.
6. Myocardial pathology
At the end, the rats were killed. Then the hearts were collected, fixed, dehydrated, transparent, paraffin, paraffin embedding, slicing, HE staining, Masson staining, and Sirius red staining in order to observe the myocardial tissue structure, cell morphology, collagen distribution et al. The frozen myocardial tissue specimens were stained with oil red O. The myocardial tissues were fixed with3%glutaraldehyde and transmission electron microscope and myocardial collagen contents were calculated and determinated with hydroxyproline content.
7. TUNEL Staining
The detection of apoptotic cells in paraffin sections by immunohisto chemistry. The percentage of the numbers of apoptotic cells were observed by microscope, and then the positive stained cells were counted by statistical analysis. The specific operation methods were dealed with the reference TUNEL apoptosis Kit (Calbiochem) steps.
8. Immunohistochemical Staining
The inflammatory factor TNF-a and IL-6, type Ⅰ and Ⅲ collagens and collagen degrading enzyme MMP-1and9, and RIP3expressions in the paraffin sections of left ventricular myocardial tissues by immunohistochemical detection were observed and then comprehensive analysis were done well.
9. Real-time quantitative RT-PCR
The total RNAs of myocardial Trizol were extracted from the myocardial tissues reserved at-80℃. The expression levels of RIP3mRNA were detected with real-time quantitative RT-PCR.
10. Western-blot
The total protein was extracted from the myocardial tissues cryopre served at-80℃. The GLP-1content and expression of GLP-1receptors, the inflammatory factor TNF-α and IL-6, type Ⅰ and Ⅲ collagens and collagen degrading enzyme MMP-1and9, and RIP3expressions were detected with Western-Blot method.
11. Statistical analysis
The data used by SPSS17.0statistical software for analysis. Numerical variable data were expressed by mean±standard deviation. The Comparison between the two groups was calculated with the small sample t test. The Comparison in different groups was calculated by One-Way ANOVA analysis of variance and P<0.05is regarded as significant difference.
Results
1. General situation of all the rats
During the course of the establishment of the type2diabetic rats models, a total of four is eliminated because of not up to standard and five rats died in the whole experiment. Finally, fifty-eight rats were involved in this experiment, and were divided into four groups:the Control group including10rats; the Diabetic group including15rats; the DM+DPP-Ⅳi (Low) group including16rats; and the DM+DPP-Ⅳi (High) group including17rats. The Control group had a good mental state, weight increased significantly, quick reaction, hair color white and shiny. However, polydipsia, polyuria and weight loss of symptoms occurred in DM groups, and poor spirit, slow weight gain or even declining, fur dirty and dull. The results compared with the DM group were slightly better in the treatment groups with different doses of DPP-Ⅳ inhibitors in DM rats. At the beginning of the experiment, no differences exist in the heart rate, blood pressure and body weight of rats in each group (P>0.05). But at the end, compared with the control group, the body weight decreased (P<0.01-0.05), the heart rate, blood pressure increased (P<0.01-0.05), and after different doses of DPP-Ⅳ inhibitors treatment, body weight increased, blood pressure and heart rate decreased in different degrees(P<0.01-0.05).
2. Results of the blood examination
2.1Comparison of metabolism of glucose and lipids in different groups
The phenomena of insulin resistance exist in all diabetic subgroups after four weeks with high-fat feeding before establishment of the models. During the whole process of the experiment, compared with the control group, the serum levels of FBG, blood glucose at10:00am and HbA1c, increased significantly (P<0.01) in DM group. Compared with the DM groups, all the indexes above in the DM groups with different doses of DPP-Ⅳ inhibitors decreased (P<0.01-0.05). No significant difference exists in FINS, but ISI decreased significantly (P<0.05) after one week establishing the models. However, at the end, compared with the control group and different doses of drug intervention groups, FINS decreased significantly (P<0.01-0.05) in DM group and from NO.5to21week, ISI decreased significantly (P<0.05) in DM group compared with the control group. The serum levels of TC, TG, LDL-C, and FFA increased significantly (P<0.01-0.05) in all the DM subgroups during the whole process of diabetes, and all these issues decreased in different degree (P<0.01-0.05) after oral different doses of DPP-Ⅳ inhibitors compared with the DM group.
2.2Serum levels of GLP-1examinated in fasting and oral glucose after30min
Diabetic model was successful and stable model, was treated for eighth weeks, DM compared with the control group, respectively, after oral glucose30min fasting plasma levels of GLP-1were decreased in different degree (P<0.01-0.05); different doses of DPP-Ⅳ inhibitors treatment, compared with the DM group, in the13-21weeks, fasting, oral glucose30min, GLP-1elevated plasma levels in varying degrees (P<0.01-0.05), especially the DM group were given high doses of DPP-Ⅳ inhibitor treatment, compared with the control group, no significant difference (P>0.05).
3. Results of ECG
ECG of the control group demonstrated P wave, QRS wave, T wave were visible and amplitude consistency of QRS waves. Abnormal ECG were found in ten diabetic rats, accounted for66.67%, and QRS "electrical alternans" phenomena were seen more and also including super ventricular tachycardia, ventricular tachycardia, slow heartbeat and other complex arrhythmia in the DM group. However, abnormal ECG were found in only fourteen diabetic rats with DPP-Ⅳ inhibitors, accounted for42.42%, in addition, no complicated arrhythmia occurred and "QRS alternans" reduced in the DM groups treated with DPP-IV inhibitors, suggesting that DPP-IV inhibitors improved the electrical activity of the heart.
4. Cardiac structure and function measured by echocardiology
Structure changes of left ventricule:at the beginning, no significant difference was found in the size of left heart chambers, LVIDs and LVIDd (P>0.05). However, at the end, compared with the control group, LVIDs and LVIDd of the DM group increased significantly (P<0.05). Compared with the DM group, LVIDs and LVIDd of the group treated with different doses of DPP-Ⅳ inhibitors decreased significantly (P<0.05).
Systolic function of the left ventricule:at the beginning, no significant differences of FS and EF existed in each group (P>0.05). However, at the end, compared with the control group, FS and LVEF decreased significantly (P<0.01-0.05) and compared with the DM group, FS and LVEF elevated significantly (P<0.01-0.05).
Diastolic function of the left ventricule:at the beginning, all indexes were showed no significant difference (P>0.05). Compared with the control group, during the period of13w-terminal21w, E/A, E'/A' ratio decreased (P<0.01-0.05) and E/E'ratio increased (P<0.01-0.05). Compared to the DM group, E/A and E'/A' ratio increased (P<0.01-0.05) and E/E 'decreased (P<0.01-0.05) were found in the DM group treated with DPP-Ⅳ inhibitors.
5. Parameters of left ventricular function measured by Millar catheter
Compared with the control group, LVSP decreased significantly (P<0.01), LVEDP increased (P<0.01),+dp/dt Max and-dp/dt Max absolute value decreased (P<0.01-0.05). Compared with the DM group, LVSP increased significantly in the DM treated with high doses of DPP-Ⅳ inhibitors (P<0.05),+dp/dt max (mmHg/s) increased significantly in different doses of DPP-Ⅳ inhibitors (P<0.05). LVEDP decreased significantly (P<0.01-0.05),-dp/dt max (mmHg/s) increased (P<0.05) and Tau decreased (P<0.05) in the DM group treated with different doses of DPP-Ⅳ inhibitors.
6. Effects of DPP-Ⅳ inhibitors on myocardial remodeling of left ventricle in diabetic rats
6.1Gross pathology and HE staining
Comparison the control group, cardiac gross morphological features demonstrated large sizes, high HW/BW (mg/g)(P<0.05) and Left ventricular cavity enlargement and ventricular wall hypertrophy occurred and cellular cross-sectional area increased significantly (498.37±14.31vs347.84±12.27μM3, P<0.01). HE staining:myocardial cell hypertrophy, muscle fibers derange ment or fracture phenomenon, irregular shape, uneven size of nuclei occurred in the DM group. Comparison the DM group, cardiac sizes, HW/BW (mg/g) ratio became smaller (P<0.05), left ventricular hypertrophy alleviated, cardio myocyte cross-sectional area decreased significantly (p<0.01-0.05) and HE staining:myo cardial cellular sizes reduced, cells in a regular arrangement, nuclear uniform size in the DM group treated with DPP-IV inhibitors.
6.2Effect of DPP-IV inhibitors on the content of GLP-1and the expression of GLP-1Receptor in diabetic myocardial tissue
Western-Blot:at the end, compared with the control group, the content of GLP-1(P<0.01) and the expression of GLP-1R (p<0.01) decreased significant ly in diabetic myocardial tissues. Compared with the DM group, the content of GLP-1and the expression of GLP-IR increased in the DM group treated with DPP-IV inhibitors.6.3Effects of DPP-IV inhibitors on myocardial collagen metabolism
Masson staining:compared with the control, the collagen fibers arranged in disorder, rough, uneven distribution, sedimentary increased in the DM group, and compared with DM group, the myocardial collagen deposition reduction, arrayed orderly, distribution was uniform in the DM group treated with different doses of DPP-Ⅳ inhibitors.
Sirius red staining:compared with the control group, collagen fibers signi ficantly increased, disorganized, and irregular reticular collagen fibers around small arteries in the DM group and the myocardial collagen deposition decreased in the group treated with different doses of DPP-Ⅳ inhibitors.
Compared with the control group, the collagen content in diabetic myocar dial tissue (15.86±0.43vs7.06±0.31μg/mg, P<0.01), CVF%(4.8±0.49vs0.47±0.06, P<0.01-0.05) and perivascular collagen area/lumen area ratio increased (PVCA/LA)(2.74±0.37vs0.5±0.02,P<0.01-0.05) in the DM group. Compared with the DM group, the collagen content of left ventricular tissue decreased in in the DM group treated with DPP-Ⅳ inhibitors.
Immunohistochemical staining:compared with the control group, the positive expression of type Ⅰ and Ⅲ collagen increased significantly in diabetic myocar dial tissues, and brown granules decreased after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of type Ⅰ and Ⅲ collagen in diabetic myocardial tissue increased significantly (P<0.01) and expression decreased significantly (P<0.01-0.05) after treatment with DPP-IV inhibitors.
6.4MMP-1,9reduced in DM myocardial tissues treated with DPP-IV inhibitors
Immunohistochemical staining:compared with the control group, the positive expression of MMP-I, MMP-9increased significantly in diabetic myocardial tissues and brown granules decreased after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of MMP-1, MMP-9in diabetic myocardial tissues increased significantly (P<0.05) and expression decreased significantly (P<0.01-0.05) after treatment with DPP-Ⅳ inhibitors.
6.5Inflammatory factors TNF-a and IL-6reduced in DM myocardial tissues treated with DPP-Ⅳ inhibitors
Immunohistochemical staining:a few sparse pale or brown granules in the control group and with more densely dark or brown granules in myocardial cellular cytoplasm. Brown particles, scattered distribution in sparse reduced after treatment with DPP-Ⅳ inhibitors.
Western-Blot:compared with the control group, the expression of TNF-a and IL-6in diabetic myocardial tissue increased significantly (P<0.01) and expression decreased significantly (P<0.05) after treatment with DPP-Ⅳ inhibitors.
6.6Fatty deposition reduced in diabetic myocardial and epicardial tissues treated with DPP-IV inhibitors by Oil red O staining
Compared with the control group, lipids content of diabetic myocardium (7.03±0.31vs0.32±0.01, P<0.01) and that of epicardial adipose tissue (12.76±0.48vs0.32±0.01, P<0.01) increased significantly. Compared with the DM group, lipid content, epicardial adipose tissue reduced after treatment of different doses of DPP-IV inhibitors (P<0.01-0.05).
6.7The expression of RIP3in diabetic myocardial tissues and DM group treated with DPP-Ⅳ inhibitors
Immunohistochemical staining:compared with the control group, RIP3brown positive particles increased and distributed in the nucleus around more in diabetic myocardial tissues, and brown positive particles in the cell decreased after treatment with different doses of DPP-Ⅳ inhibitors.
Quantitative real-time RT-PCR and Western-Blot:compared with the control group, mRNA level and protein expression of RIP3increased significantly (P<0.01-0.05) in diabetic myocardial tissues, and compared with the DM group, mRNA level and protein expression of RIP3decreased (P<0.01-0.05) after treatment with DPP-Ⅳ inhibitors.
6.8Effects of DPP-Ⅳ inhibitors on DM myocardial ultrastructure by TEM observation
Myocardial irregular nuclei, nuclear pyknosis, chromatin aggregation, early presentation of apoptotic cells in the DM group. Myocardial cellular nucleolus more clear, nuclear membrane smooth and complete were seen in the control group. No obvious aggregation of chromatin, no nuclear pyknosis and nuclear membrane still intact, smooth were seen in the DM rats after treatment with DPP-Ⅳ inhibitors.
6.9Effects of DPP-Ⅳ inhibitors on apoptosis of diabetic myocardial tissues by TUNEL staining
Compared with the control group, the percentage of apoptotic cells increased significantly (P<0.01) and compared with DM group, the apoptosis rate reduced significantly (P<0.01) after after treatment with DPP-IV inhibitors.
6.10Analysis of the relationships between the expression RIP3mRNA and cellular apoptotic rate in diabetic myocardial tissues
Combined with the relationships between the expression RIP3mRNA and cellular apoptotic rate in diabetic myocardial tissues, Pearson correlation coefficient r=0.795(P0.001), the percentage of apoptosis positive related to RIP3mRNA in diabetic myocardial tissues.
Conclusions
1. DPP-IV inhibitors could exactly impove the pathological remodeling including inflammation, metabolism of collagen network, lipids content and apoptosis of myocardial cells in type2diabetic rats.
2. The expressions of RIP3were up-regulated and positively correlated with apoptosis of the cells in diabetic myocardial tissues and DPP-IV inhibitors could down-regulate the expression of RIP3and inhibit apoptotic cells of the myocardial tissues.
3. DPP-IV inhibitors could significantly reduce the E/E'ratio in diabetic rats, improve systolic blood pressure (LVSP), reduced left ventricular end diastolic pressure (LVEDP),±dp/dt max and Tau. DPP-IV inhibitors could significantly improve both systolic and diastolic functions of the left ventricle in type2diabetic rats.
Background
Apoptosis is a pathophysiological process regulated by gene control, active, and programmed cell death, and is extremely important for maintaining homeostasis. Apoptosis of myocardial cells is one of the main pathologic changes in diabetic myocardial lesions and increased with the increase of apoptotic cells, eventually myocardial tissue remodeling and cardiac dysfunc tion occurred.
Recent studies demonstrated that RIPs (receptor-interacting protein) family as a kind of regulation of cell survival, apoptosis, necrosis of important signal molecules has attracted increasing attention, in which RIP3has autophosphoryla tion, induction of apoptosis and activation of NF-κB. Overexpression of RIP3in various cell lines, would undergo apoptosis. Zhang DW et al. reported that RIP3was TNF-a induced apoptosis and necrosis of mutual conversion "molecular switch", and RIP3was closely related to mitochondrial energy metabolism of cells and ROS released. RIP3regulated enzyme interactions related to energy metabolism, and overproduction of ROS induced programmed cell necrosis. Therefore, RIP3was involved in many intracellular signal pathways, and play an important physiological function. Combined with the research of the first part of this study, up-regulated expression of RIP3was found in diabetic myocardial tissues, and significantly positive correlated with myocardial apoptosis rate. However, in high glucose conditions, mitochondrial dysfunction, cellular energy supply disorder, RIP3was involved in energic metabolism related enzyme action, activity, effect of mitochondrial function and membrane potential changes of myocardial cells mediated apoptosis has not been reported.
Recent reports, glucagon-like peptide-1(GLP-1) in addition to hypoglycemic effect, still could inhibit myocardial apoptosis after ischemia/reperfusion or myocardial infarct tion, improve heart failure. GLP-1might inhibit pancreatic islet cells apoptosis via activating P13K. MAPK signaling. First part of this study demonstrated that DPP-1V inhibitors inhibited RIP3expression in diabetic myocardium and decreased myocardial cellular apoptosis by increasing the level of GLP-1. Previous study showed that, high glucose might directly stimulate or cause changes of Bax, Bcl-2expression, start the mitochondrial damage pathway, leading to increased mitochondrial membrane permeability, cytochrome C released from mitochondrial into the cytoplasm, start the caspase cascade reaction, cell apoptosis. However, in high glucose environment, research on the regulation of RIP3expression is involved in the apoptosis pathway via GLP-1is no reported till now.
Hence, study on the previous reports and first task based on the animal levels, this part will study and put forward possible hypotheses:over-expression of RIP3participates in the apoptotic pathway of diabetic myocardial cells via TNF-a mediated death receptor, also involved in mitochondrial injury. RIP3directly or indirectly (through the process of adjusting the energy to produce excess of ROS) cause the mitochondrial membrane permeability changes, mitochondrial cytochrome C releasing into the cytosol. activated Caspase-3expression, cell apoptosis, while RIP3silence could improve the pathological change. GLP-1might inhibit overexpression of RIP3via multiple signaling pathways. To further verify this process, this part adopts high glucose-induced apoptosis process, to explore changes of RIP3expression in this process and effect of GLP-1and the possible mechanisms, and provide a theoretical basis for the description of GLP-1, RIP3in high glucose induced apoptosis in ventricular myocytes and the possible signal pathway.
Objectives
1. To verify stimulation with high glucose can induce ventricular myocardial apopto sis, and apoptosis associated with expression of RIP3.
2. To investigate the apoptotic process of ventricular myocardiocytes induced by high glucose, RIP3may play a key role in apoptosis via the mitochondrial damage.
3. To explore the possible signal pathway of effects of GLP-1on the expression of RIP3in high glucose conditions.
Methods
1. Culture and identification of primary myocardial cells
1-3days of newborn Wistar rats isolated left ventricular posterior shear, trypsin digestion, differential centrifugation to obtain myocardial cells, the pulsation of myocardial cells, and the primary myocardial cells cultured. The troponin T monoclonal antibodies by immunofluorescence staining, fluorescence microscope and identified as the primary cultured myocardial cells.
2. Experimental groups and intervention
In vitro physiological state, diabetes status, adding stimulus to different factors, prognosis of myocardial cells. Cells were treated separately:myocardial cells were treated with different concentrations of glucose (5.5,25,33.3,50mmol/L). To identify suitable for the required concentration, time stimulation (33.3mmol/L,72h) for high (HG) the research object. HG group with Control siRNA, RIP3siRNA and GLP-1of different concentrations after incubation of cells, and design corresponding control group.
siRNA Technology:inhibition of RIP3expression by siRNA technology. RIP3siRNA pretreatment of24h with myocardial cells induced by high glucose, and72h, expression of charged cells by flow cytometry and Western-Blot technique for the detection of cell apoptosis and the target protein.
3. Myocardial apoptosis was detected by flow cytometry
Using Annexin V/PI flow cytometry staining method, detection of myocardial cellular apoptosis occurrence, incidence rate of the myocardial cell apoptosis.
4. Detection of mitochondrial membrane potential
Using the JC-1method, by laser confocal microscopy, fluorescence detection of mitochondrial membrane potential change in value, detection of mitochondrial membrane potential (AΨm) changes.
5. real-time quantitative RT-PCR detection
Cells were collected, total RNA was extracted by reverse transcription reaction, cDNA, β-actin as a reference, through the detection of RIP3expression of mRNA by RT-PCR.
6. Western-Blot detection
All the cells were collected, extracted total protein, respectively by SDS-poly acrylamide gel electrophoresis separation, membrane protein, mark, color and other steps, to detect the expression of RIP3, cytochrome C, Caspase-3, cleaved caspase3.
Results
1. Myocardial apoptosis induced by Selection of the glucose concentrations and choices of time
5.5mmol/L[NG],25%[HG25],33.3%[HG33.3],50%[HG50] of different concentra tions of glucose and mannitol in control medium with mannitol Group was0,24,48cells,72h, flow cytomctry apoptosis is shown, NG and mannitol than in control group, HG33.3, HG50stimulation of48h (P<0.05),72h (P<0.01) could significantly increase cellular apoptosis, HG25stimulation of72h (P<0.05) can increase cellular apoptosis. Therefore, the high concentration of glucose can induce the apoptosis of myocardial cells, combined with myocardial cellular viability results, with33.3%glucose for72hours as the object of study.
2. Relationships between the expression of RIP3and apoptosis of myocardial cells induced by high glucose
2.1Overexpression of RIP3during the course of apoptosis induced by high glucose in myocardial cells
(1) Myocardial cells after stimulation of72h with a high concentration of glucose (33.3%)[HG], collected. The results were displayed by real-time quantitative RT-PCR and Western-Blot assay:compared with5.5%concentrations of glucose[NG], both mRNA (P<0.05) and protein (P<0.05) levels of the expressions of RIP;, elevated significantly in HG group.
(2) RIP3siRNA, Control siRNA were transfected into primary cultured cardiomyo cytes after24h, then the cells stimulated with high glucose for72h. siRNA transfection efficiency was verified by RT-PCR and Western-Blot. Compared with the HG group and control siRNA transfection group, RIP3siRNA transfection group demonstrated that the expressions of both mRNAs (P<0.05) and protein (P<0.05) decreased significantly, suggesting that RIP3siRNA inhibited the expression of RIP3effe
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