当归芍药散含药血清及其有效成分对肾间质纤维化大鼠的作用及机制

Effects of Danggui Shaoyao San-medicated serum and its active ingredients on renal interstitial fibrosis rats

目的 探讨当归芍药散(DSS)含药血清及其有效成分阿魏酸(FA)、芍药苷(PF)对肾间质纤维化(RIF)大鼠的作用及机制。方法 制备当归芍药散药液,以低剂量(0.05 mL DSS药液/10 g体质量)、中剂量(0.1 mL DSS药液/10 g体质量)、高剂量(0.2 mL DSS药液/10 g体质量)每日同一时间灌胃雄性Sprague Dawley大鼠,持续7 d,腹主动脉取血获得相应低、中、高剂量DSS血清(DSSS)。将大鼠肾小管上皮细胞(NRK-52E)分成对照组、模型组(TGF-β处理)、siRNA转染组(TGF-β+siRNA处理)、FA组(TGF-β+FA处理)、PF组(TGF-β+PF处理)及DSSS低、中、高剂量组(TGF-β+DSSS-L、TGF-β+DSSS-M、TGF-β+DSSS-H分别处理)。筛选并确定DSSS、FA、PF、TGF-β对NRK-52E细胞的干预浓度,对以上各组NRK-52E细胞进行干预。采用Western blotting法检测各组NLRP3、半胱氨酸蛋白酶1(CASP-1)、Gasdermin D(GSDMD)、白细胞介素-18(IL-18)、纤连蛋白(FN)、E型钙黏蛋白(E-Cadherin)、N型钙黏蛋白(N-Cadherin)的蛋白表达变化。结果 相较于模型组,DSSS、FA、PF治疗组细胞焦亡相关蛋白NLRP3、CASP-1、GSDMD、IL-18表达均降低,差异均有统计学意义(P均<0.05)。DSSS中、高剂量及FA、PF治疗组相较于模型组纤维化相关黏附蛋白FN、E-Cadherin、N-Cadherin表达降低,差异均有统计学意义(P均<0.05)。结论 当归芍药散含药血清及有效成分FA、PF可抑制TGF-β诱导的NRK-52E细胞纤维化模型中NLRP3介导的焦亡蛋白激活,并能调节黏附蛋白表达和上皮-间质转化进展,缓解纤维化。

Objective To investigate the interventional effects of Danggui Shaoyao San(DSS)-medicated serum and its active ingredients ferulic acid(FA) and paeoniflorin(PF) on renal interstitial fibrosis(RIF) rats and the mechanism.Methods DSS solution was prepared and administered to male Sprague Dawley rats by gavage at the same time of the day at low(0.05 mL DSS solution/10 g body mass),medium(0.1 mL DSS solution/10 g body mass),and high(0.2 mL DSS solution/10 g body mass) doses for 7 d.Blood was obtained from the abdominal aorta to obtain the equivalent low-,medium-,and high-dose of DSS serum(DSSS).Rat renal tubular epithelial cells(NRK-52E) were divided into the control group(Control),the model group(treated with TGF-β),the siRNA transfection group(treated with TGF-β+siRNA),the FA group(treated with TGF-β+FA),the PF group(treated with TGF-β+PF),and the low-,medium-,and high-dose DSSS groups(treated with TGF-β+DSS-L,TGF β+DSS-M,and TGF-β+DSS-H),respectively.The intervention concentrations of DSSS,FA,PF and TGF-β on NRK-52E cells were screened and determined for NRK-52E cells in the above groups.Western blotting was used to detect the changes in the protein levels of NLRP3,cysteine protease 1(CASP1),Gasdermin D(GSDMD),interleukin-18(IL-18),fibronectin(FN),E-Cadherin,and N-Cadherin in each group.Results Compared with the model group,the expression levels of pyroptosis-related proteins NLRP3,CASP1,GSDMD,and IL-18 decreased in the DSSS,FA,and PF groups,and the difference was statistically significant(all P<0.05).Compared with the model group,the expression levels of fibrosis-related adhesion proteins FN,E-Cadherin and NCadherin decreased in the medium-dose and high-dose DSSS and FA and PF groups,and the difference was statistically significant(all P<0.05).Conclusion DSS-medicated serum and its active ingredients,paeoniflorin and ferulic acid,can inhibit the activation of NLRP3-associated pyroptosis proteins in the TGF-β-induced fibrosis model of NRK-52E cells and regulate the expression of adhesion proteins and the progression of epithelial-mesenchymal transdifferentiation to alleviate the fibrosis.