大黄有效部位及伟素对慢性环孢素肾病大鼠的防治研究
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摘要
环孢霉素A (cyclosporine A, CsA)是由真菌产生的环状11肽活性物质。自1983年FDA批准用于实体器官移植后,迅速成为器官移植抗排斥和自身免疫性疾病治疗的一线药物,临床应用较多,疗效确切。但该药的副作用特别是慢性肾毒性限制了它的广泛应用。由CsA肾毒性导致的慢性肾功能进行性衰退的慢性环孢素肾病(chronic cyclosporine nephropathy, CCN)以渐进性肾功能不全、入球小动脉透明样变性、炎症细胞浸润、条索状间质纤维化和肾性免疫原性增加为主要特征性病理改变。Mihatsch等对肾移植患者的肾活检资料进行分析,证实CsA所致肾小管中毒的发生率为9%～37%,急性肾功能衰竭伴弥漫性间质纤维化的发生率为0%～19%,条索状间质纤维化以及CsA相关动脉病变的发生率分别为5%～50%和5%～30%。研究认为,CCN的病因与肾小球缺血、肾内肾素血管紧张素系统(renin-agiotensin system, RAS)激活、细胞凋亡内质网应激和自体吞噬、单核巨噬细胞(monocyte/macrophage, MC/MP)浸润及炎症、转化生长因子β1(transforming growth factor-betor 1, TGF-β1)与基质金属蛋白酶(matrix metalloproteinases, MMPs)/金属蛋白酶抑制因子(tissue inhibitor of metalloproteinases, TIMPs)调节失衡、骨桥蛋白、核因子-kappa B (nuclear factor-kappa B, NF-κB)、Toll-样受体等有关。针对其发病机制,人们进行了大量的临床和实验研究,并试图应用西药、中药或采取减量甚至停用CsA的方式来防治CCN。
大黄是我国传统中药之一,大黄有效部位(Rhubarb's effective parts,Rep)为大黄醇提液过大孔吸附树脂得到的洗脱物,包含以下五种成分：大黄素、大黄酚、大黄素甲醚、大黄酸、芦荟大黄素。文献报道其在改善脑缺血、脑代谢及抗排斥反应方面效果显著。伟素(舒洛地特,sulodexide)是经过高度纯化的生物制品,与肝素同属糖胺聚糖(glycosaminoglycans,GAGs)类药物。GAG通常指一大类分子,包括肝素、低分子肝素、硫酸类肝素、硫酸类皮肤素以及糖胺聚糖类化合物,它们具有共同的生物学活性即保护肾脏的活性。伟素由两种GAG组成,在实验模型中具有预防糖尿病肾病(dibetic nephropathy,DN)的活性,能够抑制TGF-β1过度表达以及肾小球细胞高浓度葡萄糖诱导的基质合成,修复内皮细胞机能障碍。但Rep和伟素是否可以通过改善肾脏缺血和代谢,对抗肾排斥反应及保护肾脏活性等机制影响CCN的发生和发展,目前尚未可知,亦无相关文献报道。为此,我们复制了CCN大鼠模型,观察不同剂量Rep和伟素在不同时间点对模型大鼠的干预和治疗作用,并对其可能的作用机理进行探讨。
本论文分以下2部分。
第1部分Rep及伟素对CCN大鼠的干预作用
目的：
观察Rep及伟素对CCN的预防效果。
方法：
1.实验分组及生化指标测定：SPF级健康雄性Wistar大鼠54只(180～200g),给予低盐饲料,自由饮水,适应性喂养1周后,将动物随机分为9组,分别为：第一组正常对照组(VH3：橄榄油5ml·kg-1·d-1×28 d);第二组模型组(MX3:CsA 25mg·kg-1·d-1×28 d)；第三组阳性对照组(YDY:CsA 25mg·kg-1·d-1+贝那普利5mg·kg-1·d-1×28d)；第四组Rep干预1组(DY1：Rep 50mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28 d,二药灌胃时间间隔2h以上)；第五组Rep干预2组(DY2:CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28d,二药灌胃时间间隔2h以上)；第六组Rep干预3组(DY3:CsA 25mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1 +Rep 50mg·kg-1·d-1×21d,二药灌胃时间间隔2h以上)；第七组伟素干预1组(WY1：伟素10mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×28d);第八组伟素干预2组(WY2:CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×28d);第九组伟素干预3组(WY3:CsA 25mg·kg-1·d-1×7 d,后给予CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×21d)。按实验设计,CsA及Rep为灌胃给药,伟素为腹腔注射。所有大鼠均于第28天处死。实验期间在用药前和用药后的每周最后一天测量大鼠体重并调整灌胃和腹腔注射剂量。同时采集大鼠尾静脉血,测定血肌酐(serum creatinine, Scr)和血尿素氮(blood urea nitrogen, BUN)。
2.取肾组织行HE、PAS及Masson染色观察病理变化,分析各组小动脉透明样变性、肾间质纤维化(renal interstitial fibrosis, RIF)情况并通过半定量积分评估其程度。入球小动脉透明样变性通过计数入球及球旁小动脉病变来判断。每个标本至少检查50个小动脉,进行下列评分：0=无透明样变性；1=早期透明样变性；2=明显透明样变性。RIF通过计数每个视野损伤面积的百分比来评估：0=正常肾间质,0.5=<5%,1=5%到15%,1.5=16%到25%,2=26%到35%,2.5=36%到45%,3=>45%肾间质面积损伤。
3.同时取肾组织行电镜检查观察超微结构变化。
4.冰上分离肾脏并采取免疫组织化学法检测肾组织CD68、Ⅲ型胶原及Ⅳ型胶原的表达,逆转录—聚合酶链反应(RT-PCR)法检测肾组织TGF-β1和TIMP-1 mRNA表达,ELISA法检测TGF-β1、TIMP-1和MMP-9蛋白表达。
结果：
1.体重、BUN、Scr:
1.1体重：给予CsA后,MX3组大鼠1周后增长延缓,2周后体重下降,第2-4周与VH3相比,体重下降(P<0.05)；所有干预组及YDY组均高于MX3组(P<0.05),其中DY3、WY3、YDY低于VH3组(P<0.05),其它组与VH3组相近(P>0.05)。
1.2 BUN:MX3组大鼠第1周血BUN水平开始上升,第2-4周,与VH3组相比,差异显著(P<0.05); DY1、WY1、WY2组大鼠BUN水平无上升(P>0.05),均低于MX3组(P<0.05),与VH3组相近(P>0.05)。YDY、DY3、WY3组于1周后仍见上升,3周后逐渐下降且均高于VH3组、低于MX3组(P<0.05),而DY2逐渐上升,于4周开始下降,均低于其他三组(P<0.05),其它三组无差别(P>0.05)。
1.3 Scr：与VH3组相比,第1～4周,MX3组大鼠Scr水平上升(P<0.05)。第2-4周所有干预组及YDY组均低于MX3组(P<0.05)；与VH3组比,DY1组较低(P<0.05), DY2、WY1与之相近(P>0.05),其它干预组及YDY组上升(P<0.05)。WY2组低于YDY、DY3、WY3组(P<0.05),而DY3、WY3组于4周下降(P<0.05)均低于YDY组(P<0.05)。
2.光镜结果：VH3组大鼠肾组织结构正常。MX3组大鼠于四周后肾小球毛细血管袢开放不佳,近曲小管细胞内空泡变性坏死,小管萎缩同时伴单核细胞浸润,可见灶状及条带状RIF。肾内小动脉管壁肌性增厚,入球小动脉透明样变性及损伤,偶见局灶性肾小球硬化。而干预组及YDY组大鼠的上述病变明显减轻,其小动脉透明样变性及RIF也明显减轻。
2.1小动脉透明样变性评分：各干预组及YDY组评分均高于VH3组,低于MX3组(P<0.05)；干预组中DY1、DY2、DY3、WY3高于YDY组(P<0.05),其它干预组与YDY组相近(P>0.05)。伟素与Rep比较：伟素各个时间点评分均低于Rep组(P<0.05),而且第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)；Rep各个时间点间无差别(P>0.05)。
2.2 RIF评分：各干预组及YDY组均高于VH3组,低于MX3组(P<0.05)：干预组中DY3、WY3高于YDY组(P<0.05),其它干预组与YDY组相近(P>0.05)。伟素与Rep比较：在第1个时间点伟素低于Rep组(P<0.05),其余2个时间点二者无差别(P>0.05)。二药均显示第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)。
3.肾组织超微结构变化：VH3组肾组织超微结构正常。MX3组大鼠肾小球基底膜增厚,系膜基质增宽、增多,足细胞足突广泛融合、微绒毛稀疏；小管基底膜不规则增厚、断裂,上皮细胞大量空泡、大小不等,细胞核固缩,小管微绒毛脱落,上皮细胞内溶酶体、线粒体明显增多,间质见较多胶原纤维。DY1、DY2、WY1、WY2组大鼠上述病变明显减轻,偶见足突融合,系膜基质节段轻度增多,小管可见少量空泡,溶酶体、线粒体较少,小管基膜完整。YDY、DY3、WY3组病变较重但较MX3组轻。
4.肾组织免疫组织化学：各组大鼠肾间质CD68、胶原Ⅲ、胶原Ⅳ表达变化趋势与HE染色显示的炎性细胞浸润趋势相似。给药4周后,MX3组CD68、胶原Ⅲ、胶原Ⅳ的表达高于对照组和预防组(P<0.05)。
4.1 CD68表达：DY1、DY2、WY1、WY2组CD68表达水平与VH3组相近(P>0.05), YDY、DY3、WY3组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；YDY组与DY3、WY3组相近(P>0.05),均高于其它用药干预组(P<0.05)。伟素与Rep比较：在第3个时间点Rep低于伟素组(P<0.05),其余2个时间点二者无差别(P>0.05)。两药均显示第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)。
4.2胶原Ⅲ表达：DY2、WY2组胶原Ⅲ表达水平与VH3组相近(P>0.05),其它干预组及YDY组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；而与YDY组相比,WY1组与之相近(P>0.05),DY3、WY3组较之增高(P<0.05),其它干预组均降低(P<0.05)。伟素与Rep比较：在第3个时间点伟素低于Rep组(P<0.05),其余2个时间点二者无差别(P>0.05)。两药均显示时间点3>2>1(P<0.05)。
4.3胶原Ⅳ表达：WY1与VH3组胶原Ⅳ表达相近(P>0.05),其它干预组及YDY组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；而与YDY组比,WY1组降低(P<0.05),DY3、WY3组升高(P<0.05),其它干预组与之相近(P>0.05)。伟素与Rep比较：在第1、3个时间点伟素低于Rep组(P<0.05),第2个时间点二者无差别(P>0.05)。两药均显示第3个时间点高于前2个时间点(P<0.05),伟素第2个时间点又高于第1个时间点(P<0.05),而Rep前两个时间点间无差别(P>0.05)。
5.TGF-β1和TIMP-1mRNA表达
5.1 TGF-β1mRNA表达：VH3组有少量TGF-β1mRNA表达,除WY1组外,MX3以及其它干预组表达增加(P<0.05),干预组及YDY组的表达要低于MX3组(P<0.05),而各干预组中的表达要低于YDY组(P<0.05)。伟素与Rep比较：伟素各个时间点均低于Rep (P<0.05),且Rep在时间点3>2>1(P<0.05),而伟素在时间点1、2效果相近(P>0.05),但低于时间点3(P<0.05)。
5.2 TIMP-1mRNA表达：VH3组有少量TIMP-1mRNA表达,MX3、YDY以及干预组表达增加(P<0.05),干预组及YDY组表达均低于MX3组(P<0.05),而各干预组的表达要低于YDY组(P<0.05)。伟素与Rep比较：伟素各个时间点均低于Rep组(P<0.05),而二者TIMP-1mRNA表达水平均随着用药时间的延迟而逐渐增高(P<0.05)。
6. TGF-β1、TIMP-1和MMP-9蛋白表达
6.1 TGF-β1蛋白表达：WY1、WY2组TGF-β1蛋白表达与VH3组相近(P>0.05), DY1、DY2、WY3组比之降低(P<0.05), DY3、YDY组比之升高(P<0.05)；各组均高于MX3组(P<0.05),低于YDY组(P<0.05)。伟素与Rep比较：各组之间差异均有显著性(P<0.05),在前两个时间点用药Rep低于伟素组,而在时间点3,正好相反。Rep组在时间点3高于2、1(P<0.05),后二时间点间无差别(P>0.05)；而伟素组在时间点1、2干预用药效果相近(P>0.05),但高于时间点3(P<0.05)。
6.2 TIMP-1蛋白表达：DY3、WY1、WY2组TIMP-1蛋白表达与VH3组相近(P>0.05),DY1、DY2、WY3组比之降低(P<0.05),YDY组升高(P<0.05)；各干预组均低于MX3组【除DY3(P=0.05)外P值均<0.05】,而YDY组高于MX3组(P<0.05)；各干预组均低于YDY组(P<0.05)。伟素与Rep比较：在时间点1、2Rep低于伟素组(P<0.05),在时间点3伟素低于Rep组(P<0.05)。伟素组在时间点3低于时间点2、1(P<0.05),Rep在时间点3高于时间点2、1(P<0.05),而各自在时间点2与1比差异无显著性(P>0.05)。
6.3 MMP-9蛋白表达：MX3、WY1组MMP-9蛋白表达水平与VH3组相近(P>0.05),而其它干预组及YDY组均增高(P<0.05),以YDY组为最高(P<0.05)。伟素与Rep比较：在时间点1和3伟素低于Rep组(P<0.05),而在时间点2 Rep低于伟素组(P<0.05)；Rep组在各个时间点基本一致(P>0.05),而伟素组各个时间点MMP-9蛋白表达水平由低到高为1<3<2(P<0.05)。
结论：1.伟素和Rep单独干预用药均可减轻CsA肾毒性,甚至可消除其部分毒性,早期干预效果更好。2.采用伟素或Rep预防CsA肾毒性优于贝那普利。3.伟素在改善小动脉透明样变性方面优于Rep；改善RIF方面,提前给药时伟素优于Rep,而同时及后期给药干预时效果相当,但干预机理不同。
第2部分Rep及伟素对CCN大鼠的治疗作用
目的：
复制CCN模型,并于2周后停用CsA,观察停用后肾毒性能否自行消除；同时观察Rep及伟素对CCN是否存在治疗作用；并评估其疗效。
方法：
1.实验分组：SPF级健康雄性Wistar大鼠78只(180～200 g),给予低盐饲料,自由饮水,适应性喂养1周后,将动物随机分为9组分别为：第一组正常对照组(VH1：为对照1组,n=6,给予橄榄油5ml·kg-1·d-1×14d,第14天处死；VH2：为对照2组,给予橄榄油5ml·kg-1·d-1×14d,后改为注射用水5ml·kg-1·d-1×14d,第28天处死；VH3：为对照3组,给予橄榄油5ml·kg-1·d-1×28d,第28天处死)；第二组模型组(MX1：为模型1组,CsA 25mg·kg-1·d-1×14d,第14天处死；MX2：为模型2组,CsA 25mg·kg-1·d-1×14d,后改为注射用水5ml·kg-1·d-1×14d,第28天处死；MX3：为模型3组,给予CsA25mg·kg-1·d-1×28d,第28天处死)；第三组阳性对照组(YDZ：CsA25mg·kg-1·d-1×14d,后改为贝那普利5mg·kg-1·d-1×14d)；第四组Rep治疗1组(DZ1：CsA 25mg·kg-1·d-1×14d,后改为Rep 25mg·kg-1·d-1×14d)；第五组Rep治疗2组(DZ2：CsA 25mg·kg-1·d-1×14d,后改为Rep50mg·kg-1·d-1×14d)；第六组Rep治疗3组(DZ3：CsA 25mg·kg-1·d-1×14d,后改为Rep 100mg·kg-1·d-1×14d)；第七组伟素治疗1组(WZ1：CsA25mg·kg-1·d-1×14d,后改为伟素10mg·kg-1·d-1×14d)；第八组伟素治疗2组(WZ2：CsA 25mg·kg-1·d-1×14d,后改为伟素20mg·kg-1·d-1×14d)；第九组伟素治疗3组(WZ3：CsA 25mg·kg-1·d-1×14d,后改为伟素40mg·kg-1·d-1×14d)。按实验设计CsA及Rep为灌胃给药,伟素为腹腔注射。第三至八组均于第28天处死。第九组因有2例死亡而退出实验。
2.实验观察及所有指标检测同第1部分。
结果：
1.体重、BUN、Scr:
1.1体重：除第九组大鼠外,其余组大鼠均存活并纳入最后统计。第1天各组大鼠体重差异无显著性(P>0.05)；第14天模型组及治疗组、YDZ组大鼠体重差异无显著性(P>0.05),而与正常对照组大鼠比体重下降(P<0.05)；第21天,MX3组大鼠体重继续下降,其它组(除VH1、MX1已处死外)大鼠体重均上升(P<0.05),至28天各组体重均较前上升,以MX3、WZ2组与前相比差异无显著性(P>0.05),其它组均有显著差异(P<0.05)。第28天以VH2、VH3组体重最重,所有组与MX3组(除VH1、MX1已处死)比均升高(P<0.05),各治疗组与YDZ及MX2组比差异无显著性(P>0.05)。
1.2 BUN、Scr:第1天,各组BUN、Scr比较差异无显著性(P>0.05)；第7天、14天,各组与正常对照组相比BUN、Scr均上升(P<0.05)；第28天各组(除VH1、MX1已处死外)与MX3组相比BUN、Scr下降(P<0.05)；与MX2组相比,DZ1、DZ2组BUN上升(P<0.05),DZ1、DZ2、WZ2组Scr上升(P<0.05),而其它治疗组及YDZ组无变化(P>0.05)；各治疗组及YDZ与正常对照组相比无差别(P>0.05)；与YDZ组相比,DZ1、DZ2组BUN上升(P<0.05), DZ1、DZ2、WZ2 Scr上升(P<0.05),其它治疗组无变化(P>0.05)。
2.光镜结果：正常对照组大鼠肾组织结构正常。MX3组改变同第1部分。MX1组大鼠肾小球毛细血管袢开放不佳,间质见单核细胞浸润,散在条带状间质纤维化,部分入球小动脉透明样变性,但上述改变均轻于MX3组。而YDZ组及MX2组大鼠的上述病变明显减轻,治疗组趋于正常。小动脉透明样变性及RIF见下：
2.1小动脉透明样变性评分：各治疗组及YDZ组与正常对照组比无改变(P>0.05)；与模型MX1、MX3组比减轻(P<0.05)；与MX2组比无差别(P>0.05)；而治疗组与YDZ组比亦无差别(P>0.05)。
2.2 RIF评分：正常对照组见少量RIF；模型组均加重以MX3组最重,MX1次之,MX2最轻(P<0.05)；而YDZ与MX2组相似(P>0.05)；治疗组均轻于YDZ、MX2组(P<0.05),重于正常对照组(包括VH1、VH2、VH3)(P<0.05)。伟素与Rep比较：各组由轻到重依次为WZ10.05)。
3.肾组织超微结构变化：正常对照组肾组织超微结构正常。MX3组改变同第1部分。MX1组大鼠病变稍轻,足突部分融合,系膜基质轻度增多,小管可见大量空泡、上皮细胞核缘不规整、染色质增多、可见溶酶体、线粒体,小管基膜厚度不一,节段断裂,偶见间质胶原纤维。MX2组病变轻微,YDZ及治疗组基本正常。
4.免疫组织化学
4.1 CD68表达：CD68表达由低到高为VH1、VH2、VH30.05); DZ3、WZ1、WZ2均增高(P<0.05)。伟素与Rep比较：各组以DZ1表达最低,接下来依次为DZ2、WZ1、WZ2、DZ3(除WZ2与DZ3之间比较P>0.05,余P<0.05)。
4.2胶原Ⅲ表达：其表达以正常对照组(VH1、VH2、VH3)、DZ1、DZ2、DZ3最低(互相比较P>0.05)；模型组均增高(P<0.05) (MX3>MX2>MX1, P<0.05); YDZ、WZ1、WZ2与MX2相近(P>0.05),高于MX1(P<0.05),低于MX3(P<0.05)。伟素与Rep比较：Rep各组均低于伟素组(P<0.05),而同种药物不同剂量间无差别(P>0.05)。
4.3胶原Ⅳ表达：表达在正常对照组(VH1、VH2、VH3)、MX2、DZ2、WZ1相近(P>0.05); MX1、MX3、YDZ、DZ1、DZ3、WZ2增高(P<0.05),而以MX3组为最高(P<0.05),其次为YDZ(P<0.05),其余四组最低(P<0.05)(四组间P>0.05)。伟素与Rep比较：各组以DZ2、WZ1表达较低(二者之间差异无显著性,P>0.05),接下来依次为DZ3、WZ2、DZ1(三者之间差异无显著性P>0.05,与前二者相比P<0.05)。
5.TGF-β1和TIMP-1mRNA表达
5.1 TGF-β1mRNA表达：表达以DZ20.05)0.05)表达较低,DZ3WZ2表达(二者之间P>0.05)增高(与前三组相比P<0.05)。
5.2 TIMP-1mRNA表达：表达以WZ10.05) 6. TGF-β1、TIMP-1和MMP-9蛋白表达
6.1 TGF-β1蛋白表达：撤退CsA后MX2组恢复至正常水平,治疗组及YDZ组均增高(P<0.05), WZ1、DZ2、DZ1组表达高于YDZ组(P<0.05),而WZ2、DZ3组表达水平与YDZ组相近(P>0.05)。伟素与Rep比较：除DZ3与WZ1、WZ2组差异无显著性(P>0.05),其余各组相互比较差异均有显著性(P<0.05)。各组以WZ2、DZ3表达最低,接下来依次为WZ1、DZ2、DZ1。
6.2 TIMP-1蛋白表达：撤退CsA后MX2组恢复至正常水平,而除贝那普利外,其它药物治疗均使其表达增高(P<0.05)。伟素与Pep比较：DZ3、WZ1、WZ2(三者之间P>0.05)组最低,其次为DZ2组,DZ1组表达最高(P<0.05)。
6.3 MMP-9蛋白表达：表达以WZ2最高；MX1、DZ1、DZ2、WZ1(这几组间差异无显著性P>0.05)次之；VH3、MX2、MX3、DZ3、VH1、VH2、YDZ(这几组间差异无显著性P>0.05)最低(P<0.05)。伟素与Rep比较：表达以WZ2组最高,DZ1、DZ2、WZ1较高,DZ3最低。DZ3分别与DZ1、DZ2、WZ2组比差异有显著性(P<0.05),其它组间相比P>0.05。
结论：撤退CsA后,大鼠一般生化指标及肾组织小动脉透明样变性可自行恢复正常；RIF较前有所改善,但应用伟素或Rep后改善更明显,分别在应用10mg·kg-1·d-1, 50mg·kg-1·d-1时效果最好,二者作用相当。
As active substance of mycetogenetic circular peptide 11, cyclosporine A(CsA) had become to be first-line drug to treat rejection reaction after organ transplantation and autoimmune diseases, quickly put into clinical use and then achieved exact therapeutic effects since it was given approval to be immunosuppressive agent for organ transplantation by FDA in 1983. However, the adverse effect especially chronic nephrotoxicity limited its use. Chronic CsA nephrotoxicity(CCN) is characterized by progressive renal dysfunction,afferent arteriolopathy,inflammatory cell influx,striped tubulointerstitial fibrosis,and increased intrarenal immunogenecity. Mihatsch etc. analyzed kidney biopsy data of kidney transplantation patients, verified that incidence of kidney tubules poisoning due to CsA was 9%-37%, incidence of acute kidney function failure accompanied with diffuse interstitial fibrosis was 0%-19%, while incidence of stripe-like interstitial fibrosis and CsA-related arterial diseases were 5%-50% and 5%-30% respectively. The exact mechanism of this complication is not well understood,and multiple factors are implicated. The pathogenesis of CCN was considered to be relevant with glomerulus ischemia, rennin angiotensin system (RAS) activation, apoptosis, endoplasmic reticulum stress, autophagy, mononuclear macrophage(MC/MP) infiltration and inflammation, regulation disbalance of TGF-β1 and MMPs/TIMPs, osteopontin, nuclear factor-kappa B (NF-κB), Toll-like receptor and so on. Focus on its pathogenesis, a lot of researches had tried to intervent and treat CCN by using western durgs, Chinese drugs as well as reduction or even interruption of CsA.
As one of traditional Chinese drugs, Rhubarb's effective parts (Rep) is eluate produced by rhababerone extraction passing through macroporous adsorptive resin which includes five ingredients, emodin, chrysophanol, emodin monomethyl ether, rhein and aloe-emodin. Satisfactory therapeutic effects of Rep were reported for improving cerebral ischemia, cerebral metabolism and resisting rejection reaction. Another drug—Sulodexide is a kind of highly purified biological product. It belongs to glycosaminoglycans(GAGs) drugs with biological activity of protecting kidney including heparin, low molecular heparin, heparin sulphate, sulphuric dematan and glycosaminoglycan mixture. Sulodexide consists of two kinds of GAG with the activity of preventing diabetic nephropathy(DN) in experimental models. It was found to resist the overexpression of TGF-β1 and substrate synthesis induced by high-concentration glucose in kidney glomerulus cells, and repair disfunction of endothelial cell during the study of DN. Although both Rep and Sulodexide had the prevention activity for kidney, no document reported the therapeutic effect of two drugs to treat CCN. So we established CCN rat model, using Sulodexide and Rep to treat CCN at different timepoint and different dosage and explore the protective and therapeutic mechanism meanwhile.
The article was divided into two parts.
Part 1 Intervention effect of Rep and Sulodexide on CCN rats
Objective:To observe whether Rep and Sulodexide have protective role for CCN and to explore the fundamental mechanism.
Methods:
1. Experimental grouping and biochemical indicator determination:54 SPF healthy male Wistar rats (180～200g) were divided into 9 groups randomly after 1 week's adaptive feeding with low salt diet and free drinking:Group 1, normal control group (VH3:olive oil 5ml·kg-1·d-1×28 d); Group 2, model group (MX3:CsA 25mg·kg-1·d-1×28 d); Group 3, positive control group (YDY:CsA 25mg·kg-1·d-1+ benazepril 5mg·kg-1·d-1×28 d, the two drugs administered over 2 hours intervals); Group 4, Rep intervention group 1 (DY1:after Rep 50mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28d, the two drugs administered over 2 hours intervals); Group 5, Rep intervention group 2 (DY2:CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28 d, the two drugs administered over 2 hours intervals); Group 6, Rep intervention group 3 (DY3:after CsA 25mg·kg-1·d-1×7d,CsA 25mg·kg-1·d-1+ Rep 50mg·kg-1·d-1×21d, the two drugs administered over 2 hours intervals); Group 7, Sulodexide intervention group 1 (WY1:after Sulodexide 10mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×28d); Group 8, Sulodexide intervention group 2(WY2:CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×28d); Group 9, Sulodexide intervention group 3 (WY3:after CsA 25mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×21d). Based on experimental design, CsA and Rep were taken by intragastric administration, Sulodexide was given by intraperitoneal injection.All rats were killed on day 28th. During the experimental period, rats in each group were raised freely. Rat's body weight was measured at the last day in every week before and after medication, intragastric administration and abdominal injection dosage would be adjusted. At the same time, venous blood from rat tail was collected to measure serum creatinine(Scr) and blood urea nitrogen(BUN).
2. Harvested kidney tissues were fixed in periodate-lysine-paraformaldehyde solution and embedded in wax.After dewaxing,2-μm sections were processed and stained with HE,PAS and Masson. Pathological changes,hyaline degeneration of small arteries and renal interstitial fibrosis(RIF) were observed. A semiquantitative score was used to assess the extend of changes in each category.Hyalinosis of small arteries was determined by counting afferent and the juxtaglomerular arterioles available for examination with a minimum of 50 glomeruli per biopsy assessed:0=no hyalinosis; 1 =early hyaline changes; 2=pronounced hyalinosis.RIF was estimated by counting the percentage of injured areas per field and scored as follows:0=normal interstitium,0.5 =<5% of interstitium areas injured,1-5% to 15%,1.5=16% to 25%,2=26% to 35%, 2.5=36% to 45%, and 3=>45%.
3. The ultrastructural changes of kidney tissues were also observed by electron microscope.
4. Kidney was isolated from rat on ice and the following indicators of the kidney tissues were examined.
4.1 Immunohistochemical detection:CD68, collagenⅢand collagenⅣexpression;
4.2 Reverse transcription-polymerase chain reaction (RT-PCR) assay:TGF-β1 and TIMP-1 mRNA expression;
4.3 ELISA detection:TGF-β1, TIMP-1 and MMP-9 protein expression.
Results:
1.Body weight, BUN, Scr
1.1 Body weight:1 week after CsA administration, the rat in MX3 group growed delay, the weight decreased after 2 weeks vs VH3; 2-4 weeks,the rat weight of YDY and all intervention groups was higher than that of MX3 group (P<0.05), DY3, WY3, YDY group lower than VH3 group(P<0.05), other group was similar to VH3 group (P>0.05).
1.2 BUN:1 week after CsA administration, the BUN level of MX3 group began to increase,after 2 weeks,it was higher (P<0.05) than VH3 group; two weeks later the BUN level of DY1, WY1, WY2 group didn't increase (P>0.05), lower than MX3 group(P<0.05), similar to VH3 group(P> 0.05).1 week after, the BUN level of YDY, DY3, WY3 increased apparently,after 3 weeks it began to decline, but higher than VH3 lower than MX3 group(P<0.05);however,the rat BUN level of DY2 group was lower than the above-mentioned three groups (P<0.05),it was similar among the three groups (P> 0.05).
1.3 Scr:After 1 week, the rat Scr level of MX3 group increased (P<0.05) vs VH3. At 2-4 weeks that of YDY and all intervention groups was lower than MX3 group.Compared with VH3 group, DY1 group decreased (P<0.05), DY2, WY1 group no changes (P>0.05), YDY and other intervention groups increased (P<0.05). The Scr level of WY2 was lower than YDY, DY3, WY3 group(P<0.05).The Scr level of DY3, WY3 at 4 weeks decreased (P<0.05) and lower than YDY group (P<0.05).
2. Light microscopy:In normal control group, light microscopy showed normal structure of kidney tissues. In MX3 group, capillary loop opened badly in kidney glomerulus, cellular and intercellular vacuolization,tubular collapse and dilation.Striped interstistial fibrosis was present with thickening of the tubular basement membranes and Bowman's capsule.Muscular thickening of intrarenal arteriolar vessel wall,arterilar hyalinosis and injury appeared, and focal glomerulosclerosis was found occasionally.The above-mentioned pathological changes of YDY and intervention groups significantly reduced. The hyaline degeneration and RIF of erery group were as follows.
2.1 Hyalinosis of small arteries score:The score was higher in YDY and intervention groups than VH3 group,lower than MX3 group (P<0.05), higher in DY1, DY2, DY3, WY3 group than YDY group (P<0.05). Rep vs Sulodexide:The score in Sulodexide groups in various time points was lower than Rep groups(P<0.05), and increased at the third time point as well as higher than at the previous two time points (P<0.05), no difference was found between the first two time points (P>0.05).There was no difference among the various time points in Rep groups(P>0.05).
2.2 RIF score:The score in YDY and intervention groups was higher than VH3 group (P<0.05), lower than MX3 group (P<0.05), in which the score of DY3, WY3 group was higher than YDY group (P<0.05), similar in the other intervention groups with YDY group (P>0.05). Rep vs Sulodexide:At the first time point it was lower in Sulodexide group than Rep group (P<0.05), no difference was found between the two medicine groups at the other two time points (P>0.05). In both medicine groups it was higher at the third time point than that at the front two time points (P<0.05), no difference was found between the front two time points (P>0.05).
3. Renal ultrastructure changes:The structure was normal in normal control groups.In rat kidney tissues of MX3 group, glomerular basement membrane thickened, mesangium matrix broadened and increased, podocyte foot process fused extensively and microvillus became sparsed; tubular basement membrane thickened irregularly and broke, epithelial cell appeared a lot of vacuoles, nucleus edge changed to be irregular,chromatin thickened and concentrated, pyknosis,tubular microvillus exfoliated, epithelial cell endolysosome and mitochondria increased significantly, collagen fiber increased in interstitium. The rat lesions of DY1,DY2,WY1,WY2 groups significantly lightened:podocyte foot processes fused occasionly; segmental mesangial matrix mild increased; tubular showed a little bubble, lysosome, mitochondria and the basement membrane intact.Pathological changes of YDY,DY3,WY3 group deteriorated but improved much compared with MX3 group.
4. Kidney immunohistochemistry:CD68, collagenⅢand collagenⅣexpression levels in every group showed a similar trend with the inflammatory cell infiltration in HE staining. After 4 weeks, those were higher in model group than in control and intervention groups.
4.1 CD68 expression:The expression level was similar in DY1, DY2, WY1, WY2 with VH3 group(P>0.05), lower in YDY, DY3, WY3 than VH3 group (P<0.05).The level was lower in YDY and all intervention groups than MX3 group (P<0.05); it was similar in YDY with DY3, WY3 group (P>0.05), but higher than that in the other intervention groups (P<0.05). Rep vs Sulodexide:At the third time point in Rep group it was lower than Sulodexide group (P<0.05), there was no difference between the two medicine groups at the other two time points (P>0.05). Both medicines showed higher level at the third time point than the front two time points (P<0.05), no difference between the front two time points (P>0.05).
4.2 CollagenⅢexpression:Compared with VH3 group, collagenⅢexpression level was similar in DY2, WY2 groups (P>0.05), higher in YDY and the other intervention groups (P<0.05). Compared with MX3 group,it was lower in YDY and all intervention groups (P<0.05). While compared with YDY group, the level in WY1 group had no change (P>0.05), DY3, WY3 group increased (P<0.05), other intervention groups decreased (P<0.05). Rep vs Sulodexide:At the third time point it was lower in Sulodexide than Rep group(P<0.05), there was no difference between the two medicine groups at the other two time points (P>0.05). Both medicines showed it was higher at the third time point than the front two time points (P<0.05), and higher at the first time point than the second time point (P<0.05).
4.3 CollagenⅣexpression:Vs VH3 group, collagenⅣexpression level in WY1 group had no change(P>0.05), the other intervention groups increased (P<0.05). Compared with MX3 group, the level of YDY and all intervention groups decreased(P <0.05); compared with YDY group,the expression level in WY1 group decreased (P <0.05), that in DY3, WY3 group increased(P<0.05), other intervention groups had no difference (P>0.05). Rep vs Sulodexide:The level was lower in Sulodexide group at the first and third time point than Rep group (P<0.05), there was no difference between the two medicine groups at the second time point (P>0.05). Both medicines showed higher at the third time point than the front two time points (P<0.05), in Sulodexide group the level was higher at the second time point than the first one (P <0.05), and in Rep group there was no difference between the front two time points (P >0.05).
5. TGF-β1 and TIMP-1mRNA expression
5.1 TGF-β1mRNA expression:In VH3 group, there was only a small amount of TGF-β1mRNA expression, it increased significantly in model,YDY and other intervention groups except WY1 group (P<0.05), the expression of rats in YDY and all intervention groups was lower than MX3 group (P<0.05), that was lower in Rep and Sulodexide groups than YDY group (P<0.05). Rep vs Sulodexide:Compared with Rep groups, it decreased in Sulodexide group (P<0.05), and the expression level in Rep groups was 3>2>1(P<0.05).There was no difference between the first and second time point(P>0.05) in Sulodexide groups,but they were all lower than at the third time point (P<0.05).
5.2 TIMP-1 mRNA expression:In VH3 group, there was only a small amount of expression, it increased in MX3,YDY and intervention groups(P<0.05), the expression of rats in YDY and all intervention groups was lower than in MX3 group (P<0.05), lower in Rep and Sulodexide groups than in YDY group(P<0.05). Rep vs Sulodexide: The expression level was lower in Sulodexide groups at any time point than in Rep groups(P<0.05), while both medicine groups showed gradually increase with the time extending (P<0.05).
6. TGF-β1 TIMP-1 and MMP-9 protein expression
6.1 TGF-β1 protein expression:Compared with VH3 group,the expression was similar in WY1, WY2 group (P>0.05), decreased in DY1, DY2, WY3 group (P<0.05), increased in DY3, YDY group (P<0.05). The level in each other group was higher than that in MX3 group(P<0.05), lower than VH3 group(P<0.05). Rep vs Sulodexide: There was significant difference among all groups (P<0.05), at the front two time points the expression level in Rep was lower than that in Sulodexide group, and at the third time point, just the opposite.It was higher at the third time point than the other two time points(P<0.05) in Rep groups,no difference between the two ones (P>0.05); and the expression level in Sulodexide groups was similar at the first and second time point(P>0.05), but both higher than the third one (P<0.05).
6.2 TIMP-1 protein expression:Compared with VH3 group,the expression level was similar in DY3, WY1, WY2 group (P>0.05); decreased in DY1,DY2, WY3 group (P<0.05); increased in YDY group (P<0.05).It was lower in all the other groups except DY3 group (P=0.05) compared with MX3 group (P<0.05). The TIMP-1 protein expression level in each other group was lower than YDY group (P<0.05). Rep vs Sulodexide:At the front two time points the expression in Rep decreased compared with that in Sulodexide group(P<0.05), but at the third time point,it decreased in Sulodexide compared with that in Rep group(P<0.05).It was higher at the third time point than the other two time points in sulodexide groups(P<0.05); however, lower at the third time point than the other two time points in Rep groups(P<0.05).There was no difference between the front two ones (P>0.05) both in Rep and Sulodexide groups.
6.3 MMP-9 protein expression:Compared with VH3 group, no change was found in MX3, WY1 group (P>0.05), the expression increased in YDY group and other intervention groups (P<0.05), among those groups the expression of MMP-9 protein in YDY increased the highest (P<0.05).Rep vs Sulodexide:At the first and third time point the expression level in Sulodexide group was lower than in Rep group (P<0.05), but at the second time point,it was lower in Rep than in Sulodexide group.There was no difference among the three time points (P>0.05) in Rep groups,but the level in Sulodexide groups was 2>3>1(P<0.05).
Conclusion:
Cyclosporine nephrotoxicity could be significantly reduced,even some toxicity can be eliminated by Sulodexide or Rep intervention. Early intervention,better result would be obtained.The effect of Rep and Sulodexide was better than benazepril in decreasing CsA nephrotoxicity.The comparison between Sulodexide and Rep groups found that administration at the first time point,the effect of Sulodexide was better than that of Rep;however,the effect of two medicines administration at the other two time points matched.
Part 2 The therapeutic use of Rep and Sulodexide in CCN rats
Objective:To establish CCN model and investigate whether nephrotoxicity could be eliminated by itself 2 weeks after CsA withdrawal; To observe whether Rep or Sulodexide has the therapeutic effect on CCN simultaneously and to evaluate the curative effect.
Methods:
1. Experimental grouping:78 SPF healthy male Wistar rats (180-200g) were divided into 9 groups randomly after 1 week's adaptive feeding with low salt diet and free drinking:Group 1, normal control group (VH1:n=6, olive oil 5ml·kg-1·d-1×14d and sacrificed at 14 d; VH2:n=6, olive oil 5ml·kg-1·d-1×14d, later water for injection 5ml·kg-1·d-1×14d and sacrificed at 28 d; VH3:n=6, olive oil 5ml·kg-1·d-1×28 d and sacrificed at 28 d); Group 2, model group (MX1:n=6, CsA 25mg·kg-1×14d and sacrificed at 14 d; MX2:n=6, CsA 25mg·kg-1·d-1×14d, later water for injection 5ml·kg-1·d-1×14 d and sacrificed at 28 d; MX3:n=6, CsA 25mg·kg-1·d-1×28 d and sacrificed at 28 d); Group 3, positive control group (YDZ CsA 25mg·kg-1·d-1×14d, later benazepril 5mg·kg-1·d-1×14d); Group 4:Rep therapy group 1(DZ1:CsA 25mg·kg-1·d-1×14d, later Rep 25mg·kg-1·d-1×14d); Group 5, Rep therapy group 2 (DZ2:CsA 25mg·kg-1·d-1×14d, later Rep 50mg·kg-1·d-1×14 d); Group 6, Rep therapy group 3(DZ3:CsA 25mg·kg-1·d-1×14d, later Rep 100mg·kg-1·d-1×14d); Group 7, Sulodexide therapy group 1 (WZ1:CsA 25mg·kg-1·d-1×14d, later Sulodexide 10mg·kg-1·d-1×14d); Group 8, Sulodexide therapy group 2 (WZ2:CsA 25mg·kg-1·d-1×14d, later Sulodexide 20 mg·kg-1·d-1×14 d); Group 9, Sulodexide therapy group 3 (WZ3:CsA 25mg·kg-1·d-1×14d, later Sulodexide 40 mg·kg-1·d-1×14d). Based on experimental design, CsA and Rep were taken by intragastric administration, Sulodexide was given by intraperitoneal injection. The rats in group 3-8 were all killed at 28d.Group 9 failed to finish the experiment because of two rats were dead.
2. Rats observation and all indexes checking methods were as same as Part 1.
Results:
1.Body weight,BUN and Scr
1.1 Body weight:Except group 9, rats in the other groups survived and were included in the last statistics. At the first day, there were no significant differences of rat's body weights among various groups(P>0.05); At 14d, rats'body weights among MX groups,YDZ and therapy groups had no difference(P>0.05), while body weights in above-mentioned groups decreased compared to those in VH group(P<0.05). At 21d, rats'body weights continued to decline in MX3 group, but increased in other groups(except VH1, MX1 group because rats had been sacrificed). To 28d, all rats' body weights were improved. Compared with the results at 21d, body weights in MX3 and WZ2 groups had no significant differences, but those in other groups improved(P <0.05). At 28d, body weights in VH2 and VH3 group increased to the highest level. The difference among MX3 and the other groups was significant (P<0.05). Body weights in all therapy groups had no significance compared with those in YDZ and MX2 groups(P>0.05).
1.2 BUN,Scr:At the first day, there were no differences of BUN, Scr among all groups(P>0.05); At 7 d and 14 d, level of BUN, Scr in all MX groups,YDZ and therapy groups increased compared to those of normal control group(P<0.05); At 28 d, level of BUN, Scr in the experimental groups except VH1 and MX1 group(rats had been sacrificed) decreased compared with those in MX3 group(P<0.05); Compared to MX2 group,BUN results in DZ1, DZ2 groups and Scr results in DZ1, DZ2,WZ2 groups increased, while other therapy and YDZ group had no changes(P>0.05). Compared to YDZ group, BUN level in DZ1 and DZ2 groups and Scr level in DZ1, DZ2 and WZ2 groups increased (P<0.05), while other therapy groups had no changes(P>0.05).
2. Light microscopy results:In normal control group, light microscopy showed normal structure of kidney tissues. The pathology changes of MX3 group were as same as those in Part1. In MX1 group, the results showed the capillary loop opened badly, monocyte infiltration in interstitial tissue, interspersed striped interstitial fibrosis, partial afferent arterioles hyalinization. However, the above-mentioned pathological changes in MX2 and YDZ group improved significantly, and nearly restored to normal appearance in therapy groups. Hyalinosis and RIF in each group were as follows:
2.1 Hyalinosis of small arteries score:No obvious hyalinosis was found in therapy and YDZ groups compared to normal control and MX2 groups (P>0.05), while the hyalinosis was relieved compared to MX1, MX3 groups(P<0.05); the results in therapy groups had no differences with the generation in YDZ group(P>0.05).
2.2 RIF score:Slight RIF could be seen in normal control group; model groups(MX1, MX2 and MX3) showed significantly aggravation which most serious RIF in MX3, slightest in MX2 and moderate in MX1 group(P<0.05).The serious degree of RIF was similar in YDZ and MX2 groups(P>0.05). RIF in therapy groups was less serious than YDZ and MX2 group(P<0.05) while more serious than normal control groups(VH1, VH2 and VH3) (P<0.05). Rep vs Sulodexide:RIF in WZ1 group was slightest, and RIF in DZ1, DZ3 and WZ2 was most serious and RIF degree in DZ2 was moderate (P<0.05) in which there were no statistical differences among DZ1, DZ3 and WZ2 groups(P>0.05).
3. Ultrastructure changes in kidney tissues:The structure was normal in normal control groups.The changes of MX3 group were as same as those in Part1.The changes in MX1 group showed slighter, podocyte foot process fused partially, mesangium matrix increased slightly, vacuoles, lysosome and mitochondria could be found in tubule, thickness of tubular basement membrane was discrepant, segment broke, nuclear chromatin concentrated and interstitial collagen fiber appeared occasionally. Pathological changes in MX2 group improved obviously compared to the results in MX1 and MX3 groups, and the pathology was nearly normal in YDZ and therapy groups.
4.Immunohistochemistry results in kidney tissues
4.1 CD68 expression:The level of CD68 expression from low to high was VH1, VH2,VH30.05). Rep vs Sulodexide:Results in DZ1 group were lowest, and subsequently increased in DZ2, WZ1, WZ2, DZ3 group in turn(P<0.05, except the difference between those of WZ2 and DZ3 group).
4.2 CollagenⅢexpression:CollagenⅢexpression level was lowest in normal control group (VH1, VH2 and VH3 group), DZ1, DZ2 and DZ3 group(P>0.05). The results in model groups increased (MX3>MX2>MX1,P<0.05).Changes in YDZ, WZ1 and WZ2 groups were similar to those of MX2 group(P>0.05), while higher than MX1 group(P<0.05) and lower than MX3 group(P<0.05). Rep vs Sulodexide:Level in Rep therapy groups was lower than Sulodexide therapy groups(P<0.05), while no difference was found in different dosage groups using the same therapeutic drug.
4.3 CollagenⅣexpression:There were no statistical differences among collagenⅣexpression level in normal control group(VH1, VH2 and VH3 group), MX2, DZ2 and WZ1 group(P>0.05); Level in MX1, MX3, YDZ, DZ1, DZ3 and WZ2 increased (P<0.05) which was highest in MX3 group(P<0.05),higher in YDZ group(P<0.05) and the lowest in the other four groups(P<0.05, level in the four groups was similar, P >0.05). Rep vs Sulodexide:Results in DZ2, WZ1 were comparable low(level in the two groups was similar, P>0.05), and subsequently increased in DZ3, WZ2 and DZ1 group in turn (P<0.05, level of the above three groups was similar, P>0.05).
5. TGF-β1 and TIMP-1mRNA expression
5.1 TGF-β1mRNA expression:TGF-β1mRNA expression level was lowest in DZ2 group, and subsequently increased in VH1, VH2, VH3, DZ1, DZ3 and WZ1 group in turn, and the level was even higher in WZ2, YDZ, MX2, MX1 group, and level of MX3 group was highest(P<0.05). Rep vs Sulodexide:Results in DZ2, WZ1 and DZ1 group were lower(level in the three groups was similar, P>0.05), level in DZ3 and WZ2 groups (level in the two groups was similar, P>0.05) increased compared to the above-mentioned three groups(P<0.05).
5.2 TIMP-1mRNA expression:TIMP-1mRNA expression level was lowest in WZ1 group, lower in WZ2 group, and subsequently in VH1, VH2, VH3 and DZ1 group, then increased in MX2, DZ2, YDZ, DZ3, MX1 group in turn, level in MX3 group was highest(P<0.05). Rep vs Sulodexide:The group sequence according to TIMP-1mRNA expression level from low to high was WZ1, WZ2, DZ1, DZ2 and DZ3(P<0.05), and level in Sulodexide therapy groups was all lower than that in Rep therapy groups(P<0.05).
6. TGF-β1,TIMP-1 and MMP-9 protein Expression
6.1 TGF-β1 protein expression:TGF-β1 protein expression restored to normal level in MX2 after CsA withdrawal and increased in YDZ and all drug therapy groups(P<0.05), results in WZ1, DZ2 and DZ1 group were higher than those of YDZ group(P<0.05), while level in WZ2 and DZ3 was similar to that of YDZ group(P >0.05). Rep vs Sulodexide:No difference was found among DZ3, WZ1 and WZ2 groups(P>0.05), the differences among the other groups had statistical significance(P <0.05) which level in WZ2 and DZ3 groups lowest, and subsequently WZ1, DZ2 and DZ1 in turn.
6.2 TIMP-1 protein expression:TIMP-1 protein expression restored to normal level in MX2 after CsA withdrawal, and increased in drug therapy groups (P<0.05) except benazepril group. Rep vs Sulodexide:Level in DZ3, WZ1 and WZ2 was lowest(three groups'results were similar, P>0.05), lower in DZ2 group and highest in DZ1 group(P<0.05).
6.3 MMP-9 protein expression:MMP-9 protein expression was highest in WZ2 group, lower in MX1, DZ1, DZ2 and WZ1 group (level in these groups was similar, P >0.05), lowest in VH3, MX2, MX3, DZ3, VH1, VH2 and YDZ(P<0.05) which level in these groups was similar(P>0.05). MMP-9 protein expression restored to normal level in MX2 after CsA withdrawal(compared to normal control group, P>0.05). Results in therapeutic groups except DZ3 and YDZ group increased compared to normal control group and MX2, MX3, MX1 group(P<0.05). Rep vs Sulodexide:Level in WZ2 group was highest, that in DZ1, DZ2 and WZ1 was comparable higher while level in DZ3 group was lowest. There was difference between DZ3 and DZ1, DZ2, WZ2 groups (P<0.05),while the differences among level in other groups had no statistical significance(P>0.05).
Conclusions:
After CsA withdrawal,common indexes such as body weight,BUN,Scr and small artery hyalinosis of rat kidney tissues could be restored, RIF could be improved slightly,but the administration of Sulodexide or Rep could improved RIF obviously.When 10 mg·kg-1·d-1 Sulodexide or 50mg·kg-1·d-1 Rep was administered,the best result appeared.The two medcines'effect matched each other.
大黄是我国传统中药之一,大黄有效部位(Rhubarb's effective parts,Rep)为大黄醇提液过大孔吸附树脂得到的洗脱物,包含以下五种成分：大黄素、大黄酚、大黄素甲醚、大黄酸、芦荟大黄素。文献报道其在改善脑缺血、脑代谢及抗排斥反应方面效果显著。伟素(舒洛地特,sulodexide)是经过高度纯化的生物制品,与肝素同属糖胺聚糖(glycosaminoglycans,GAGs)类药物。GAG通常指一大类分子,包括肝素、低分子肝素、硫酸类肝素、硫酸类皮肤素以及糖胺聚糖类化合物,它们具有共同的生物学活性即保护肾脏的活性。伟素由两种GAG组成,在实验模型中具有预防糖尿病肾病(dibetic nephropathy,DN)的活性,能够抑制TGF-β1过度表达以及肾小球细胞高浓度葡萄糖诱导的基质合成,修复内皮细胞机能障碍。但Rep和伟素是否可以通过改善肾脏缺血和代谢,对抗肾排斥反应及保护肾脏活性等机制影响CCN的发生和发展,目前尚未可知,亦无相关文献报道。为此,我们复制了CCN大鼠模型,观察不同剂量Rep和伟素在不同时间点对模型大鼠的干预和治疗作用,并对其可能的作用机理进行探讨。
本论文分以下2部分。
第1部分Rep及伟素对CCN大鼠的干预作用
目的：
观察Rep及伟素对CCN的预防效果。
方法：
1.实验分组及生化指标测定：SPF级健康雄性Wistar大鼠54只(180～200g),给予低盐饲料,自由饮水,适应性喂养1周后,将动物随机分为9组,分别为：第一组正常对照组(VH3：橄榄油5ml·kg-1·d-1×28 d);第二组模型组(MX3:CsA 25mg·kg-1·d-1×28 d)；第三组阳性对照组(YDY:CsA 25mg·kg-1·d-1+贝那普利5mg·kg-1·d-1×28d)；第四组Rep干预1组(DY1：Rep 50mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28 d,二药灌胃时间间隔2h以上)；第五组Rep干预2组(DY2:CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28d,二药灌胃时间间隔2h以上)；第六组Rep干预3组(DY3:CsA 25mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1 +Rep 50mg·kg-1·d-1×21d,二药灌胃时间间隔2h以上)；第七组伟素干预1组(WY1：伟素10mg·kg-1·d-1×7d,后给予CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×28d);第八组伟素干预2组(WY2:CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×28d);第九组伟素干预3组(WY3:CsA 25mg·kg-1·d-1×7 d,后给予CsA 25mg·kg-1·d-1+伟素10mg·kg-1·d-1×21d)。按实验设计,CsA及Rep为灌胃给药,伟素为腹腔注射。所有大鼠均于第28天处死。实验期间在用药前和用药后的每周最后一天测量大鼠体重并调整灌胃和腹腔注射剂量。同时采集大鼠尾静脉血,测定血肌酐(serum creatinine, Scr)和血尿素氮(blood urea nitrogen, BUN)。
2.取肾组织行HE、PAS及Masson染色观察病理变化,分析各组小动脉透明样变性、肾间质纤维化(renal interstitial fibrosis, RIF)情况并通过半定量积分评估其程度。入球小动脉透明样变性通过计数入球及球旁小动脉病变来判断。每个标本至少检查50个小动脉,进行下列评分：0=无透明样变性；1=早期透明样变性；2=明显透明样变性。RIF通过计数每个视野损伤面积的百分比来评估：0=正常肾间质,0.5=<5%,1=5%到15%,1.5=16%到25%,2=26%到35%,2.5=36%到45%,3=>45%肾间质面积损伤。
3.同时取肾组织行电镜检查观察超微结构变化。
4.冰上分离肾脏并采取免疫组织化学法检测肾组织CD68、Ⅲ型胶原及Ⅳ型胶原的表达,逆转录—聚合酶链反应(RT-PCR)法检测肾组织TGF-β1和TIMP-1 mRNA表达,ELISA法检测TGF-β1、TIMP-1和MMP-9蛋白表达。
结果：
1.体重、BUN、Scr:
1.1体重：给予CsA后,MX3组大鼠1周后增长延缓,2周后体重下降,第2-4周与VH3相比,体重下降(P<0.05)；所有干预组及YDY组均高于MX3组(P<0.05),其中DY3、WY3、YDY低于VH3组(P<0.05),其它组与VH3组相近(P>0.05)。
1.2 BUN:MX3组大鼠第1周血BUN水平开始上升,第2-4周,与VH3组相比,差异显著(P<0.05); DY1、WY1、WY2组大鼠BUN水平无上升(P>0.05),均低于MX3组(P<0.05),与VH3组相近(P>0.05)。YDY、DY3、WY3组于1周后仍见上升,3周后逐渐下降且均高于VH3组、低于MX3组(P<0.05),而DY2逐渐上升,于4周开始下降,均低于其他三组(P<0.05),其它三组无差别(P>0.05)。
1.3 Scr：与VH3组相比,第1～4周,MX3组大鼠Scr水平上升(P<0.05)。第2-4周所有干预组及YDY组均低于MX3组(P<0.05)；与VH3组比,DY1组较低(P<0.05), DY2、WY1与之相近(P>0.05),其它干预组及YDY组上升(P<0.05)。WY2组低于YDY、DY3、WY3组(P<0.05),而DY3、WY3组于4周下降(P<0.05)均低于YDY组(P<0.05)。
2.光镜结果：VH3组大鼠肾组织结构正常。MX3组大鼠于四周后肾小球毛细血管袢开放不佳,近曲小管细胞内空泡变性坏死,小管萎缩同时伴单核细胞浸润,可见灶状及条带状RIF。肾内小动脉管壁肌性增厚,入球小动脉透明样变性及损伤,偶见局灶性肾小球硬化。而干预组及YDY组大鼠的上述病变明显减轻,其小动脉透明样变性及RIF也明显减轻。
2.1小动脉透明样变性评分：各干预组及YDY组评分均高于VH3组,低于MX3组(P<0.05)；干预组中DY1、DY2、DY3、WY3高于YDY组(P<0.05),其它干预组与YDY组相近(P>0.05)。伟素与Rep比较：伟素各个时间点评分均低于Rep组(P<0.05),而且第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)；Rep各个时间点间无差别(P>0.05)。
2.2 RIF评分：各干预组及YDY组均高于VH3组,低于MX3组(P<0.05)：干预组中DY3、WY3高于YDY组(P<0.05),其它干预组与YDY组相近(P>0.05)。伟素与Rep比较：在第1个时间点伟素低于Rep组(P<0.05),其余2个时间点二者无差别(P>0.05)。二药均显示第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)。
3.肾组织超微结构变化：VH3组肾组织超微结构正常。MX3组大鼠肾小球基底膜增厚,系膜基质增宽、增多,足细胞足突广泛融合、微绒毛稀疏；小管基底膜不规则增厚、断裂,上皮细胞大量空泡、大小不等,细胞核固缩,小管微绒毛脱落,上皮细胞内溶酶体、线粒体明显增多,间质见较多胶原纤维。DY1、DY2、WY1、WY2组大鼠上述病变明显减轻,偶见足突融合,系膜基质节段轻度增多,小管可见少量空泡,溶酶体、线粒体较少,小管基膜完整。YDY、DY3、WY3组病变较重但较MX3组轻。
4.肾组织免疫组织化学：各组大鼠肾间质CD68、胶原Ⅲ、胶原Ⅳ表达变化趋势与HE染色显示的炎性细胞浸润趋势相似。给药4周后,MX3组CD68、胶原Ⅲ、胶原Ⅳ的表达高于对照组和预防组(P<0.05)。
4.1 CD68表达：DY1、DY2、WY1、WY2组CD68表达水平与VH3组相近(P>0.05), YDY、DY3、WY3组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；YDY组与DY3、WY3组相近(P>0.05),均高于其它用药干预组(P<0.05)。伟素与Rep比较：在第3个时间点Rep低于伟素组(P<0.05),其余2个时间点二者无差别(P>0.05)。两药均显示第三个时间点高于前两个时间点(P<0.05),前两个时间点间无差别(P>0.05)。
4.2胶原Ⅲ表达：DY2、WY2组胶原Ⅲ表达水平与VH3组相近(P>0.05),其它干预组及YDY组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；而与YDY组相比,WY1组与之相近(P>0.05),DY3、WY3组较之增高(P<0.05),其它干预组均降低(P<0.05)。伟素与Rep比较：在第3个时间点伟素低于Rep组(P<0.05),其余2个时间点二者无差别(P>0.05)。两药均显示时间点3>2>1(P<0.05)。
4.3胶原Ⅳ表达：WY1与VH3组胶原Ⅳ表达相近(P>0.05),其它干预组及YDY组高于VH3组(P<0.05)；各干预组及YDY组均低于MX3组(P<0.05)；而与YDY组比,WY1组降低(P<0.05),DY3、WY3组升高(P<0.05),其它干预组与之相近(P>0.05)。伟素与Rep比较：在第1、3个时间点伟素低于Rep组(P<0.05),第2个时间点二者无差别(P>0.05)。两药均显示第3个时间点高于前2个时间点(P<0.05),伟素第2个时间点又高于第1个时间点(P<0.05),而Rep前两个时间点间无差别(P>0.05)。
5.TGF-β1和TIMP-1mRNA表达
5.1 TGF-β1mRNA表达：VH3组有少量TGF-β1mRNA表达,除WY1组外,MX3以及其它干预组表达增加(P<0.05),干预组及YDY组的表达要低于MX3组(P<0.05),而各干预组中的表达要低于YDY组(P<0.05)。伟素与Rep比较：伟素各个时间点均低于Rep (P<0.05),且Rep在时间点3>2>1(P<0.05),而伟素在时间点1、2效果相近(P>0.05),但低于时间点3(P<0.05)。
5.2 TIMP-1mRNA表达：VH3组有少量TIMP-1mRNA表达,MX3、YDY以及干预组表达增加(P<0.05),干预组及YDY组表达均低于MX3组(P<0.05),而各干预组的表达要低于YDY组(P<0.05)。伟素与Rep比较：伟素各个时间点均低于Rep组(P<0.05),而二者TIMP-1mRNA表达水平均随着用药时间的延迟而逐渐增高(P<0.05)。
6. TGF-β1、TIMP-1和MMP-9蛋白表达
6.1 TGF-β1蛋白表达：WY1、WY2组TGF-β1蛋白表达与VH3组相近(P>0.05), DY1、DY2、WY3组比之降低(P<0.05), DY3、YDY组比之升高(P<0.05)；各组均高于MX3组(P<0.05),低于YDY组(P<0.05)。伟素与Rep比较：各组之间差异均有显著性(P<0.05),在前两个时间点用药Rep低于伟素组,而在时间点3,正好相反。Rep组在时间点3高于2、1(P<0.05),后二时间点间无差别(P>0.05)；而伟素组在时间点1、2干预用药效果相近(P>0.05),但高于时间点3(P<0.05)。
6.2 TIMP-1蛋白表达：DY3、WY1、WY2组TIMP-1蛋白表达与VH3组相近(P>0.05),DY1、DY2、WY3组比之降低(P<0.05),YDY组升高(P<0.05)；各干预组均低于MX3组【除DY3(P=0.05)外P值均<0.05】,而YDY组高于MX3组(P<0.05)；各干预组均低于YDY组(P<0.05)。伟素与Rep比较：在时间点1、2Rep低于伟素组(P<0.05),在时间点3伟素低于Rep组(P<0.05)。伟素组在时间点3低于时间点2、1(P<0.05),Rep在时间点3高于时间点2、1(P<0.05),而各自在时间点2与1比差异无显著性(P>0.05)。
6.3 MMP-9蛋白表达：MX3、WY1组MMP-9蛋白表达水平与VH3组相近(P>0.05),而其它干预组及YDY组均增高(P<0.05),以YDY组为最高(P<0.05)。伟素与Rep比较：在时间点1和3伟素低于Rep组(P<0.05),而在时间点2 Rep低于伟素组(P<0.05)；Rep组在各个时间点基本一致(P>0.05),而伟素组各个时间点MMP-9蛋白表达水平由低到高为1<3<2(P<0.05)。
结论：1.伟素和Rep单独干预用药均可减轻CsA肾毒性,甚至可消除其部分毒性,早期干预效果更好。2.采用伟素或Rep预防CsA肾毒性优于贝那普利。3.伟素在改善小动脉透明样变性方面优于Rep；改善RIF方面,提前给药时伟素优于Rep,而同时及后期给药干预时效果相当,但干预机理不同。
第2部分Rep及伟素对CCN大鼠的治疗作用
目的：
复制CCN模型,并于2周后停用CsA,观察停用后肾毒性能否自行消除；同时观察Rep及伟素对CCN是否存在治疗作用；并评估其疗效。
方法：
1.实验分组：SPF级健康雄性Wistar大鼠78只(180～200 g),给予低盐饲料,自由饮水,适应性喂养1周后,将动物随机分为9组分别为：第一组正常对照组(VH1：为对照1组,n=6,给予橄榄油5ml·kg-1·d-1×14d,第14天处死；VH2：为对照2组,给予橄榄油5ml·kg-1·d-1×14d,后改为注射用水5ml·kg-1·d-1×14d,第28天处死；VH3：为对照3组,给予橄榄油5ml·kg-1·d-1×28d,第28天处死)；第二组模型组(MX1：为模型1组,CsA 25mg·kg-1·d-1×14d,第14天处死；MX2：为模型2组,CsA 25mg·kg-1·d-1×14d,后改为注射用水5ml·kg-1·d-1×14d,第28天处死；MX3：为模型3组,给予CsA25mg·kg-1·d-1×28d,第28天处死)；第三组阳性对照组(YDZ：CsA25mg·kg-1·d-1×14d,后改为贝那普利5mg·kg-1·d-1×14d)；第四组Rep治疗1组(DZ1：CsA 25mg·kg-1·d-1×14d,后改为Rep 25mg·kg-1·d-1×14d)；第五组Rep治疗2组(DZ2：CsA 25mg·kg-1·d-1×14d,后改为Rep50mg·kg-1·d-1×14d)；第六组Rep治疗3组(DZ3：CsA 25mg·kg-1·d-1×14d,后改为Rep 100mg·kg-1·d-1×14d)；第七组伟素治疗1组(WZ1：CsA25mg·kg-1·d-1×14d,后改为伟素10mg·kg-1·d-1×14d)；第八组伟素治疗2组(WZ2：CsA 25mg·kg-1·d-1×14d,后改为伟素20mg·kg-1·d-1×14d)；第九组伟素治疗3组(WZ3：CsA 25mg·kg-1·d-1×14d,后改为伟素40mg·kg-1·d-1×14d)。按实验设计CsA及Rep为灌胃给药,伟素为腹腔注射。第三至八组均于第28天处死。第九组因有2例死亡而退出实验。
2.实验观察及所有指标检测同第1部分。
结果：
1.体重、BUN、Scr:
1.1体重：除第九组大鼠外,其余组大鼠均存活并纳入最后统计。第1天各组大鼠体重差异无显著性(P>0.05)；第14天模型组及治疗组、YDZ组大鼠体重差异无显著性(P>0.05),而与正常对照组大鼠比体重下降(P<0.05)；第21天,MX3组大鼠体重继续下降,其它组(除VH1、MX1已处死外)大鼠体重均上升(P<0.05),至28天各组体重均较前上升,以MX3、WZ2组与前相比差异无显著性(P>0.05),其它组均有显著差异(P<0.05)。第28天以VH2、VH3组体重最重,所有组与MX3组(除VH1、MX1已处死)比均升高(P<0.05),各治疗组与YDZ及MX2组比差异无显著性(P>0.05)。
1.2 BUN、Scr:第1天,各组BUN、Scr比较差异无显著性(P>0.05)；第7天、14天,各组与正常对照组相比BUN、Scr均上升(P<0.05)；第28天各组(除VH1、MX1已处死外)与MX3组相比BUN、Scr下降(P<0.05)；与MX2组相比,DZ1、DZ2组BUN上升(P<0.05),DZ1、DZ2、WZ2组Scr上升(P<0.05),而其它治疗组及YDZ组无变化(P>0.05)；各治疗组及YDZ与正常对照组相比无差别(P>0.05)；与YDZ组相比,DZ1、DZ2组BUN上升(P<0.05), DZ1、DZ2、WZ2 Scr上升(P<0.05),其它治疗组无变化(P>0.05)。
2.光镜结果：正常对照组大鼠肾组织结构正常。MX3组改变同第1部分。MX1组大鼠肾小球毛细血管袢开放不佳,间质见单核细胞浸润,散在条带状间质纤维化,部分入球小动脉透明样变性,但上述改变均轻于MX3组。而YDZ组及MX2组大鼠的上述病变明显减轻,治疗组趋于正常。小动脉透明样变性及RIF见下：
2.1小动脉透明样变性评分：各治疗组及YDZ组与正常对照组比无改变(P>0.05)；与模型MX1、MX3组比减轻(P<0.05)；与MX2组比无差别(P>0.05)；而治疗组与YDZ组比亦无差别(P>0.05)。
2.2 RIF评分：正常对照组见少量RIF；模型组均加重以MX3组最重,MX1次之,MX2最轻(P<0.05)；而YDZ与MX2组相似(P>0.05)；治疗组均轻于YDZ、MX2组(P<0.05),重于正常对照组(包括VH1、VH2、VH3)(P<0.05)。伟素与Rep比较：各组由轻到重依次为WZ1
3.肾组织超微结构变化：正常对照组肾组织超微结构正常。MX3组改变同第1部分。MX1组大鼠病变稍轻,足突部分融合,系膜基质轻度增多,小管可见大量空泡、上皮细胞核缘不规整、染色质增多、可见溶酶体、线粒体,小管基膜厚度不一,节段断裂,偶见间质胶原纤维。MX2组病变轻微,YDZ及治疗组基本正常。
4.免疫组织化学
4.1 CD68表达：CD68表达由低到高为VH1、VH2、VH3
4.2胶原Ⅲ表达：其表达以正常对照组(VH1、VH2、VH3)、DZ1、DZ2、DZ3最低(互相比较P>0.05)；模型组均增高(P<0.05) (MX3>MX2>MX1, P<0.05); YDZ、WZ1、WZ2与MX2相近(P>0.05),高于MX1(P<0.05),低于MX3(P<0.05)。伟素与Rep比较：Rep各组均低于伟素组(P<0.05),而同种药物不同剂量间无差别(P>0.05)。
4.3胶原Ⅳ表达：表达在正常对照组(VH1、VH2、VH3)、MX2、DZ2、WZ1相近(P>0.05); MX1、MX3、YDZ、DZ1、DZ3、WZ2增高(P<0.05),而以MX3组为最高(P<0.05),其次为YDZ(P<0.05),其余四组最低(P<0.05)(四组间P>0.05)。伟素与Rep比较：各组以DZ2、WZ1表达较低(二者之间差异无显著性,P>0.05),接下来依次为DZ3、WZ2、DZ1(三者之间差异无显著性P>0.05,与前二者相比P<0.05)。
5.TGF-β1和TIMP-1mRNA表达
5.1 TGF-β1mRNA表达：表达以DZ2
5.2 TIMP-1mRNA表达：表达以WZ1
6.1 TGF-β1蛋白表达：撤退CsA后MX2组恢复至正常水平,治疗组及YDZ组均增高(P<0.05), WZ1、DZ2、DZ1组表达高于YDZ组(P<0.05),而WZ2、DZ3组表达水平与YDZ组相近(P>0.05)。伟素与Rep比较：除DZ3与WZ1、WZ2组差异无显著性(P>0.05),其余各组相互比较差异均有显著性(P<0.05)。各组以WZ2、DZ3表达最低,接下来依次为WZ1、DZ2、DZ1。
6.2 TIMP-1蛋白表达：撤退CsA后MX2组恢复至正常水平,而除贝那普利外,其它药物治疗均使其表达增高(P<0.05)。伟素与Pep比较：DZ3、WZ1、WZ2(三者之间P>0.05)组最低,其次为DZ2组,DZ1组表达最高(P<0.05)。
6.3 MMP-9蛋白表达：表达以WZ2最高；MX1、DZ1、DZ2、WZ1(这几组间差异无显著性P>0.05)次之；VH3、MX2、MX3、DZ3、VH1、VH2、YDZ(这几组间差异无显著性P>0.05)最低(P<0.05)。伟素与Rep比较：表达以WZ2组最高,DZ1、DZ2、WZ1较高,DZ3最低。DZ3分别与DZ1、DZ2、WZ2组比差异有显著性(P<0.05),其它组间相比P>0.05。
结论：撤退CsA后,大鼠一般生化指标及肾组织小动脉透明样变性可自行恢复正常；RIF较前有所改善,但应用伟素或Rep后改善更明显,分别在应用10mg·kg-1·d-1, 50mg·kg-1·d-1时效果最好,二者作用相当。
As active substance of mycetogenetic circular peptide 11, cyclosporine A(CsA) had become to be first-line drug to treat rejection reaction after organ transplantation and autoimmune diseases, quickly put into clinical use and then achieved exact therapeutic effects since it was given approval to be immunosuppressive agent for organ transplantation by FDA in 1983. However, the adverse effect especially chronic nephrotoxicity limited its use. Chronic CsA nephrotoxicity(CCN) is characterized by progressive renal dysfunction,afferent arteriolopathy,inflammatory cell influx,striped tubulointerstitial fibrosis,and increased intrarenal immunogenecity. Mihatsch etc. analyzed kidney biopsy data of kidney transplantation patients, verified that incidence of kidney tubules poisoning due to CsA was 9%-37%, incidence of acute kidney function failure accompanied with diffuse interstitial fibrosis was 0%-19%, while incidence of stripe-like interstitial fibrosis and CsA-related arterial diseases were 5%-50% and 5%-30% respectively. The exact mechanism of this complication is not well understood,and multiple factors are implicated. The pathogenesis of CCN was considered to be relevant with glomerulus ischemia, rennin angiotensin system (RAS) activation, apoptosis, endoplasmic reticulum stress, autophagy, mononuclear macrophage(MC/MP) infiltration and inflammation, regulation disbalance of TGF-β1 and MMPs/TIMPs, osteopontin, nuclear factor-kappa B (NF-κB), Toll-like receptor and so on. Focus on its pathogenesis, a lot of researches had tried to intervent and treat CCN by using western durgs, Chinese drugs as well as reduction or even interruption of CsA.
As one of traditional Chinese drugs, Rhubarb's effective parts (Rep) is eluate produced by rhababerone extraction passing through macroporous adsorptive resin which includes five ingredients, emodin, chrysophanol, emodin monomethyl ether, rhein and aloe-emodin. Satisfactory therapeutic effects of Rep were reported for improving cerebral ischemia, cerebral metabolism and resisting rejection reaction. Another drug—Sulodexide is a kind of highly purified biological product. It belongs to glycosaminoglycans(GAGs) drugs with biological activity of protecting kidney including heparin, low molecular heparin, heparin sulphate, sulphuric dematan and glycosaminoglycan mixture. Sulodexide consists of two kinds of GAG with the activity of preventing diabetic nephropathy(DN) in experimental models. It was found to resist the overexpression of TGF-β1 and substrate synthesis induced by high-concentration glucose in kidney glomerulus cells, and repair disfunction of endothelial cell during the study of DN. Although both Rep and Sulodexide had the prevention activity for kidney, no document reported the therapeutic effect of two drugs to treat CCN. So we established CCN rat model, using Sulodexide and Rep to treat CCN at different timepoint and different dosage and explore the protective and therapeutic mechanism meanwhile.
The article was divided into two parts.
Part 1 Intervention effect of Rep and Sulodexide on CCN rats
Objective:To observe whether Rep and Sulodexide have protective role for CCN and to explore the fundamental mechanism.
Methods:
1. Experimental grouping and biochemical indicator determination:54 SPF healthy male Wistar rats (180～200g) were divided into 9 groups randomly after 1 week's adaptive feeding with low salt diet and free drinking:Group 1, normal control group (VH3:olive oil 5ml·kg-1·d-1×28 d); Group 2, model group (MX3:CsA 25mg·kg-1·d-1×28 d); Group 3, positive control group (YDY:CsA 25mg·kg-1·d-1+ benazepril 5mg·kg-1·d-1×28 d, the two drugs administered over 2 hours intervals); Group 4, Rep intervention group 1 (DY1:after Rep 50mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28d, the two drugs administered over 2 hours intervals); Group 5, Rep intervention group 2 (DY2:CsA 25mg·kg-1·d-1+Rep 50mg·kg-1·d-1×28 d, the two drugs administered over 2 hours intervals); Group 6, Rep intervention group 3 (DY3:after CsA 25mg·kg-1·d-1×7d,CsA 25mg·kg-1·d-1+ Rep 50mg·kg-1·d-1×21d, the two drugs administered over 2 hours intervals); Group 7, Sulodexide intervention group 1 (WY1:after Sulodexide 10mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×28d); Group 8, Sulodexide intervention group 2(WY2:CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×28d); Group 9, Sulodexide intervention group 3 (WY3:after CsA 25mg·kg-1·d-1×7d, CsA 25mg·kg-1·d-1+Sulodexide 10mg·kg-1·d-1×21d). Based on experimental design, CsA and Rep were taken by intragastric administration, Sulodexide was given by intraperitoneal injection.All rats were killed on day 28th. During the experimental period, rats in each group were raised freely. Rat's body weight was measured at the last day in every week before and after medication, intragastric administration and abdominal injection dosage would be adjusted. At the same time, venous blood from rat tail was collected to measure serum creatinine(Scr) and blood urea nitrogen(BUN).
2. Harvested kidney tissues were fixed in periodate-lysine-paraformaldehyde solution and embedded in wax.After dewaxing,2-μm sections were processed and stained with HE,PAS and Masson. Pathological changes,hyaline degeneration of small arteries and renal interstitial fibrosis(RIF) were observed. A semiquantitative score was used to assess the extend of changes in each category.Hyalinosis of small arteries was determined by counting afferent and the juxtaglomerular arterioles available for examination with a minimum of 50 glomeruli per biopsy assessed:0=no hyalinosis; 1 =early hyaline changes; 2=pronounced hyalinosis.RIF was estimated by counting the percentage of injured areas per field and scored as follows:0=normal interstitium,0.5 =<5% of interstitium areas injured,1-5% to 15%,1.5=16% to 25%,2=26% to 35%, 2.5=36% to 45%, and 3=>45%.
3. The ultrastructural changes of kidney tissues were also observed by electron microscope.
4. Kidney was isolated from rat on ice and the following indicators of the kidney tissues were examined.
4.1 Immunohistochemical detection:CD68, collagenⅢand collagenⅣexpression;
4.2 Reverse transcription-polymerase chain reaction (RT-PCR) assay:TGF-β1 and TIMP-1 mRNA expression;
4.3 ELISA detection:TGF-β1, TIMP-1 and MMP-9 protein expression.
Results:
1.Body weight, BUN, Scr
1.1 Body weight:1 week after CsA administration, the rat in MX3 group growed delay, the weight decreased after 2 weeks vs VH3; 2-4 weeks,the rat weight of YDY and all intervention groups was higher than that of MX3 group (P<0.05), DY3, WY3, YDY group lower than VH3 group(P<0.05), other group was similar to VH3 group (P>0.05).
1.2 BUN:1 week after CsA administration, the BUN level of MX3 group began to increase,after 2 weeks,it was higher (P<0.05) than VH3 group; two weeks later the BUN level of DY1, WY1, WY2 group didn't increase (P>0.05), lower than MX3 group(P<0.05), similar to VH3 group(P> 0.05).1 week after, the BUN level of YDY, DY3, WY3 increased apparently,after 3 weeks it began to decline, but higher than VH3 lower than MX3 group(P<0.05);however,the rat BUN level of DY2 group was lower than the above-mentioned three groups (P<0.05),it was similar among the three groups (P> 0.05).
1.3 Scr:After 1 week, the rat Scr level of MX3 group increased (P<0.05) vs VH3. At 2-4 weeks that of YDY and all intervention groups was lower than MX3 group.Compared with VH3 group, DY1 group decreased (P<0.05), DY2, WY1 group no changes (P>0.05), YDY and other intervention groups increased (P<0.05). The Scr level of WY2 was lower than YDY, DY3, WY3 group(P<0.05).The Scr level of DY3, WY3 at 4 weeks decreased (P<0.05) and lower than YDY group (P<0.05).
2. Light microscopy:In normal control group, light microscopy showed normal structure of kidney tissues. In MX3 group, capillary loop opened badly in kidney glomerulus, cellular and intercellular vacuolization,tubular collapse and dilation.Striped interstistial fibrosis was present with thickening of the tubular basement membranes and Bowman's capsule.Muscular thickening of intrarenal arteriolar vessel wall,arterilar hyalinosis and injury appeared, and focal glomerulosclerosis was found occasionally.The above-mentioned pathological changes of YDY and intervention groups significantly reduced. The hyaline degeneration and RIF of erery group were as follows.
2.1 Hyalinosis of small arteries score:The score was higher in YDY and intervention groups than VH3 group,lower than MX3 group (P<0.05), higher in DY1, DY2, DY3, WY3 group than YDY group (P<0.05). Rep vs Sulodexide:The score in Sulodexide groups in various time points was lower than Rep groups(P<0.05), and increased at the third time point as well as higher than at the previous two time points (P<0.05), no difference was found between the first two time points (P>0.05).There was no difference among the various time points in Rep groups(P>0.05).
2.2 RIF score:The score in YDY and intervention groups was higher than VH3 group (P<0.05), lower than MX3 group (P<0.05), in which the score of DY3, WY3 group was higher than YDY group (P<0.05), similar in the other intervention groups with YDY group (P>0.05). Rep vs Sulodexide:At the first time point it was lower in Sulodexide group than Rep group (P<0.05), no difference was found between the two medicine groups at the other two time points (P>0.05). In both medicine groups it was higher at the third time point than that at the front two time points (P<0.05), no difference was found between the front two time points (P>0.05).
3. Renal ultrastructure changes:The structure was normal in normal control groups.In rat kidney tissues of MX3 group, glomerular basement membrane thickened, mesangium matrix broadened and increased, podocyte foot process fused extensively and microvillus became sparsed; tubular basement membrane thickened irregularly and broke, epithelial cell appeared a lot of vacuoles, nucleus edge changed to be irregular,chromatin thickened and concentrated, pyknosis,tubular microvillus exfoliated, epithelial cell endolysosome and mitochondria increased significantly, collagen fiber increased in interstitium. The rat lesions of DY1,DY2,WY1,WY2 groups significantly lightened:podocyte foot processes fused occasionly; segmental mesangial matrix mild increased; tubular showed a little bubble, lysosome, mitochondria and the basement membrane intact.Pathological changes of YDY,DY3,WY3 group deteriorated but improved much compared with MX3 group.
4. Kidney immunohistochemistry:CD68, collagenⅢand collagenⅣexpression levels in every group showed a similar trend with the inflammatory cell infiltration in HE staining. After 4 weeks, those were higher in model group than in control and intervention groups.
4.1 CD68 expression:The expression level was similar in DY1, DY2, WY1, WY2 with VH3 group(P>0.05), lower in YDY, DY3, WY3 than VH3 group (P<0.05).The level was lower in YDY and all intervention groups than MX3 group (P<0.05); it was similar in YDY with DY3, WY3 group (P>0.05), but higher than that in the other intervention groups (P<0.05). Rep vs Sulodexide:At the third time point in Rep group it was lower than Sulodexide group (P<0.05), there was no difference between the two medicine groups at the other two time points (P>0.05). Both medicines showed higher level at the third time point than the front two time points (P<0.05), no difference between the front two time points (P>0.05).
4.2 CollagenⅢexpression:Compared with VH3 group, collagenⅢexpression level was similar in DY2, WY2 groups (P>0.05), higher in YDY and the other intervention groups (P<0.05). Compared with MX3 group,it was lower in YDY and all intervention groups (P<0.05). While compared with YDY group, the level in WY1 group had no change (P>0.05), DY3, WY3 group increased (P<0.05), other intervention groups decreased (P<0.05). Rep vs Sulodexide:At the third time point it was lower in Sulodexide than Rep group(P<0.05), there was no difference between the two medicine groups at the other two time points (P>0.05). Both medicines showed it was higher at the third time point than the front two time points (P<0.05), and higher at the first time point than the second time point (P<0.05).
4.3 CollagenⅣexpression:Vs VH3 group, collagenⅣexpression level in WY1 group had no change(P>0.05), the other intervention groups increased (P<0.05). Compared with MX3 group, the level of YDY and all intervention groups decreased(P <0.05); compared with YDY group,the expression level in WY1 group decreased (P <0.05), that in DY3, WY3 group increased(P<0.05), other intervention groups had no difference (P>0.05). Rep vs Sulodexide:The level was lower in Sulodexide group at the first and third time point than Rep group (P<0.05), there was no difference between the two medicine groups at the second time point (P>0.05). Both medicines showed higher at the third time point than the front two time points (P<0.05), in Sulodexide group the level was higher at the second time point than the first one (P <0.05), and in Rep group there was no difference between the front two time points (P >0.05).
5. TGF-β1 and TIMP-1mRNA expression
5.1 TGF-β1mRNA expression:In VH3 group, there was only a small amount of TGF-β1mRNA expression, it increased significantly in model,YDY and other intervention groups except WY1 group (P<0.05), the expression of rats in YDY and all intervention groups was lower than MX3 group (P<0.05), that was lower in Rep and Sulodexide groups than YDY group (P<0.05). Rep vs Sulodexide:Compared with Rep groups, it decreased in Sulodexide group (P<0.05), and the expression level in Rep groups was 3>2>1(P<0.05).There was no difference between the first and second time point(P>0.05) in Sulodexide groups,but they were all lower than at the third time point (P<0.05).
5.2 TIMP-1 mRNA expression:In VH3 group, there was only a small amount of expression, it increased in MX3,YDY and intervention groups(P<0.05), the expression of rats in YDY and all intervention groups was lower than in MX3 group (P<0.05), lower in Rep and Sulodexide groups than in YDY group(P<0.05). Rep vs Sulodexide: The expression level was lower in Sulodexide groups at any time point than in Rep groups(P<0.05), while both medicine groups showed gradually increase with the time extending (P<0.05).
6. TGF-β1 TIMP-1 and MMP-9 protein expression
6.1 TGF-β1 protein expression:Compared with VH3 group,the expression was similar in WY1, WY2 group (P>0.05), decreased in DY1, DY2, WY3 group (P<0.05), increased in DY3, YDY group (P<0.05). The level in each other group was higher than that in MX3 group(P<0.05), lower than VH3 group(P<0.05). Rep vs Sulodexide: There was significant difference among all groups (P<0.05), at the front two time points the expression level in Rep was lower than that in Sulodexide group, and at the third time point, just the opposite.It was higher at the third time point than the other two time points(P<0.05) in Rep groups,no difference between the two ones (P>0.05); and the expression level in Sulodexide groups was similar at the first and second time point(P>0.05), but both higher than the third one (P<0.05).
6.2 TIMP-1 protein expression:Compared with VH3 group,the expression level was similar in DY3, WY1, WY2 group (P>0.05); decreased in DY1,DY2, WY3 group (P<0.05); increased in YDY group (P<0.05).It was lower in all the other groups except DY3 group (P=0.05) compared with MX3 group (P<0.05). The TIMP-1 protein expression level in each other group was lower than YDY group (P<0.05). Rep vs Sulodexide:At the front two time points the expression in Rep decreased compared with that in Sulodexide group(P<0.05), but at the third time point,it decreased in Sulodexide compared with that in Rep group(P<0.05).It was higher at the third time point than the other two time points in sulodexide groups(P<0.05); however, lower at the third time point than the other two time points in Rep groups(P<0.05).There was no difference between the front two ones (P>0.05) both in Rep and Sulodexide groups.
6.3 MMP-9 protein expression:Compared with VH3 group, no change was found in MX3, WY1 group (P>0.05), the expression increased in YDY group and other intervention groups (P<0.05), among those groups the expression of MMP-9 protein in YDY increased the highest (P<0.05).Rep vs Sulodexide:At the first and third time point the expression level in Sulodexide group was lower than in Rep group (P<0.05), but at the second time point,it was lower in Rep than in Sulodexide group.There was no difference among the three time points (P>0.05) in Rep groups,but the level in Sulodexide groups was 2>3>1(P<0.05).
Conclusion:
Cyclosporine nephrotoxicity could be significantly reduced,even some toxicity can be eliminated by Sulodexide or Rep intervention. Early intervention,better result would be obtained.The effect of Rep and Sulodexide was better than benazepril in decreasing CsA nephrotoxicity.The comparison between Sulodexide and Rep groups found that administration at the first time point,the effect of Sulodexide was better than that of Rep;however,the effect of two medicines administration at the other two time points matched.
Part 2 The therapeutic use of Rep and Sulodexide in CCN rats
Objective:To establish CCN model and investigate whether nephrotoxicity could be eliminated by itself 2 weeks after CsA withdrawal; To observe whether Rep or Sulodexide has the therapeutic effect on CCN simultaneously and to evaluate the curative effect.
Methods:
1. Experimental grouping:78 SPF healthy male Wistar rats (180-200g) were divided into 9 groups randomly after 1 week's adaptive feeding with low salt diet and free drinking:Group 1, normal control group (VH1:n=6, olive oil 5ml·kg-1·d-1×14d and sacrificed at 14 d; VH2:n=6, olive oil 5ml·kg-1·d-1×14d, later water for injection 5ml·kg-1·d-1×14d and sacrificed at 28 d; VH3:n=6, olive oil 5ml·kg-1·d-1×28 d and sacrificed at 28 d); Group 2, model group (MX1:n=6, CsA 25mg·kg-1×14d and sacrificed at 14 d; MX2:n=6, CsA 25mg·kg-1·d-1×14d, later water for injection 5ml·kg-1·d-1×14 d and sacrificed at 28 d; MX3:n=6, CsA 25mg·kg-1·d-1×28 d and sacrificed at 28 d); Group 3, positive control group (YDZ CsA 25mg·kg-1·d-1×14d, later benazepril 5mg·kg-1·d-1×14d); Group 4:Rep therapy group 1(DZ1:CsA 25mg·kg-1·d-1×14d, later Rep 25mg·kg-1·d-1×14d); Group 5, Rep therapy group 2 (DZ2:CsA 25mg·kg-1·d-1×14d, later Rep 50mg·kg-1·d-1×14 d); Group 6, Rep therapy group 3(DZ3:CsA 25mg·kg-1·d-1×14d, later Rep 100mg·kg-1·d-1×14d); Group 7, Sulodexide therapy group 1 (WZ1:CsA 25mg·kg-1·d-1×14d, later Sulodexide 10mg·kg-1·d-1×14d); Group 8, Sulodexide therapy group 2 (WZ2:CsA 25mg·kg-1·d-1×14d, later Sulodexide 20 mg·kg-1·d-1×14 d); Group 9, Sulodexide therapy group 3 (WZ3:CsA 25mg·kg-1·d-1×14d, later Sulodexide 40 mg·kg-1·d-1×14d). Based on experimental design, CsA and Rep were taken by intragastric administration, Sulodexide was given by intraperitoneal injection. The rats in group 3-8 were all killed at 28d.Group 9 failed to finish the experiment because of two rats were dead.
2. Rats observation and all indexes checking methods were as same as Part 1.
Results:
1.Body weight,BUN and Scr
1.1 Body weight:Except group 9, rats in the other groups survived and were included in the last statistics. At the first day, there were no significant differences of rat's body weights among various groups(P>0.05); At 14d, rats'body weights among MX groups,YDZ and therapy groups had no difference(P>0.05), while body weights in above-mentioned groups decreased compared to those in VH group(P<0.05). At 21d, rats'body weights continued to decline in MX3 group, but increased in other groups(except VH1, MX1 group because rats had been sacrificed). To 28d, all rats' body weights were improved. Compared with the results at 21d, body weights in MX3 and WZ2 groups had no significant differences, but those in other groups improved(P <0.05). At 28d, body weights in VH2 and VH3 group increased to the highest level. The difference among MX3 and the other groups was significant (P<0.05). Body weights in all therapy groups had no significance compared with those in YDZ and MX2 groups(P>0.05).
1.2 BUN,Scr:At the first day, there were no differences of BUN, Scr among all groups(P>0.05); At 7 d and 14 d, level of BUN, Scr in all MX groups,YDZ and therapy groups increased compared to those of normal control group(P<0.05); At 28 d, level of BUN, Scr in the experimental groups except VH1 and MX1 group(rats had been sacrificed) decreased compared with those in MX3 group(P<0.05); Compared to MX2 group,BUN results in DZ1, DZ2 groups and Scr results in DZ1, DZ2,WZ2 groups increased, while other therapy and YDZ group had no changes(P>0.05). Compared to YDZ group, BUN level in DZ1 and DZ2 groups and Scr level in DZ1, DZ2 and WZ2 groups increased (P<0.05), while other therapy groups had no changes(P>0.05).
2. Light microscopy results:In normal control group, light microscopy showed normal structure of kidney tissues. The pathology changes of MX3 group were as same as those in Part1. In MX1 group, the results showed the capillary loop opened badly, monocyte infiltration in interstitial tissue, interspersed striped interstitial fibrosis, partial afferent arterioles hyalinization. However, the above-mentioned pathological changes in MX2 and YDZ group improved significantly, and nearly restored to normal appearance in therapy groups. Hyalinosis and RIF in each group were as follows:
2.1 Hyalinosis of small arteries score:No obvious hyalinosis was found in therapy and YDZ groups compared to normal control and MX2 groups (P>0.05), while the hyalinosis was relieved compared to MX1, MX3 groups(P<0.05); the results in therapy groups had no differences with the generation in YDZ group(P>0.05).
2.2 RIF score:Slight RIF could be seen in normal control group; model groups(MX1, MX2 and MX3) showed significantly aggravation which most serious RIF in MX3, slightest in MX2 and moderate in MX1 group(P<0.05).The serious degree of RIF was similar in YDZ and MX2 groups(P>0.05). RIF in therapy groups was less serious than YDZ and MX2 group(P<0.05) while more serious than normal control groups(VH1, VH2 and VH3) (P<0.05). Rep vs Sulodexide:RIF in WZ1 group was slightest, and RIF in DZ1, DZ3 and WZ2 was most serious and RIF degree in DZ2 was moderate (P<0.05) in which there were no statistical differences among DZ1, DZ3 and WZ2 groups(P>0.05).
3. Ultrastructure changes in kidney tissues:The structure was normal in normal control groups.The changes of MX3 group were as same as those in Part1.The changes in MX1 group showed slighter, podocyte foot process fused partially, mesangium matrix increased slightly, vacuoles, lysosome and mitochondria could be found in tubule, thickness of tubular basement membrane was discrepant, segment broke, nuclear chromatin concentrated and interstitial collagen fiber appeared occasionally. Pathological changes in MX2 group improved obviously compared to the results in MX1 and MX3 groups, and the pathology was nearly normal in YDZ and therapy groups.
4.Immunohistochemistry results in kidney tissues
4.1 CD68 expression:The level of CD68 expression from low to high was VH1, VH2,VH3
4.2 CollagenⅢexpression:CollagenⅢexpression level was lowest in normal control group (VH1, VH2 and VH3 group), DZ1, DZ2 and DZ3 group(P>0.05). The results in model groups increased (MX3>MX2>MX1,P<0.05).Changes in YDZ, WZ1 and WZ2 groups were similar to those of MX2 group(P>0.05), while higher than MX1 group(P<0.05) and lower than MX3 group(P<0.05). Rep vs Sulodexide:Level in Rep therapy groups was lower than Sulodexide therapy groups(P<0.05), while no difference was found in different dosage groups using the same therapeutic drug.
4.3 CollagenⅣexpression:There were no statistical differences among collagenⅣexpression level in normal control group(VH1, VH2 and VH3 group), MX2, DZ2 and WZ1 group(P>0.05); Level in MX1, MX3, YDZ, DZ1, DZ3 and WZ2 increased (P<0.05) which was highest in MX3 group(P<0.05),higher in YDZ group(P<0.05) and the lowest in the other four groups(P<0.05, level in the four groups was similar, P >0.05). Rep vs Sulodexide:Results in DZ2, WZ1 were comparable low(level in the two groups was similar, P>0.05), and subsequently increased in DZ3, WZ2 and DZ1 group in turn (P<0.05, level of the above three groups was similar, P>0.05).
5. TGF-β1 and TIMP-1mRNA expression
5.1 TGF-β1mRNA expression:TGF-β1mRNA expression level was lowest in DZ2 group, and subsequently increased in VH1, VH2, VH3, DZ1, DZ3 and WZ1 group in turn, and the level was even higher in WZ2, YDZ, MX2, MX1 group, and level of MX3 group was highest(P<0.05). Rep vs Sulodexide:Results in DZ2, WZ1 and DZ1 group were lower(level in the three groups was similar, P>0.05), level in DZ3 and WZ2 groups (level in the two groups was similar, P>0.05) increased compared to the above-mentioned three groups(P<0.05).
5.2 TIMP-1mRNA expression:TIMP-1mRNA expression level was lowest in WZ1 group, lower in WZ2 group, and subsequently in VH1, VH2, VH3 and DZ1 group, then increased in MX2, DZ2, YDZ, DZ3, MX1 group in turn, level in MX3 group was highest(P<0.05). Rep vs Sulodexide:The group sequence according to TIMP-1mRNA expression level from low to high was WZ1, WZ2, DZ1, DZ2 and DZ3(P<0.05), and level in Sulodexide therapy groups was all lower than that in Rep therapy groups(P<0.05).
6. TGF-β1,TIMP-1 and MMP-9 protein Expression
6.1 TGF-β1 protein expression:TGF-β1 protein expression restored to normal level in MX2 after CsA withdrawal and increased in YDZ and all drug therapy groups(P<0.05), results in WZ1, DZ2 and DZ1 group were higher than those of YDZ group(P<0.05), while level in WZ2 and DZ3 was similar to that of YDZ group(P >0.05). Rep vs Sulodexide:No difference was found among DZ3, WZ1 and WZ2 groups(P>0.05), the differences among the other groups had statistical significance(P <0.05) which level in WZ2 and DZ3 groups lowest, and subsequently WZ1, DZ2 and DZ1 in turn.
6.2 TIMP-1 protein expression:TIMP-1 protein expression restored to normal level in MX2 after CsA withdrawal, and increased in drug therapy groups (P<0.05) except benazepril group. Rep vs Sulodexide:Level in DZ3, WZ1 and WZ2 was lowest(three groups'results were similar, P>0.05), lower in DZ2 group and highest in DZ1 group(P<0.05).
6.3 MMP-9 protein expression:MMP-9 protein expression was highest in WZ2 group, lower in MX1, DZ1, DZ2 and WZ1 group (level in these groups was similar, P >0.05), lowest in VH3, MX2, MX3, DZ3, VH1, VH2 and YDZ(P<0.05) which level in these groups was similar(P>0.05). MMP-9 protein expression restored to normal level in MX2 after CsA withdrawal(compared to normal control group, P>0.05). Results in therapeutic groups except DZ3 and YDZ group increased compared to normal control group and MX2, MX3, MX1 group(P<0.05). Rep vs Sulodexide:Level in WZ2 group was highest, that in DZ1, DZ2 and WZ1 was comparable higher while level in DZ3 group was lowest. There was difference between DZ3 and DZ1, DZ2, WZ2 groups (P<0.05),while the differences among level in other groups had no statistical significance(P>0.05).
Conclusions:
After CsA withdrawal,common indexes such as body weight,BUN,Scr and small artery hyalinosis of rat kidney tissues could be restored, RIF could be improved slightly,but the administration of Sulodexide or Rep could improved RIF obviously.When 10 mg·kg-1·d-1 Sulodexide or 50mg·kg-1·d-1 Rep was administered,the best result appeared.The two medcines'effect matched each other.
引文
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