雷公藤乙素对糖尿病肾病足细胞损伤自噬作用及免疫代谢的影响

Effect of triptolide on autophagy and immunity of DKD podocyte injury

目的:探讨雷公藤乙素对糖尿病肾病(DKD)足细胞损伤自噬作用及DKD患者树突状细胞、T淋巴细胞、糖代谢的影响。方法:以体外培养小鼠足细胞系为研究对象,将足细胞分为正常对照组、高糖组和高糖+雷公藤乙素组。Western blot检测三组足细胞特异性指标肾病蛋白(Nephrin)、肾小球足细胞裂隙膜蛋白(Podocin)及自噬相关蛋白LC3、p62、哺乳动物雷帕霉素靶蛋白(mTOR)表达情况;RT-PCR法检测三组足细胞炎症因子白介素-1α(IL-1α)、白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6) mRNA表达水平。收集健康者和DKD患者外周静脉血单核细胞向树突状细胞(DC)分化,混合淋巴细胞反应。将其分为正常对照组、DKD组和DKD+雷公藤乙素组。流式细胞仪检测DC表面分子HLA-DR MHCⅡ、CD86、CD54水平,T淋巴细胞CD3^(+)、CD4^(+)、CD8^(+)水平以及用HLA-DR MHCⅡ标记的DC细胞葡萄糖转运蛋白-1(GLUT-1)水平。结果:与高糖组比较,高糖+雷公藤乙素组Nephrin、mTOR蛋白表达升高,p62表达降低(均P<0.05),LC3、Podocin蛋白表达虽升高,但差异无统计学意义(均P>0.05)。与高糖组比较,高糖+雷公藤乙素组IL-1α、TNF-α mRNA表达水平明显改善(均P<0.05),IL-1β、IL-6 mRNA表达水平虽有改善,但是差异无统计学意义(均P>0.05)。与DKD组比较,DKD+雷公藤乙素组DC表达HLA-DR MHCⅡ、CD86、CD54平均荧光值较高(均P<0.05)。与DKD组比较,DKD+雷公藤乙素组T淋巴细胞CD3^(+)、CD4^(+)和CD8^(+)表达升高(均P<0.05)。与DKD组比较,DKD+雷公藤乙素组GLUT-1表达量升高(P<0.05)。结论:雷公藤乙素能够减轻足细胞损伤,激活自噬的活性,能够使DC有效激活T淋巴细胞,改善免疫功能,并能够调节糖代谢。

Objective:To investigate the effect of triptolide on podocyte injury and autophagy in diabetic kidney disease(DKD),and the effects of triptolide on dendritic cells,T lymphocytes and glucose metabolism in DKD patients.Methods:Mouse podocyte cultured in vitro were divided into normal control group,high glucose group and high glucose+triptolide group.Western blot was used to detect the expression of podocyte specific indicators,Nephrin,Podocin and autophagy related proteins LC3,p62,mTOR.The mRNA expression levels of inflammatory cytokines interleukin-1α(IL-1α),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the three groups were detected by RT-PCR.Collect peripheral venous blood monocytes from healthy people and DKD patients to differentiate into DC,and mix lymphocyte reaction,and they were divided into normal control group,DKD group,and DKD+triptolide group.Flow cytometry was used to detect the expression of HLA-DR MHCⅡ,CD86,and CD54 on the surface of DC cells,as well as the expression of CD3+,CD4+,and CD8+on T lymphocytes,as well as the expression of glucose transporter-1(GLUT-1)in DC cells labeled with HLA-DR MHCⅡ.Results:Compared with the high glucose group,the protein expressions of Nephrin and mTOR in the high glucose+triptolide group were increased,while the expression of p62 was decreased(all P<0.05);The protein expressions of LC3 and Podocin were increased,but the differences were not statistically significant(all P>0.05).Compared with high glucose group,mRNA expression levels of IL-1αand TNF-αin high glucose+triptolide group were significantly improved(all P<0.05),the mRNA expression levels of IL-1βand IL-6 were improved,but the differences were not statistically significant(all P>0.05).Compared with DKD group,DKD+triptolide group expressed higher mean fluorescence values of MHCⅡ,CD86,CD54(all P<0.05).Compared with DKD group,expressions of CD3+,CD4+and CD8+in T lymphocytes of DKD+triptolide group were increased(all P<0.05).Compared with DKD group,GLUT-1 expression in DKD+triptolide group increased(P<0.05).Conclusion:Triptolide can alleviate podocyte damage,activate autophagy activity,effectively activate T lymphocytes through dendritic cells,improve immune function,and regulate glucose metabolism.