加味真武汤通过调控miR-377表达水平对糖尿病肾病的影响

Effect of Jiawei Zhenwu decoction on diabetic nephropathy by regulating the expression of miR-377

目的 探讨加味真武汤通过调控miR-377表达水平对糖尿病肾病(DN)细胞炎症水平和免疫功能的影响及分子机制.方法 选取40只SPF级雄性大鼠,对其中30只大鼠进行腹腔注射链脲佐菌素,建立大鼠DN模型,并随机分为模型对照组、加味真武汤低剂量组、加味真武汤高剂量组,每组各10只.同时设置空白对照组(10只).建立HK-2细胞的DN模型,分为miR-NC+DN组(将miR-NC转染至建模后的HK-2细胞)、miR-377+DN组(将miR-377转染至建模后的HK-2细胞)、anti-miR-NC+DN组(将anti-miR-NC转染至建模后的HK-2细胞)、an-ti-miR-377+DN组(将anti-miR-377转染至建模后的HK-2细胞)、加味真武汤+miR-NC+DN组(将miR-NC转染至建模后的HK-2细胞,再用高剂量加味真武汤处理)、加味真武汤+miR-377+DN 组(将miR-377转染至建模后的HK-2细胞,再用高剂量加味真武汤处理).采用实时荧光定量PCR检测miR-377表达水平;采用酶联免疫吸附试验检测肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)水平;采用流式细胞仪检测CD4^(+)、CD8^(+)和Th1水平.结果 与空白对照组比较,模型对照组的TNF-α、IL-6、IL-8、CD4^(+)、miR-377水平升高,CD8^(+)和Th1水平降低,差异均有统计学意义(均P<0.001);与模型对照组比较,加味真武汤低剂量组、加味真武汤高剂量组的TNF-α、IL-6、IL-8、CD4^(+)、miR-377水平降低,CD8^(+)和Th1水平升高,差异均有统计学意义(均P<0.001).与miR-NC+DN组比较,miR-377+DN组的TNF-α、IL-6、IL-8水平以及CD4^(+)水平升高,CD8^(+)和Th1水平降低(均P<0.001);与anti-miR-NC+DN组比较,anti-miR-377+DN组的TNF-α、IL-6、IL-8水平以及CD4^(+)水平降低,CD8^(+)和Th1水平升高(均P<0.001).与加味真武汤+miR-NC+DN组比较,加味真武汤+miR-377+DN组的TNF-α、IL-6、IL-8水平以及CD4^(+)水平降低,CD8^(+)和Th1水平升高(均P<0.001).结论 加味真武汤通过抑制miR-377表达减轻DN细胞炎症水平,增强患者的免疫功能,值得临床推广应用.

Objective Jiawei Zhenwu decoction regulates the effect of miR-377 on the inflammation level and immune function of diabetic nephropathy(DN)cells and its molecular mechanism.Methods Forty SPF male rats were selected,30 of them were intraperitoneally injected with streptozotocin to establish a rat model of DN.The rats were randomly divided into model control group,Jiawei Zhenwu decoction low-dose group,and Jiawei Zhenwu decoction high-dose group,10 rats in each group.At the same time,blank control group(10 rats)was set up.The DN model of HK-2 cells was established.The cells were divided into miR-NC+DN group(transfect miR-NC to HK-2 cells after modeling),miR-377+DN group(miR-377 transfected into HK-2 cells after modeling),anti-miRNC+DN group(transfected anti-miR-NC into HK-2 cells after modeling),anti-miR-377+DN group(transfected anti-miR-377 to HK-2 cells after modeling),Jiawei Zhenwu decoction+miR-NC+DN group(transfected miR-NC into HK-2 cells after modeling,then treated with Jiawei Zhenwu decoction high-dose),Jiawei Zhenwu decoction+miR-377+DN group(transfected miR-377 into modeled HK-2 cells,and then treated with Jiawei Zhenwu decoction high-dose).Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression level of miR-377;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of TNF-α,IL-6,and IL-8;the levels of CD4^(+),CD8^(+)and Thl were detected by flow cytometry.Results Compared with the blank control group,the levels of TNF-α,IL-6 and IL-8,CD4^(+)and miR-377 in the model control group were significantly increased,and the levels of CD8^(+)and Thl were significantly decreased(all P<0.001);compared with the model control group,the levels of TNF-α,IL-6 and IL-8,CD4^(+)and miR-377 in the Jiawei Zhenwu decoction low-dose group and the Jiawei Zhenwu high-dose decoction group were significantly reduced,and the levels of CD8^(+)and Thl were significantly increased(all P<0.001).Compared with the miRNC+DN group,the levels of TNF-α,IL-6,IL-8 and CD4^(+)in the miR-377+DN group were significantly increased,and the levels of CD8^(+)and Thl were significantly reduced(all P<0.001).Compared with anti-miR-NC+DN group,the levels of TNF-α,IL-6,IL-8 and CD4^(+)in the anti-miR-377+DN group were significantly reduced,and the levels of CD8^(+)and Thl were significantly increased(all P<0.001).Compared with the Jiawei Zhenwu decoction+miR-NC+DN group,the levels of TNF-α,IL-6,IL-8 and CD4^(+)in the Jiawei Zhenwu decoction+miR-377+DN group were significantly decreased,while the levels of CD8^(+)and Thl increased significantly(all P<0.001).Conclusions Jiawei Zhenwu decoction can reduce the inflammation level of DN cells and enhance immune function of patients by inhibiting the expression of miR-377,which is worthy of clinical application.