肾康注射液对CAPD小鼠腹膜间皮细胞的保护作用及机制研究

Protective effect and its mechanism of Shenkang injection on peritoneal mesothelial cells in CAPD mice

目的探讨肾康注射液对连续性不卧床腹膜透析(CAPD)小鼠腹膜间皮细胞(PMCs)的保护作用及可能机制。方法 40只ICR小鼠均分为空白对照(A)组,阳性对照腹透(B)组(2.5%腹透液),低(C)、中(D)、高(E)剂量肾康干预组(2.5%腹透液+5、10及20 mL/kg肾康注射液),观察至4周。采用生化法检测空腹血糖、总胆固醇、三酰甘油、C-反应蛋白(CRP)水平;采用酶联免疫吸附试验检测血清及透出液中肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)、结缔组织生长因子(CTGF)水平;HE染色观察腹膜组织病理改变;分别以免疫组化染色和Real-time PCR检测腹膜组织中TNF-α、TGF-β1、VEGF、CTGF的蛋白质表达和m RNA转录水平。结果各组小鼠体质量、空腹血糖、总胆固醇、三酰甘油水平差异无统计学意义。与B组比较,C、E组差异均无统计学意义,但D组CRP水平显著减低,无论是血清还是腹腔透出液中,C、D组TNF-α、TGF-β1、VEGF及CTGF表达水平均降低,而E组TNF-α、TGF-β1升高(P<0.05),但E组VEGF及CTGF差异均无统计学意义。与E组比较,除C组腹腔液中CTGF差异无统计学意义外,血清及腹腔透出液中C、D组TNF-α、TGF-β1、VEGF、CTGF均降低(P<0.05)。B组可见腹膜间皮细胞均有损害;C、D、E组有不同程度改善。与B组比较,C、D、E组TNF-α、TGF-β1、VEGF、CTGF蛋白及m RNA水平呈逐渐降低趋势(P<0.05)。结论一定浓度的肾康注射液可通过抑制CAPD小鼠PMCs的TNF-α、TGF-β1、VEGF、CTGF的表达,保护PMCs,从而控制腹膜纤维化的发生发展。

Objective To study the protective effect of Shenkang injection on peritoneal mesothelial cells(PMCs)ofcontinuous ambulatory peritoneal dialysis(CAPD)mice,and explore the possible mechanism.Methods Forty ICR micewere randomly divided into blank control(A)group,peritoneal dialysis(B)group,low dose of Shenkang(C)group(2.5%dialysate+5mL/kg Shenkang injection),medium(D)group(2.5%dialysate+10mL/kg Shenkang injection)and high(E)group(2.5%dialysate+20mL/kg Shenkang injection).Mice were observed for4weeks.Fasting blood glucose,totalcholesterol,triglyceride and C-reactive protein(CRP)were detected by biochemical assay.Tumor necrosis factor-alpha(TNF-α),transforming growth factor-beta1(TGF-β1),vascular endothelial growth factor(VEGF)and connective tissuegrowth factor(CTGF)levels of serum and dialysate were detected by ELISA.Pathological changes of peritoneal tissue wereobserved by HE staining.Expression and mRNA transcription levels of these four cytokines in the peritoneal tissue weredetected by immunohistochemical staining and real-time PCR respectively.Results There were no significant differencesin body weight,fasting blood glucose,total cholesterol and triglyceride between5groups of mice(P>0.05).Compared withgroup B,there was no significant difference in CRP level between group C and group E,but which was significantlydecreased in group D(P<0.05).The serum and dialysate levels of TNF-α,TGF-β1,VEGF and CTGF were decreased ingroup C and group D.The serum and dialysate levels of TNF-αand TGF-β1were significantly increased in group E(P<0.05),but there was no significant difference between VEGF and CTGF in group E.Compared with group E,except for CTGF in dialysate of group C,the serum and dialysate levels of TNF-α,TGF-β1,VEGF and CTGF were significant decreased ingroup C and group D(P<0.05).Damaged PMCs were found in group B,which were improved in various degrees in group C,group D and group E.Compared with group B,the protein expression and mRNA relative transcription levels of TNF-α,TGF-β1,VEGF and CTGF tended to decrease gradually in group C,group D and group E(P<0.05).Conclusion A certainconcentration of Shenkang injection can protect PMCs by inhibiting the expression of TNF-α,TGF-β1,VEGF and CTGF inCAPD mice,so as to control the occurrence and development of peritoneal fibrosis.