灯盏花素对腹膜透析效能及腹膜间皮细胞TLR4信号传导影响的实验研究
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摘要
慢性肾脏病在全球范围内的发病率呈逐年上升的趋势,继心脑血管疾病、肿瘤、糖尿病之后又一威胁人类健康的重要疾病。在发达国家,普通人群的发病率6.5%～10%,而我国初步流行性调查显示,40岁以上人群的患病率为8%～9%,其中大多数患者发展至尿毒症。腹膜透析作为治疗慢性肾功能衰竭的主要手段之一,由于其在治疗时对肾小球滤过影响小,心血管功能稳定性好,操作简便,价格相对便宜,以及病患回归社会的机会高等特点,越来越受到重视。全世界大约有10%～15%终末期肾脏病(ESRD)患者接受腹膜透析技术治疗,人数超过15万,不同国家使用率差异较大,香港70%ESRD患者使用腹膜透析,而日本仅仅4%。我国从上世纪60年代开展腹膜透析治疗,随着我国人民生活水平提高,对于健康的要求越来越高,且腹膜透析技术日臻完善,采取腹膜透析技术的ESRD患者迅速增加。尽管腹膜透析技术发展很快,由于导管功能障碍和生物相容性问题、营养失衡、腹膜炎、腹透液生物相容性以及社会心理因素,影响其发展。其中随着透析时间的延长,腹膜纤维化,腹膜超滤功能和转运功能下降,甚至衰竭即腹膜透析超滤失败(ultrafiltration failure UFF),成为腹膜透析终止的主要因素。腹膜透析进行18个月的病人,UFF的发生率是14%,而腹膜透析治疗8年以上因UFF退出的占51%。故研究UFF的发生机制,寻求有效的防治措施和治疗药物,是一个具有重要意义的课题。
腹膜透析腹膜纤维化的发病机制十分复杂,多数研究者认为腹膜纤维化的发生与透析液的性质、腹膜炎、细胞因子等因素有关。腹透液的低PH值、高糖、乳酸盐和葡萄糖降解产物(GDP)等生物不相容因素,直接导致间皮细胞损伤,同时对于腹腔内白细胞产生明显抑制作用,降低防御能力;糖基化终产物可导致新生血管增生,促进纤维化。腹膜透析还存在许多易感途径和易感因素。腹膜炎一旦发生,引起一系列炎症反应,在补体参与下,趋化因子释放,粒细胞聚集,炎性介质释放,引起血管扩张,间皮细胞损伤,细胞外基质沉积,纤维化发生。近几年研究表明细胞因子在UFF发病中的作用不容忽视,涉及到腹膜回吸收增加、慢性腹腔炎症、糖基化终产物和氧自由基损伤等诸多方面。
菊科植物灯盏花[erigeron breviscapus(vaniot) Hand Mazz]又名灯盏细辛,有效成分为灯盏花素(erigeron)、黄酮类等,是黄苓素的4位接上一个羟基,从而对蛋白激酶C(protein kinase,PKC)有很好的抑制作用。PKC在细胞分泌、释放、溶解、膜运动、受体结合、平滑肌收缩、细胞增殖分化和癌变过程中均起重要作用。大量试验证实灯盏花素能够对抗缺血再灌注损伤,抑制氧化应激与脂质过氧化,防止纤维化的发生。还能够防止糖尿病肾病细胞外基质的积聚。对于糖尿病肾组织中单核细胞趋化蛋白-1(MCP-1)和细胞间粘附分子-1(ICAM-1)对巨噬细胞较强的趋化与激活作用以及细胞与细胞外基质之间的粘附作用均有抑制作用。总之,灯盏花素具有防止组织纤维化,减轻细胞外基质的积聚以及炎症反应的作用,就此推测灯盏花素对UFF有一定的改善作用。
UFF是腹膜透析严重并发症,是终止腹膜透析治疗的原因之一,但发病机理和防治尚有待进一步研究。TLRs是在人和小鼠细胞上发现的一种介导天然免疫的跨膜信号传递受体家族,迄今至少发现有11种人类跨膜蛋白的基因。Toll受体介导的免疫和炎症信号传导通路依赖于TIR结构域向细胞转导其识别信号,诱导很多快速反应基因的活化,产生诸如炎症细胞因子、主要组织相容性复合体(major histocompatibility complex,MHC)、共刺激分子、趋化因子等,参与机体的防御反应。TLR4在组织器官损伤和炎症的作用均有报道,但在腹膜透析失超滤的作用尚未见报道,因此研究腹膜透析时TLR4信号传导通路的变化,无疑有助于揭示UFF发病的分子机制。本项目通过建立腹膜透析尿毒症大鼠模型,观察腹膜透析大鼠透析效能及间皮细胞细胞TLR4、MCP-1、MIP-2的表达,探讨TLRs信号传导通路在腹膜透析UFF发生发展中的作用,开辟腹膜透析研究的新途径。同时研究灯盏花素对上述途径的影响,探讨灯盏花素对于腹膜间皮细胞保护,腹膜透析效能的提高,避免腹膜透析UFF发生的分子机制。
第一部分自制腹透管尿毒症大鼠腹膜透析模型的建立
目的:建立腺嘌呤尿毒症大鼠模型,在此基础上研究自制腹透管和手术方式建立尿毒症大鼠腹膜透析模型。
方法:SD大鼠用含有7500mg·kg-1腺嘌呤的颗粒饲料喂养,投放量为300mg/(kg·d),连续喂养7w,检测血清尿素氮、肌酐和肾脏病理确定尿毒症鼠模型的建立是否成功。自制腹透管采用硅胶吸痰管,外口使用一次性无菌输液接头连接;传统腹透管将头皮针改制。常规手术将腹透管置入大鼠腹腔。将上述术后大鼠分成3组,每组10只。自制腹透管1组腹透管由后背颈部皮肤穿出,自制腹透管2组腹透管由近尾背部皮肤穿出,传统头皮针组(出口同自制1组)。随后对30只大鼠用4.25%腹透液进行连续腹膜透析(CAPD),每日换液4次,每次25ml。观察管道的完整性,隧道口有无感染,管道是否包裹,腹膜炎发生情况,2周后透析排出量的变化等。
统计学方法:统计学处理:结果以(?)±s表示,采用SPSS13.0软件进行统计学分析,组内比较用配对t检验,组间比较用重复测量方差分析。样本率的比较采用χ~2检验。
结果:
1、腹透管完整性比较:自制1组、传统头皮针组大鼠腹透管完好,无管道破损和渗漏;自制2组的大鼠先后在15d内管道完全咬破损坏,无法使用。
2、出口处和隧道口感染情况:自制1组3例,自制2组2例,传统头皮针组6例发生隧道口感染。自制1组、自制2组与头皮针组存在统计学差异(P＜0.01),自制1组与自制2组则无统计学差异。
3、腹膜炎发生情况:自制1组1例,自制2组7例,传统头皮针组3例发生腹膜炎。自制1组与自制2组、传统头皮针组有统计学差异(P＜0.01,P＜0.05)。
4、腹透液排出情况:自制1组第1天与第15天相比腹膜透析出液量无统计学差异(t=2.207,P=0.055),头皮针组则有统计学意义(t=6.046,P=0.000),第1天自制1组、自制2组、头皮针组间相比无统计学差异(t=1.187,P=0.251),第15天自制1组与头皮针组相比存在统计学差异(t=2.724,P=0.014),自制2组与自制1组、头皮针组存在显著统计学差异(P=0.029)。
5、15天后自制1组腹膜透析管腹腔端被大网膜完全包裹1例、部分包裹5例,传统头皮针组完全包裹4例、部分包裹6例。
结论:
1、采用自制硅胶吸痰管作为腹透管,外口使用一次性无菌输液接头连接,腹透管出口由后背颈部皮肤穿出的手术方式构建的尿毒症大鼠腹膜透析模型,具有成功率高的特点。
2、正确的手术方式,是模型建立的基本保证,而透析管道的选材、接头稳固耐用是腹膜透析模型能否长期使用的关键。
第二部分体内实验
实验一、灯盏花素对尿毒症大鼠腹膜透析效能以及腹膜结构的影响
目的:观察灯盏花素对尿毒症大鼠腹膜透析效能以及腹膜结构的影响。
方法:将采用含腺嘌呤喂养制作尿毒症模型,随后采用自制腹透管行腹膜透析置管术制作腹膜透析模型,使用LPS加入腹腔建立尿毒症大鼠腹膜透析UFF模型。使用4.25%腹膜透析液分别加入不同浓度灯盏花素和LPS进行腹膜透析,将50只尿毒症腹膜透析模型SD大鼠平均分为5组:高浓度灯盏花素组(高浓度组)、中浓度灯盏花素组(中浓度组)、低浓度灯盏花素组(低浓度组)、对照1组(使用LPS组)、对照2组(单纯腹透液组,未加LPS),45天后行腹膜平衡试验,腹膜组织病理学检查,了解腹膜透析效能和结构的变化。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,两组间比较采用配对t检验、多组间比较采用重复测量方差分析。
结果:
1.腹膜效能及腹膜超滤量的影响:尿毒症大鼠在行腹膜透析前血肌酐无显著差异(F=0.96,P=0.345)。行腹膜透析后各加药组间D/P_(肌酐)(F=55.055,P=0.000)和超滤量(F=6.897,P=0.000)差异各不相同。对照2组的D/P_(肌酐)与高浓度组、中浓度组、低浓度组、对照1组间比较显著升高(t=-7.728,-8.696,-12.322,-9.899;P=0.000),超滤量与高浓度组、中浓度组比较无差异(t=-1.152,-1.754;P=0.273,0.100),与低浓度组、对照1组比较显著升高(t=-3.691,-3.73;P=0.003,0.005);低浓度组与对照1组间D/P_(肌酐)、超滤量比较无显著差异(P=0.361);高浓度组与中浓度组间D/P_(肌酐)、超滤量比较无显著差异(P=0.860);高浓度组、中浓度组的D/P_(肌酐)、超滤量均较低浓度组、对照1组显著升高(P＜0.001,P＜0.05)。高浓度组、中浓度组但均较对照2组显著下降(P＜0.001),但均较对照1组高(P＜0.001)。
2.大鼠腹膜组织病理变化:肉眼及光镜下观察:对照2组大鼠腹膜壁层、脏层表面光滑,无结节、渗出、充血。对照1组和低浓度组可见腹膜散在皱折,部分出现增厚,有渗出和充血现象。高浓度组、中浓度组腹膜增厚不明显,散在存在充血现象。光镜下见对照2组间皮细胞完整,细胞间连续性良好,间皮下基质无明显增多;对照1组间皮细胞肿胀、变形,部分皱缩,无完整的连续性,间皮下基质大量积聚,伴有大量炎性细胞的浸润和毛细血管增生;低浓度组间皮细胞存在肿胀、变形,细胞间连续性差,较多的间皮细胞下基质增多,可见炎性细胞浸润和毛细血管的增生;中浓度组可见间皮细胞肿胀,细胞数量减少,细胞间连续性良好,间皮下基质少量积聚,少见炎性细胞浸润,可见毛细血管增生;高浓度组间皮细胞完整,细胞连续性良好,细胞下少量基质沉积,少量炎性细胞浸润,毛细血管增生不明显。
结论:灯盏花素具有防止腹膜间皮细胞损伤,维持腹膜结构的完整性,改善局部微循环,提高腹膜的超滤效能等作用。以高、中浓度灯盏花素组为好。
实验二、灯盏花素对大鼠腹膜间皮细胞TLR4基因表达及信号通路的影响
目的:探讨TLR4及其信号通路在腹膜失超滤中所起的作用,并且观察灯盏花素对其干预作用。
方法:取实验一各组大鼠腹膜,免疫组化方法检测腹膜组织TLR4蛋白表达;采用逆转录聚合酶链反应(RT-PCR)检测TLR4 mRNA的表达情况:ELISA方法检测腹透液中MCP-1、MIP-2的浓度变化。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,两组间比较采用配对t检验、多组间比较采用重复测量方差分析;方差不齐者首先进行数据变换,若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验。检验水平a=0.05。
结果:
1.腹膜组织TLR4mRNA的表达:加有LPS的对照1组与未加LPS的对照2组腹膜组织TLRmRNA表达存在统计学差异(P=0.000)。在加有LPS的4组中腹膜TLR4mRNA相对表达量均存在显著性差异(χ~2=21.99,v=3,P=0.000)。对照1组腹膜TLR4mRNA表达量平均秩次最高;在灯盏花素干预组比较,高浓度组、中浓度组表达量平均秩次分别为8.71、7.38,低浓度组出现较高水平的表达,平均秩次为22.25,与对照1组相当。
2.组织细胞TLR4蛋白的免疫组织化学的表达:在未加LPS的对照2组,少数腹膜间皮细胞上出现TLR4蛋白表达;在加有LPS的高浓度组、中浓度组、低浓度组和对照1组中,高浓度组、中浓度组中多数的间皮细胞TLR4蛋白表达呈阳性,低浓度组、对照1组中基本上所有间皮细胞呈阳性表达(P=0.000)。
3.腹透液MCP-1、MIP-2浓度的变化(ELISA):腹透液MCP-1浓度在高浓度组、中浓度组、对照2组间无显著性差异(P=0.117,0.805,0.057);对照1组分别与高浓度组、中浓度组、低浓度组、对照2组相比存在显著性差异(P＜0.05);低浓度组腹透液MCP-1浓度高于高浓度组、中浓度组、对照2组,它们之间存在统计学意义(P＜0.05)。腹透液MIP-2浓度对照1组高于其他各组(P＜0.001);对照2组低于高浓度组、中浓度组、低浓度组(P=0.001,0.002,0.000);高浓度组与中浓度组相比、中浓度组与低浓度组相比无统计学意义(P=0.115,0.226),而高浓度组与低浓度组之间存在统计学差异(P=0.004)。
结论:
1.腹膜透析腹膜组织TLR4的表达量与腹透液中MCP-1、MIP-2浓度呈正相关,并与腹膜间皮细胞受损程度正相关。
2.灯盏花素对于TLR4的表达,以及由于TLR4激活所产生的MCP-1、MIP-2炎症因子具有下调效应。
第三部分体外实验
实验一、灯盏花素对腹膜间皮细胞生长和增殖的影响
目的:本研究旨在通过观察体外培养时灯盏花素对大鼠腹膜间皮细胞生长和增殖的影响,探讨间皮细胞受损的机制。
方法:对SD大鼠腹膜间皮细胞进行分离培养,间皮细胞分成8组,分别加入灯盏花素和LPS,包括:单纯培养基组(1组)、50ul灯盏花素组(2组)、25ul灯盏花素组(3组)、12.5ul灯盏花素组(4组)、50ul灯盏花素+10ul LPS组(5组)、25ul灯盏花素+10ul LPS组(6组)、12.5ul灯盏花素+10ul LPS组(7组)、10ul LPS组(8组)。培养48小时和72小时后通过细胞计数了解间皮细胞生长情况;培养72小时后加入MTT5mg/孔,用酶标仪490nm比色,了解间皮细胞增殖情况。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,单因素组间比较采用One Way ANOVE、SNK检验,多组间比较采用重复检验方差分析。
结果:
1.细胞数量影响:间皮细胞培养48小时和72小时后进行计数组间存在显著性差异(F=182.738,P=0.000;F=677.589,P=0.000),各个加药组间也存在显著性差异(F=606.988,P=0.000)。在未加LPS组中,3组(中等剂量)和4组(低剂量灯盏花素组)间皮细胞生长良好,与1组(对照组)相比无显著性差异(P=0.086,1.000);2组(高浓度组)与1组相比具有显著性差异(P=0.004)。而加LPS组间皮细胞生长情况差于未加组,在LPS组中,加入5组(灯盏花素高浓度组)、6组(中浓度组)和7组(低浓度组)对LPS对间皮细胞生长抑制作用具有拮抗作用(P=0.000)。
2.细胞增殖的影响:加药组间具有统计学差异(F=1265.008,P=0.000)。未加LPS组中1、3、4组的间皮细胞增殖无显著差异(P=0.64,0.929),2组的间皮细胞增殖与3、4组比较无显著性差异(P=0.705),与1组比较存在显著性差异(P=0.039)。加有LPS组中5、6组间无统计学差异(P=0.771),其余各组间存在统计学差异(P=0.000),其中8组间皮细胞增殖最差,7组次之。
结论:
1.灯盏花素对于间皮细胞的生长和增殖具有保护作用。
2.灯盏花素能够减轻LPS引起的免疫炎症造成的间皮细胞的损伤,且存在量效正相关关系。
实验二、灯盏花素对腹膜间皮细胞TLR4表达及信号通路的影响
目的:观察体外腹膜间皮细胞TLR4以及信号通路的表达情况,研究灯盏花素对TLR4的信号通路以及相关信号因子影响的分子机制。
方法:体外进行间皮细胞培养,分组同体外实验一,给予灯盏花素和LPS进行干预,72小时后采用RT-PCR法对间皮细胞TLR4 mRNA表达进行检测;采用ELISA方法对培养上清液MCP-1、MIP-2浓度进行检测。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,多组间比较采用One Way ANOVE、SNK检验;方差不齐者首先进行数据变换,若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验。检验水平a=0.05。
结果:
1.间皮细胞培养上清中MCP-1,MIP-2的浓度变化:MCP-1、MIP-2的浓度在不同组间存在显著性差异(F=80.358,P=0.000;F=320.101,P=0.000)。未加LPS组间(1～4组)MCP-1、MIP-2浓度无统计学差异(P＞0.05),加有LPS的4组中,高浓度灯盏花素组(5组)、中浓度灯盏花素组(6组)与单纯LPS组(8组)间存在统计学差异(P＜0.01),而低浓度灯盏花素组(7组)与8组间无统计学差异(P＞0.05)。
2.间皮细胞TLR4 mRNA表达检测:在未加LPS的1～4组中间皮细胞TLR4mRNA的表达量相当,无统计学差异(x~2=2.544,v=3,P=0.467);在加有LPS的5～8组中间皮细胞TLRmRNA的表达量存在的差异有显著性意义(x~2=25.63,v=3,P=0.000),8组(未加灯盏花素组)和7组(低浓度灯盏花素组)表达量相对高,平均秩次分别27.28、27.50,而5组(高浓度灯盏花素组)和6组(中浓度灯盏花素组)表达量低,平均秩次分别为9.56、9.67,可以认为不同浓度灯盏花素对TLR4mRNA的表达量的影响不同。
结论:
1.MCP-1、MIP-2对于TLR4具有依赖性,证明了TLR4信号通路的存在的。
2.灯盏花素具有下调腹膜间皮细胞TLR4 mRNA及信号通路表达的作用。
全文结论:本研究通过在尿毒症大鼠腹膜透析模型基础上进一步建立尿毒症大鼠腹膜透析UFF模型以及间皮细胞培养的实验研究,证明TLR4及其信号传导在腹膜透析腹膜发生纤维化(即腹膜超滤失败)所发挥的作用。而灯盏花素具有提高腹膜透析效能和保护腹膜间皮细胞的作用,其作用机理与下调TLR4表达,降低MCP-1、MIP-2的含量有关,为其临床推广应用于治疗腹膜透析失超滤提供了较为全面、深入的分子水平的理论依据。
The incidence of chronic kidney diseases(CKD) is rising rapidly year by year around the world,which has become one of the most important diseases listed behind cardiovascular diseases,tumor and diabetic mellitus.In developed countries,the incidence of CKD of the common crowd is 6.5～10%.In our country,the incidence of the crowd aged over 40 years old is 8～9%,among whom most people will become uraemia according to an epidemiological investigation.Peritoneal dialysis is one of the remedies for chronic renal failure.Because it has many advantages such as less influenced glomerular filtration,more steady cardiovascular function,more handling convenience,cheaper price,and higher probability for patients to come back to the society,people have attached more and more importance to it.There are about 10 or 15 percent of ESRD patients(over 150 thousands people) using peritoneal dialysis around the world.The percentage of ESRD patients using peritoneal dialysis is different among different countries.In our country,peritoneal dialysis has been used since 1960s.With the improved living standard,the people's stronger desire for healthy,and the consummate technology,the increasing number of ESRD patients are using peritoneal dialysis.But there are still many problems such as catheter dysfunction,material histoimcompatibility,nutrition imbalance,peritonitis,and social psychic:factor slowing down the progression of peritoneal dialysis,among which peritoneum fibrosis,ultrafiltrate and convery dysfunction,even ultrafiltration failure (UFF) become the main causes to stop the remedy.A report showed that the incidence of UFF was 14%in patients who had peritoneal dialysis for 18 months and 51% patients withdrew from peritoneal dialysis after eight years owing to UFF.So it is urgent to give more emphasis on finding the pathogenesis of and developing effective remedies for UFF.
The pathogenesis of peritoneal dialysis associated peritoneal fibrosis is very complicated and remains poorly understood.Most researchers think peritoneal fibrosis is relevant to the property of dialysate,peritonitis,cytokine,and so on.The histoirncompatibility factors of dialysate including low PH,high glucose,lactate and glucose degradation products damage peritoneal mesothelial cells directly,inhibit leucocytes in the abdominal cavity,cut down immune defense capability,induce glycation end products,cause angiogenesis and promote fibrosis.Peritonitis induces a series of inflammatory reaction including alexin activation,chemotatic factors release, leukocytes accumulation and inflammatory mediator release,leading to blood vessel dilatation,mesothelial cells damage,extracellular matrix accumulation and fibrosis formation.In recent years,researchers found that cytokines played important roles in UFF.UFF was related to the rising of reverse absorption by peritoneum,chronic abdominal cavity inflammation,production of glycation end products and oxygen radicals and so on.
Erigeron,4-OH-scutellaria baicalensis,is the effective component of Erigeron breviscapus(Vaniot) Hand Mazz,also names fleabane.It can inhibit protein kinase C (PKC).PKC plays significant roles in cellular secretion,release and dissolve, membranous motion,receptor combination,smooth muscle constraction,cell proliferation and differentiation,and canceration.Data proved that Erigeron could block ischemia reperfusion injury,retard oxidative stress and lipid peroxidation and prevent fibrosis proliferation.Eeigeron could also prevent extracellular matrix accumulation in diabetic nephropathy,strongly inhibit monocyte chemotactic protein- 1(MCP- 1) and intercellular adhesion molecule- 1(ICAM- 1) expression and macrophage activation in renal tissues of diabetes.In a word,Erigeron can prevent tissue fibrosis,extracellular matrix accumulation and inflammation.
UFF is a very serious complication during peritoneal dialysis and one of the reasons that give rise to peritoneal dialysis failure.However,the pathogenesis and remedy need to be investegated.Toll-like receptors(TLRs),which have been found on cells of human beings or rats,belong to transmembrane signal transduction receptor family,and can mediate natural immune.Up to now,at least eleven kinds have been found in human beings.The TLRs transduce identification signals into the cells via a specific motif,mediate immune and inflammation signal transduction pathways,activate fast reaction genes,produce inflammatory chemokines such as major histocompatibility complex(MHC),costimulatory molecules,chemotactic factors,etc and participate organism defense reaction.It was reported that TLR4 participated in tissue and organ damage and inflammation,but there is no report about the effects of TLR4 on UFF until now.To study TLR4 and its signal transduction will be helpful to reveal molecular mechanisms of UFF.
In the above context,we investigatedthe effects of TLRs signal transduction on the development of UFF by building a peritoneal dialysis model of ureamia in rats and observing peritoneal dialysis efficacy and TLR4,MCP-1,Macrophage inflammatory protein-2(MIP-2) expression.Our study will be able to open up a new way to peritoneal dialysis investigation.Meanwhile,we researched how Erigeron affect TLR4 expression and signal transduction in order to reveal the molecular mechanisms of the protecting effects of Erigeron on peritoneal mesothelium,peritoneal dialysis efficacy and UFF.
ChapterⅠTo Build a Model of Peritoneal Dialysis in Uremia Rats by Self-made Peritoneal Dialysis Catheter
Objective:To develop an improved model of peritoneal dialysis in rats that mimic human peritoneal dialysis.
Methods:Firstly,we established the uremia rat models by feeding of amidopurine according to the dose of 300 mg·kg-1 body weight.In the following seven weeks,we detected the blood serum urea nitrogen,creatinine,patho-scopy, and determined the model built.Rats were divided into three groups:experimental group 1,experimental group 2 and control group.In experimental group 1,phlegm suction catheters were used as peritoneal dialysis catheters with tie-ins made from sterile transfuse connectors.The peritoneal dialysis catheters implanted into rats were tunneled subcutaneously to the nape.Rats with different operation mode or different operation material belonged to experimental group 2 or control group,respectively.
Statistical analysis:All values were expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results of two groups before and after treatment were analyzed using paired t-test for comparisons.Compare to rate of sample usingχ~2-test.
Results:
1.Peritoneal dialysis catheters in rats in experimental group 1 were intact, whereas catheters in rats in experimental group 2 were broken.
2.Three rats in experimental group 1 and six rats in control group had tunnel opening infections.
3.One rat in experimental group 1 and three rats in control group had peritonitis.
4.In experimental group 1,the amount of exchanged solution on the first day of peritoneal dialysis was similar to that on the fifteenth day(p＞0.05),but in control group,the amount of exchanged solution on the first day was different from that on the fifteenth day(p＜0.01).
5.There were no difference between experimental group 1 and control group at the amount of exchanged solution on the first day(p＞0.05),whereas there were significant difference on the fifteenth day(p＜0.05).
6.Peritoneal dialysis catheters were completely wrapped by omentums in one rat in experimental group 1 and in 4 rats in control group.Catheters were partly wrapped in five rats in experimental group 1 and in 6 rats in control group.
Conclusion:Correct operation modes,proper operation material and firm durable catheter tie-ins are the key for developing a successful model of peritoneal dialysis.
ChapterⅡExperiments in Vivo
PartⅠEffects of Erigeron on Dialysis Efficiency and Peritoneal UItrastrueture in Uraemia Rats
Objective:To study the effect of erigeron on dialysis efficiency and peritoneal ultrastructure in uraemia rats.
Methods:Firstly,feeding amidopurine to make model of uraemia rats,then building model of peritoneal dialysis by made-self dialysis catheter.The death rate of rats was 13.33%after model created.4.25%glucose dialysate,Erigeron,LPS were used in peritoneal dialysis.50 male SD model rats were divided into five groups:high concentrate group,middle concentrate group,low concentrate group,control one, control two.Peritoneal Equilibration Tests was observed,and the changes of peritoneal structure as well.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Vet.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.
Results:
1.D/P_(creatinine) of control two was significant higher than others(P＜0.001),its intraperitoneal volume had no significant difference than high concentrate group and middle concentrate group(P＞0.05),had significant higher than low concentrate group and control one(P＜0.001).Compare to low concentrate group and control one,high concentrate group and middle concentrate group,their D/P_(creatinine) and intraperitoneal volume had no significant different(P＞0.05).
2.The extent of which integrity of peritoneal mesothelial cell destroyed,base material below cells accumulated,inflammatory cells infiltrated and blood capillary hyperplasia,there are control group one,high concentrate group,middle concertrate, low concentrate group and control group two by turns.
Conclusions:Erigeron could preserve peritoneal mesothelial cell,and improve peritoneal dialysis efficiency.
PartⅡEffects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduetion Pathways in Rats Peritoneum
Objective:The aim of this study was to bring out the effect of TLR4 gene and signal pathways on peritoneal ultrafiltration failure,also the effect of Erigeron on TLR4 gene expression.
Methods:The peritoneal tissue used of this study were from the rats in partⅠ. Immunohistochemical analysis was used to detect TLR4 of peritoneal tissue expression.Polymerase chain reaction assay was employed to detect TLR4 mRNA expression.ELISA was used to investigate MCP-1 and MIP-2.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.
Results:
1.In all groups,TLR4 mRNA expression of control two was lowest,control one was highest,high concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.
2.TLR4 protein expression of low concentrate group and control one were most significant,high concentrate group and middle concentrate group were more significant,and control two expression was lesser.
3.MCP-1 of dialysate among high concentrate group,middle concentrate group, control two were no significantly difference(P＞0.05).MCP-1 of control one was higher than low concentrate group(P＜0.05),was highest among the others.MIP-2 of control one was significantly higher than the others(P＜0.001),control two was significantly lower than all of the Erigeron group(P=0.001,0.002,0.000).Compare to high concentrate group and middle concentrate group,middle concentrate group and low concentrate group,both were no statistically difference(P=0.115,0.226). high concentrate group and low concentrate group were significantly difference(P=0. 004)
Conclusion:
1.TLR4 expression are positively associated with concentration of MCP-1 or MIP-2,also are positively associated with peritoneal mesothelial cell damage.
2.Down-regulating TLR4 expression,prohibiting MCP-1 and MIP-2 production,may be the mechanisms of Erigeron.
ChapterⅢExperiments in Vitro
PartⅠEffects of Erigeron on Peritoneal Mesothelial Cell Growth and Proliferation in Rats
Objective:The purpose of the present study is to obverse effect of erigeron on peritoneal mesothelial cells growth and proliferation in vitro,to assess the mechanism of peritoneal mesothelial cells damage.
Methods:SD rat's peritoneal mesothelial cells isolated from peritoneal cavity were cultured and maintained under the defined conditions in vitro.They were cultured with medium group(group 1),50ul erigeron(group 2),25ul erigeron(group 3), 12.5ul erigeron(group 4),50ul erigeron added 10ul LPS(group 5),25ul erigeron added 10ul LPS(group 6),12.5ul erigeron added 10ul LPS(group 7),10ul LPS(group 8).To investigate peritoneal mesothelial cells growth and proliferation by cell counts(After 48 hours and 72 hours) and by MTT(after 72 hours).
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.
Results:
1.Cell counting:Mesothelial cells of group 3 and group 4 grow well,both were no significantly than group 1(P＞0.05).three groups were significantly better than group 2(P＜0.05).Plus LPS groups were less than no LPS groups.Group 5 and group 6 were.,more advantage than group 8(P＜0.001),but group 7 was no significantly difference than group 8(P＞0.05).
2.MTT:Mesothelial cells proliferation of plus LPS groups were worse than no LPS groups.Group 3 and group 4 were better than group 2.Group 8 was worse than group 5 and group 6,but had no significantly difference than group 7.
Conclusions:
1.Erigeron has the effect of protecting mesothelial cell from damage.
2.Erigeron can lessen mesothelial cell damage which LPS leads to immune inflammation,and the extent has positive correlation.
PartⅡEffects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduction Pathways in Peritoneal Mesothelial Cells in Vitro
Objective:This study was aims at peritoneal mesothelial cells Toll-like receptor 4 gene expression and signal pathways in vitro,to assess the role of erigeron.
Methods:The mesothelial cells used of this study were from mesothelial cells cultured in partⅠ,and divided group as well.After 72 hours,polymerase chain reaction assay was used to detect TLR4 mRNA expression of mesothelial cells. ELISA was used to investigate MCP-1 and MIP-2 in supernate fluid.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.
Results:In no LPS groups,The concentration of MCP-1 or MIP-2 in supernate fluid were no significantly difference(P＞0.05),TLR4 mRNA expression was much less.In plus LPS groups,TLR4mRNA expression and the concentration of MCP-1 or MIP-2 in supernate fluid all were remarkable higher than no LPS groups,lowest, control one was highest.High concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.Group 8 was significantly difference than group 5 or group 6(P＜0.01), and no difference than group 7(P＞0.05).
Conclusion:
1.The concentration of MCP-1 or MIP-2 in supernate fluid depend on TLR4 mRNA expression.The results show TLR4 signal pathways is existing.
2.Erigeron may down-regulate TLR4 mRNA expression and signal transduction of mesothelial cells in rats.
Conclusions of the whole paper:This study was to establish a peritoneal dialysis model of uraemia rats and to culture mesothelial cells,to prove that TLR4 and its signal transduction play an important role in peritoneal fibrosis of peritoneal dialysis or ultrafiltration failure.Erigeron can improve peritoneal dialysis efficiency and inhibit mesothelial cells from damage.The mechanics may be that Erigeron can down-regulate TLR4 expression,reduce MCP-1 and MIP-2 release.And those provided overall and penetrating molecular level mechanism and laboratorial evidence for its clinic treatment on ultrafiltration failure.
腹膜透析腹膜纤维化的发病机制十分复杂,多数研究者认为腹膜纤维化的发生与透析液的性质、腹膜炎、细胞因子等因素有关。腹透液的低PH值、高糖、乳酸盐和葡萄糖降解产物(GDP)等生物不相容因素,直接导致间皮细胞损伤,同时对于腹腔内白细胞产生明显抑制作用,降低防御能力;糖基化终产物可导致新生血管增生,促进纤维化。腹膜透析还存在许多易感途径和易感因素。腹膜炎一旦发生,引起一系列炎症反应,在补体参与下,趋化因子释放,粒细胞聚集,炎性介质释放,引起血管扩张,间皮细胞损伤,细胞外基质沉积,纤维化发生。近几年研究表明细胞因子在UFF发病中的作用不容忽视,涉及到腹膜回吸收增加、慢性腹腔炎症、糖基化终产物和氧自由基损伤等诸多方面。
菊科植物灯盏花[erigeron breviscapus(vaniot) Hand Mazz]又名灯盏细辛,有效成分为灯盏花素(erigeron)、黄酮类等,是黄苓素的4位接上一个羟基,从而对蛋白激酶C(protein kinase,PKC)有很好的抑制作用。PKC在细胞分泌、释放、溶解、膜运动、受体结合、平滑肌收缩、细胞增殖分化和癌变过程中均起重要作用。大量试验证实灯盏花素能够对抗缺血再灌注损伤,抑制氧化应激与脂质过氧化,防止纤维化的发生。还能够防止糖尿病肾病细胞外基质的积聚。对于糖尿病肾组织中单核细胞趋化蛋白-1(MCP-1)和细胞间粘附分子-1(ICAM-1)对巨噬细胞较强的趋化与激活作用以及细胞与细胞外基质之间的粘附作用均有抑制作用。总之,灯盏花素具有防止组织纤维化,减轻细胞外基质的积聚以及炎症反应的作用,就此推测灯盏花素对UFF有一定的改善作用。
UFF是腹膜透析严重并发症,是终止腹膜透析治疗的原因之一,但发病机理和防治尚有待进一步研究。TLRs是在人和小鼠细胞上发现的一种介导天然免疫的跨膜信号传递受体家族,迄今至少发现有11种人类跨膜蛋白的基因。Toll受体介导的免疫和炎症信号传导通路依赖于TIR结构域向细胞转导其识别信号,诱导很多快速反应基因的活化,产生诸如炎症细胞因子、主要组织相容性复合体(major histocompatibility complex,MHC)、共刺激分子、趋化因子等,参与机体的防御反应。TLR4在组织器官损伤和炎症的作用均有报道,但在腹膜透析失超滤的作用尚未见报道,因此研究腹膜透析时TLR4信号传导通路的变化,无疑有助于揭示UFF发病的分子机制。本项目通过建立腹膜透析尿毒症大鼠模型,观察腹膜透析大鼠透析效能及间皮细胞细胞TLR4、MCP-1、MIP-2的表达,探讨TLRs信号传导通路在腹膜透析UFF发生发展中的作用,开辟腹膜透析研究的新途径。同时研究灯盏花素对上述途径的影响,探讨灯盏花素对于腹膜间皮细胞保护,腹膜透析效能的提高,避免腹膜透析UFF发生的分子机制。
第一部分自制腹透管尿毒症大鼠腹膜透析模型的建立
目的:建立腺嘌呤尿毒症大鼠模型,在此基础上研究自制腹透管和手术方式建立尿毒症大鼠腹膜透析模型。
方法:SD大鼠用含有7500mg·kg-1腺嘌呤的颗粒饲料喂养,投放量为300mg/(kg·d),连续喂养7w,检测血清尿素氮、肌酐和肾脏病理确定尿毒症鼠模型的建立是否成功。自制腹透管采用硅胶吸痰管,外口使用一次性无菌输液接头连接;传统腹透管将头皮针改制。常规手术将腹透管置入大鼠腹腔。将上述术后大鼠分成3组,每组10只。自制腹透管1组腹透管由后背颈部皮肤穿出,自制腹透管2组腹透管由近尾背部皮肤穿出,传统头皮针组(出口同自制1组)。随后对30只大鼠用4.25%腹透液进行连续腹膜透析(CAPD),每日换液4次,每次25ml。观察管道的完整性,隧道口有无感染,管道是否包裹,腹膜炎发生情况,2周后透析排出量的变化等。
统计学方法:统计学处理:结果以(?)±s表示,采用SPSS13.0软件进行统计学分析,组内比较用配对t检验,组间比较用重复测量方差分析。样本率的比较采用χ~2检验。
结果:
1、腹透管完整性比较:自制1组、传统头皮针组大鼠腹透管完好,无管道破损和渗漏;自制2组的大鼠先后在15d内管道完全咬破损坏,无法使用。
2、出口处和隧道口感染情况:自制1组3例,自制2组2例,传统头皮针组6例发生隧道口感染。自制1组、自制2组与头皮针组存在统计学差异(P＜0.01),自制1组与自制2组则无统计学差异。
3、腹膜炎发生情况:自制1组1例,自制2组7例,传统头皮针组3例发生腹膜炎。自制1组与自制2组、传统头皮针组有统计学差异(P＜0.01,P＜0.05)。
4、腹透液排出情况:自制1组第1天与第15天相比腹膜透析出液量无统计学差异(t=2.207,P=0.055),头皮针组则有统计学意义(t=6.046,P=0.000),第1天自制1组、自制2组、头皮针组间相比无统计学差异(t=1.187,P=0.251),第15天自制1组与头皮针组相比存在统计学差异(t=2.724,P=0.014),自制2组与自制1组、头皮针组存在显著统计学差异(P=0.029)。
5、15天后自制1组腹膜透析管腹腔端被大网膜完全包裹1例、部分包裹5例,传统头皮针组完全包裹4例、部分包裹6例。
结论:
1、采用自制硅胶吸痰管作为腹透管,外口使用一次性无菌输液接头连接,腹透管出口由后背颈部皮肤穿出的手术方式构建的尿毒症大鼠腹膜透析模型,具有成功率高的特点。
2、正确的手术方式,是模型建立的基本保证,而透析管道的选材、接头稳固耐用是腹膜透析模型能否长期使用的关键。
第二部分体内实验
实验一、灯盏花素对尿毒症大鼠腹膜透析效能以及腹膜结构的影响
目的:观察灯盏花素对尿毒症大鼠腹膜透析效能以及腹膜结构的影响。
方法:将采用含腺嘌呤喂养制作尿毒症模型,随后采用自制腹透管行腹膜透析置管术制作腹膜透析模型,使用LPS加入腹腔建立尿毒症大鼠腹膜透析UFF模型。使用4.25%腹膜透析液分别加入不同浓度灯盏花素和LPS进行腹膜透析,将50只尿毒症腹膜透析模型SD大鼠平均分为5组:高浓度灯盏花素组(高浓度组)、中浓度灯盏花素组(中浓度组)、低浓度灯盏花素组(低浓度组)、对照1组(使用LPS组)、对照2组(单纯腹透液组,未加LPS),45天后行腹膜平衡试验,腹膜组织病理学检查,了解腹膜透析效能和结构的变化。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,两组间比较采用配对t检验、多组间比较采用重复测量方差分析。
结果:
1.腹膜效能及腹膜超滤量的影响:尿毒症大鼠在行腹膜透析前血肌酐无显著差异(F=0.96,P=0.345)。行腹膜透析后各加药组间D/P_(肌酐)(F=55.055,P=0.000)和超滤量(F=6.897,P=0.000)差异各不相同。对照2组的D/P_(肌酐)与高浓度组、中浓度组、低浓度组、对照1组间比较显著升高(t=-7.728,-8.696,-12.322,-9.899;P=0.000),超滤量与高浓度组、中浓度组比较无差异(t=-1.152,-1.754;P=0.273,0.100),与低浓度组、对照1组比较显著升高(t=-3.691,-3.73;P=0.003,0.005);低浓度组与对照1组间D/P_(肌酐)、超滤量比较无显著差异(P=0.361);高浓度组与中浓度组间D/P_(肌酐)、超滤量比较无显著差异(P=0.860);高浓度组、中浓度组的D/P_(肌酐)、超滤量均较低浓度组、对照1组显著升高(P＜0.001,P＜0.05)。高浓度组、中浓度组但均较对照2组显著下降(P＜0.001),但均较对照1组高(P＜0.001)。
2.大鼠腹膜组织病理变化:肉眼及光镜下观察:对照2组大鼠腹膜壁层、脏层表面光滑,无结节、渗出、充血。对照1组和低浓度组可见腹膜散在皱折,部分出现增厚,有渗出和充血现象。高浓度组、中浓度组腹膜增厚不明显,散在存在充血现象。光镜下见对照2组间皮细胞完整,细胞间连续性良好,间皮下基质无明显增多;对照1组间皮细胞肿胀、变形,部分皱缩,无完整的连续性,间皮下基质大量积聚,伴有大量炎性细胞的浸润和毛细血管增生;低浓度组间皮细胞存在肿胀、变形,细胞间连续性差,较多的间皮细胞下基质增多,可见炎性细胞浸润和毛细血管的增生;中浓度组可见间皮细胞肿胀,细胞数量减少,细胞间连续性良好,间皮下基质少量积聚,少见炎性细胞浸润,可见毛细血管增生;高浓度组间皮细胞完整,细胞连续性良好,细胞下少量基质沉积,少量炎性细胞浸润,毛细血管增生不明显。
结论:灯盏花素具有防止腹膜间皮细胞损伤,维持腹膜结构的完整性,改善局部微循环,提高腹膜的超滤效能等作用。以高、中浓度灯盏花素组为好。
实验二、灯盏花素对大鼠腹膜间皮细胞TLR4基因表达及信号通路的影响
目的:探讨TLR4及其信号通路在腹膜失超滤中所起的作用,并且观察灯盏花素对其干预作用。
方法:取实验一各组大鼠腹膜,免疫组化方法检测腹膜组织TLR4蛋白表达;采用逆转录聚合酶链反应(RT-PCR)检测TLR4 mRNA的表达情况:ELISA方法检测腹透液中MCP-1、MIP-2的浓度变化。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,两组间比较采用配对t检验、多组间比较采用重复测量方差分析;方差不齐者首先进行数据变换,若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验。检验水平a=0.05。
结果:
1.腹膜组织TLR4mRNA的表达:加有LPS的对照1组与未加LPS的对照2组腹膜组织TLRmRNA表达存在统计学差异(P=0.000)。在加有LPS的4组中腹膜TLR4mRNA相对表达量均存在显著性差异(χ~2=21.99,v=3,P=0.000)。对照1组腹膜TLR4mRNA表达量平均秩次最高;在灯盏花素干预组比较,高浓度组、中浓度组表达量平均秩次分别为8.71、7.38,低浓度组出现较高水平的表达,平均秩次为22.25,与对照1组相当。
2.组织细胞TLR4蛋白的免疫组织化学的表达:在未加LPS的对照2组,少数腹膜间皮细胞上出现TLR4蛋白表达;在加有LPS的高浓度组、中浓度组、低浓度组和对照1组中,高浓度组、中浓度组中多数的间皮细胞TLR4蛋白表达呈阳性,低浓度组、对照1组中基本上所有间皮细胞呈阳性表达(P=0.000)。
3.腹透液MCP-1、MIP-2浓度的变化(ELISA):腹透液MCP-1浓度在高浓度组、中浓度组、对照2组间无显著性差异(P=0.117,0.805,0.057);对照1组分别与高浓度组、中浓度组、低浓度组、对照2组相比存在显著性差异(P＜0.05);低浓度组腹透液MCP-1浓度高于高浓度组、中浓度组、对照2组,它们之间存在统计学意义(P＜0.05)。腹透液MIP-2浓度对照1组高于其他各组(P＜0.001);对照2组低于高浓度组、中浓度组、低浓度组(P=0.001,0.002,0.000);高浓度组与中浓度组相比、中浓度组与低浓度组相比无统计学意义(P=0.115,0.226),而高浓度组与低浓度组之间存在统计学差异(P=0.004)。
结论:
1.腹膜透析腹膜组织TLR4的表达量与腹透液中MCP-1、MIP-2浓度呈正相关,并与腹膜间皮细胞受损程度正相关。
2.灯盏花素对于TLR4的表达,以及由于TLR4激活所产生的MCP-1、MIP-2炎症因子具有下调效应。
第三部分体外实验
实验一、灯盏花素对腹膜间皮细胞生长和增殖的影响
目的:本研究旨在通过观察体外培养时灯盏花素对大鼠腹膜间皮细胞生长和增殖的影响,探讨间皮细胞受损的机制。
方法:对SD大鼠腹膜间皮细胞进行分离培养,间皮细胞分成8组,分别加入灯盏花素和LPS,包括:单纯培养基组(1组)、50ul灯盏花素组(2组)、25ul灯盏花素组(3组)、12.5ul灯盏花素组(4组)、50ul灯盏花素+10ul LPS组(5组)、25ul灯盏花素+10ul LPS组(6组)、12.5ul灯盏花素+10ul LPS组(7组)、10ul LPS组(8组)。培养48小时和72小时后通过细胞计数了解间皮细胞生长情况;培养72小时后加入MTT5mg/孔,用酶标仪490nm比色,了解间皮细胞增殖情况。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,单因素组间比较采用One Way ANOVE、SNK检验,多组间比较采用重复检验方差分析。
结果:
1.细胞数量影响:间皮细胞培养48小时和72小时后进行计数组间存在显著性差异(F=182.738,P=0.000;F=677.589,P=0.000),各个加药组间也存在显著性差异(F=606.988,P=0.000)。在未加LPS组中,3组(中等剂量)和4组(低剂量灯盏花素组)间皮细胞生长良好,与1组(对照组)相比无显著性差异(P=0.086,1.000);2组(高浓度组)与1组相比具有显著性差异(P=0.004)。而加LPS组间皮细胞生长情况差于未加组,在LPS组中,加入5组(灯盏花素高浓度组)、6组(中浓度组)和7组(低浓度组)对LPS对间皮细胞生长抑制作用具有拮抗作用(P=0.000)。
2.细胞增殖的影响:加药组间具有统计学差异(F=1265.008,P=0.000)。未加LPS组中1、3、4组的间皮细胞增殖无显著差异(P=0.64,0.929),2组的间皮细胞增殖与3、4组比较无显著性差异(P=0.705),与1组比较存在显著性差异(P=0.039)。加有LPS组中5、6组间无统计学差异(P=0.771),其余各组间存在统计学差异(P=0.000),其中8组间皮细胞增殖最差,7组次之。
结论:
1.灯盏花素对于间皮细胞的生长和增殖具有保护作用。
2.灯盏花素能够减轻LPS引起的免疫炎症造成的间皮细胞的损伤,且存在量效正相关关系。
实验二、灯盏花素对腹膜间皮细胞TLR4表达及信号通路的影响
目的:观察体外腹膜间皮细胞TLR4以及信号通路的表达情况,研究灯盏花素对TLR4的信号通路以及相关信号因子影响的分子机制。
方法:体外进行间皮细胞培养,分组同体外实验一,给予灯盏花素和LPS进行干预,72小时后采用RT-PCR法对间皮细胞TLR4 mRNA表达进行检测;采用ELISA方法对培养上清液MCP-1、MIP-2浓度进行检测。
统计学方法:数据以均数±标准差((?)±s)表示。应用SPSS13.0统计软件,多组间比较采用One Way ANOVE、SNK检验;方差不齐者首先进行数据变换,若数据变换后仍不齐者多组间多重比较采用多个独立样本非参数分析Kruskal-Wallis检验。检验水平a=0.05。
结果:
1.间皮细胞培养上清中MCP-1,MIP-2的浓度变化:MCP-1、MIP-2的浓度在不同组间存在显著性差异(F=80.358,P=0.000;F=320.101,P=0.000)。未加LPS组间(1～4组)MCP-1、MIP-2浓度无统计学差异(P＞0.05),加有LPS的4组中,高浓度灯盏花素组(5组)、中浓度灯盏花素组(6组)与单纯LPS组(8组)间存在统计学差异(P＜0.01),而低浓度灯盏花素组(7组)与8组间无统计学差异(P＞0.05)。
2.间皮细胞TLR4 mRNA表达检测:在未加LPS的1～4组中间皮细胞TLR4mRNA的表达量相当,无统计学差异(x~2=2.544,v=3,P=0.467);在加有LPS的5～8组中间皮细胞TLRmRNA的表达量存在的差异有显著性意义(x~2=25.63,v=3,P=0.000),8组(未加灯盏花素组)和7组(低浓度灯盏花素组)表达量相对高,平均秩次分别27.28、27.50,而5组(高浓度灯盏花素组)和6组(中浓度灯盏花素组)表达量低,平均秩次分别为9.56、9.67,可以认为不同浓度灯盏花素对TLR4mRNA的表达量的影响不同。
结论:
1.MCP-1、MIP-2对于TLR4具有依赖性,证明了TLR4信号通路的存在的。
2.灯盏花素具有下调腹膜间皮细胞TLR4 mRNA及信号通路表达的作用。
全文结论:本研究通过在尿毒症大鼠腹膜透析模型基础上进一步建立尿毒症大鼠腹膜透析UFF模型以及间皮细胞培养的实验研究,证明TLR4及其信号传导在腹膜透析腹膜发生纤维化(即腹膜超滤失败)所发挥的作用。而灯盏花素具有提高腹膜透析效能和保护腹膜间皮细胞的作用,其作用机理与下调TLR4表达,降低MCP-1、MIP-2的含量有关,为其临床推广应用于治疗腹膜透析失超滤提供了较为全面、深入的分子水平的理论依据。
The incidence of chronic kidney diseases(CKD) is rising rapidly year by year around the world,which has become one of the most important diseases listed behind cardiovascular diseases,tumor and diabetic mellitus.In developed countries,the incidence of CKD of the common crowd is 6.5～10%.In our country,the incidence of the crowd aged over 40 years old is 8～9%,among whom most people will become uraemia according to an epidemiological investigation.Peritoneal dialysis is one of the remedies for chronic renal failure.Because it has many advantages such as less influenced glomerular filtration,more steady cardiovascular function,more handling convenience,cheaper price,and higher probability for patients to come back to the society,people have attached more and more importance to it.There are about 10 or 15 percent of ESRD patients(over 150 thousands people) using peritoneal dialysis around the world.The percentage of ESRD patients using peritoneal dialysis is different among different countries.In our country,peritoneal dialysis has been used since 1960s.With the improved living standard,the people's stronger desire for healthy,and the consummate technology,the increasing number of ESRD patients are using peritoneal dialysis.But there are still many problems such as catheter dysfunction,material histoimcompatibility,nutrition imbalance,peritonitis,and social psychic:factor slowing down the progression of peritoneal dialysis,among which peritoneum fibrosis,ultrafiltrate and convery dysfunction,even ultrafiltration failure (UFF) become the main causes to stop the remedy.A report showed that the incidence of UFF was 14%in patients who had peritoneal dialysis for 18 months and 51% patients withdrew from peritoneal dialysis after eight years owing to UFF.So it is urgent to give more emphasis on finding the pathogenesis of and developing effective remedies for UFF.
The pathogenesis of peritoneal dialysis associated peritoneal fibrosis is very complicated and remains poorly understood.Most researchers think peritoneal fibrosis is relevant to the property of dialysate,peritonitis,cytokine,and so on.The histoirncompatibility factors of dialysate including low PH,high glucose,lactate and glucose degradation products damage peritoneal mesothelial cells directly,inhibit leucocytes in the abdominal cavity,cut down immune defense capability,induce glycation end products,cause angiogenesis and promote fibrosis.Peritonitis induces a series of inflammatory reaction including alexin activation,chemotatic factors release, leukocytes accumulation and inflammatory mediator release,leading to blood vessel dilatation,mesothelial cells damage,extracellular matrix accumulation and fibrosis formation.In recent years,researchers found that cytokines played important roles in UFF.UFF was related to the rising of reverse absorption by peritoneum,chronic abdominal cavity inflammation,production of glycation end products and oxygen radicals and so on.
Erigeron,4-OH-scutellaria baicalensis,is the effective component of Erigeron breviscapus(Vaniot) Hand Mazz,also names fleabane.It can inhibit protein kinase C (PKC).PKC plays significant roles in cellular secretion,release and dissolve, membranous motion,receptor combination,smooth muscle constraction,cell proliferation and differentiation,and canceration.Data proved that Erigeron could block ischemia reperfusion injury,retard oxidative stress and lipid peroxidation and prevent fibrosis proliferation.Eeigeron could also prevent extracellular matrix accumulation in diabetic nephropathy,strongly inhibit monocyte chemotactic protein- 1(MCP- 1) and intercellular adhesion molecule- 1(ICAM- 1) expression and macrophage activation in renal tissues of diabetes.In a word,Erigeron can prevent tissue fibrosis,extracellular matrix accumulation and inflammation.
UFF is a very serious complication during peritoneal dialysis and one of the reasons that give rise to peritoneal dialysis failure.However,the pathogenesis and remedy need to be investegated.Toll-like receptors(TLRs),which have been found on cells of human beings or rats,belong to transmembrane signal transduction receptor family,and can mediate natural immune.Up to now,at least eleven kinds have been found in human beings.The TLRs transduce identification signals into the cells via a specific motif,mediate immune and inflammation signal transduction pathways,activate fast reaction genes,produce inflammatory chemokines such as major histocompatibility complex(MHC),costimulatory molecules,chemotactic factors,etc and participate organism defense reaction.It was reported that TLR4 participated in tissue and organ damage and inflammation,but there is no report about the effects of TLR4 on UFF until now.To study TLR4 and its signal transduction will be helpful to reveal molecular mechanisms of UFF.
In the above context,we investigatedthe effects of TLRs signal transduction on the development of UFF by building a peritoneal dialysis model of ureamia in rats and observing peritoneal dialysis efficacy and TLR4,MCP-1,Macrophage inflammatory protein-2(MIP-2) expression.Our study will be able to open up a new way to peritoneal dialysis investigation.Meanwhile,we researched how Erigeron affect TLR4 expression and signal transduction in order to reveal the molecular mechanisms of the protecting effects of Erigeron on peritoneal mesothelium,peritoneal dialysis efficacy and UFF.
ChapterⅠTo Build a Model of Peritoneal Dialysis in Uremia Rats by Self-made Peritoneal Dialysis Catheter
Objective:To develop an improved model of peritoneal dialysis in rats that mimic human peritoneal dialysis.
Methods:Firstly,we established the uremia rat models by feeding of amidopurine according to the dose of 300 mg·kg-1 body weight.In the following seven weeks,we detected the blood serum urea nitrogen,creatinine,patho-scopy, and determined the model built.Rats were divided into three groups:experimental group 1,experimental group 2 and control group.In experimental group 1,phlegm suction catheters were used as peritoneal dialysis catheters with tie-ins made from sterile transfuse connectors.The peritoneal dialysis catheters implanted into rats were tunneled subcutaneously to the nape.Rats with different operation mode or different operation material belonged to experimental group 2 or control group,respectively.
Statistical analysis:All values were expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results of two groups before and after treatment were analyzed using paired t-test for comparisons.Compare to rate of sample usingχ~2-test.
Results:
1.Peritoneal dialysis catheters in rats in experimental group 1 were intact, whereas catheters in rats in experimental group 2 were broken.
2.Three rats in experimental group 1 and six rats in control group had tunnel opening infections.
3.One rat in experimental group 1 and three rats in control group had peritonitis.
4.In experimental group 1,the amount of exchanged solution on the first day of peritoneal dialysis was similar to that on the fifteenth day(p＞0.05),but in control group,the amount of exchanged solution on the first day was different from that on the fifteenth day(p＜0.01).
5.There were no difference between experimental group 1 and control group at the amount of exchanged solution on the first day(p＞0.05),whereas there were significant difference on the fifteenth day(p＜0.05).
6.Peritoneal dialysis catheters were completely wrapped by omentums in one rat in experimental group 1 and in 4 rats in control group.Catheters were partly wrapped in five rats in experimental group 1 and in 6 rats in control group.
Conclusion:Correct operation modes,proper operation material and firm durable catheter tie-ins are the key for developing a successful model of peritoneal dialysis.
ChapterⅡExperiments in Vivo
PartⅠEffects of Erigeron on Dialysis Efficiency and Peritoneal UItrastrueture in Uraemia Rats
Objective:To study the effect of erigeron on dialysis efficiency and peritoneal ultrastructure in uraemia rats.
Methods:Firstly,feeding amidopurine to make model of uraemia rats,then building model of peritoneal dialysis by made-self dialysis catheter.The death rate of rats was 13.33%after model created.4.25%glucose dialysate,Erigeron,LPS were used in peritoneal dialysis.50 male SD model rats were divided into five groups:high concentrate group,middle concentrate group,low concentrate group,control one, control two.Peritoneal Equilibration Tests was observed,and the changes of peritoneal structure as well.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Vet.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.
Results:
1.D/P_(creatinine) of control two was significant higher than others(P＜0.001),its intraperitoneal volume had no significant difference than high concentrate group and middle concentrate group(P＞0.05),had significant higher than low concentrate group and control one(P＜0.001).Compare to low concentrate group and control one,high concentrate group and middle concentrate group,their D/P_(creatinine) and intraperitoneal volume had no significant different(P＞0.05).
2.The extent of which integrity of peritoneal mesothelial cell destroyed,base material below cells accumulated,inflammatory cells infiltrated and blood capillary hyperplasia,there are control group one,high concentrate group,middle concertrate, low concentrate group and control group two by turns.
Conclusions:Erigeron could preserve peritoneal mesothelial cell,and improve peritoneal dialysis efficiency.
PartⅡEffects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduetion Pathways in Rats Peritoneum
Objective:The aim of this study was to bring out the effect of TLR4 gene and signal pathways on peritoneal ultrafiltration failure,also the effect of Erigeron on TLR4 gene expression.
Methods:The peritoneal tissue used of this study were from the rats in partⅠ. Immunohistochemical analysis was used to detect TLR4 of peritoneal tissue expression.Polymerase chain reaction assay was employed to detect TLR4 mRNA expression.ELISA was used to investigate MCP-1 and MIP-2.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.
Results:
1.In all groups,TLR4 mRNA expression of control two was lowest,control one was highest,high concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.
2.TLR4 protein expression of low concentrate group and control one were most significant,high concentrate group and middle concentrate group were more significant,and control two expression was lesser.
3.MCP-1 of dialysate among high concentrate group,middle concentrate group, control two were no significantly difference(P＞0.05).MCP-1 of control one was higher than low concentrate group(P＜0.05),was highest among the others.MIP-2 of control one was significantly higher than the others(P＜0.001),control two was significantly lower than all of the Erigeron group(P=0.001,0.002,0.000).Compare to high concentrate group and middle concentrate group,middle concentrate group and low concentrate group,both were no statistically difference(P=0.115,0.226). high concentrate group and low concentrate group were significantly difference(P=0. 004)
Conclusion:
1.TLR4 expression are positively associated with concentration of MCP-1 or MIP-2,also are positively associated with peritoneal mesothelial cell damage.
2.Down-regulating TLR4 expression,prohibiting MCP-1 and MIP-2 production,may be the mechanisms of Erigeron.
ChapterⅢExperiments in Vitro
PartⅠEffects of Erigeron on Peritoneal Mesothelial Cell Growth and Proliferation in Rats
Objective:The purpose of the present study is to obverse effect of erigeron on peritoneal mesothelial cells growth and proliferation in vitro,to assess the mechanism of peritoneal mesothelial cells damage.
Methods:SD rat's peritoneal mesothelial cells isolated from peritoneal cavity were cultured and maintained under the defined conditions in vitro.They were cultured with medium group(group 1),50ul erigeron(group 2),25ul erigeron(group 3), 12.5ul erigeron(group 4),50ul erigeron added 10ul LPS(group 5),25ul erigeron added 10ul LPS(group 6),12.5ul erigeron added 10ul LPS(group 7),10ul LPS(group 8).To investigate peritoneal mesothelial cells growth and proliferation by cell counts(After 48 hours and 72 hours) and by MTT(after 72 hours).
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.
Results:
1.Cell counting:Mesothelial cells of group 3 and group 4 grow well,both were no significantly than group 1(P＞0.05).three groups were significantly better than group 2(P＜0.05).Plus LPS groups were less than no LPS groups.Group 5 and group 6 were.,more advantage than group 8(P＜0.001),but group 7 was no significantly difference than group 8(P＞0.05).
2.MTT:Mesothelial cells proliferation of plus LPS groups were worse than no LPS groups.Group 3 and group 4 were better than group 2.Group 8 was worse than group 5 and group 6,but had no significantly difference than group 7.
Conclusions:
1.Erigeron has the effect of protecting mesothelial cell from damage.
2.Erigeron can lessen mesothelial cell damage which LPS leads to immune inflammation,and the extent has positive correlation.
PartⅡEffects of Erigeron on Toll-like Receptor 4 Gene Expression and Signal Transduction Pathways in Peritoneal Mesothelial Cells in Vitro
Objective:This study was aims at peritoneal mesothelial cells Toll-like receptor 4 gene expression and signal pathways in vitro,to assess the role of erigeron.
Methods:The mesothelial cells used of this study were from mesothelial cells cultured in partⅠ,and divided group as well.After 72 hours,polymerase chain reaction assay was used to detect TLR4 mRNA expression of mesothelial cells. ELISA was used to investigate MCP-1 and MIP-2 in supernate fluid.
Statistical analysis:All values are expressed as the mean±standard deviation((?)±s).Statistical analysis was performed using the statistical package SPSS for Windows Ver.13.0.Results after treatment among multi-groups were analyzed using one-way ANOVE.If there was heterogeneity of variance,the data should be transformed first,and then analyzed.Provided that variance remained heterogeneity after data transformed,the data should be analyzed by Kruskal-Wallis H Test for multiple comparisons among multi-groups.P＜0.05 was considered to be statistically significant.
Results:In no LPS groups,The concentration of MCP-1 or MIP-2 in supernate fluid were no significantly difference(P＞0.05),TLR4 mRNA expression was much less.In plus LPS groups,TLR4mRNA expression and the concentration of MCP-1 or MIP-2 in supernate fluid all were remarkable higher than no LPS groups,lowest, control one was highest.High concentrate group and middle concentrate group,low concentrate group and control one had same expression.Compare high concentrate group and middle concentrate group with control one had significantly higher than control one.Group 8 was significantly difference than group 5 or group 6(P＜0.01), and no difference than group 7(P＞0.05).
Conclusion:
1.The concentration of MCP-1 or MIP-2 in supernate fluid depend on TLR4 mRNA expression.The results show TLR4 signal pathways is existing.
2.Erigeron may down-regulate TLR4 mRNA expression and signal transduction of mesothelial cells in rats.
Conclusions of the whole paper:This study was to establish a peritoneal dialysis model of uraemia rats and to culture mesothelial cells,to prove that TLR4 and its signal transduction play an important role in peritoneal fibrosis of peritoneal dialysis or ultrafiltration failure.Erigeron can improve peritoneal dialysis efficiency and inhibit mesothelial cells from damage.The mechanics may be that Erigeron can down-regulate TLR4 expression,reduce MCP-1 and MIP-2 release.And those provided overall and penetrating molecular level mechanism and laboratorial evidence for its clinic treatment on ultrafiltration failure.
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