姜黄素通过miR-146a抑制NF-κB信号通路保护大鼠糖尿病肾病的机制研究

Studies on protective mechanism of curcumin by inhibiting NF-kappaB signaling pathway via miR-146a in mice with diabetic nephropathy

目的探讨姜黄素对糖尿病肾病肾脏损伤的保护作用及机制研究。方法将SD大鼠随机分为对照组(N组)、糖尿病肾病模型组(DN组)和姜黄素干预治疗组(CT组)。干预治疗4周后,检测各组大鼠肾脏组织的病理改变以及Nephrin蛋白变化情况;实时定量聚合酶链反应(polymerase chain reaction,PCR)检测miR-146a和促炎因子、纤维化因子表达情况。收集不同培养条件下HBZY-1细胞,检测miR-146a mRNA表达情况;利用转染技术,将miR-146a minics转染入高糖培养条件下的HBZY-1细胞中,检测miR-146a、Traf6、促炎因子和纤维化因子mRNA表达情况以及核因子κB(nuclear factor kappa-B,NF-κB)p65蛋白变化情况。结果与N组比较,DN组血糖(blood glucose,BG)、血尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,SCr)和尿白蛋白肌酐比值(urine albumin creatine ratio,UACR)均明显升高,CT组BG、BUN、SCr和UACR含量降低,差异有统计学意义。肾脏病理切片和Western blot结果均显示,与N组比较,DN组肾脏组织损伤,而CT组肾脏组织损伤缓解。实时定量PCR结果显示,与N组相比,DN组miR-146a表达降低,促炎因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)和纤维化因子转化生长因子β(transforming growth factor-β,TGF-β)、Ⅰ型胶原蛋白(collagenⅠ)表达升高;而与DN组比较,CT组miR-146a表达则升高,细胞因子表达则降低,差异有统计学意义。分别用0、5μmol/L、15μmol/L和25μmol/L浓度姜黄素刺激HBZY-1细胞24 h后,miRNA-146a mRNA表达逐渐升高;分别用含有4.5 mmol/L(L-HC组)、15 mmol/L(M-HC组)和25 mmol/L(H-HC组)葡萄糖的培养液培养细胞,miRNA-146a mRNA表达逐渐降低。将miR-146a minics和negative control(NC)转染到高糖培养条件下HBZY-1细胞中,H-HC miR-146a组中,miR-146a mRNA表达明显高于H-HC miR-NC组,而H-HC组与H-HC miR-NC组之间差异无统计学意义;与H-HC miR-NC组比较,H-HC miR-146a组中靶基因Traf6和促炎因子(TNF-α和IL-1β)、纤维化因子(TGF-β和collagenⅠ)均降低,差异有统计学意义。H-HC miR-146a组中p65蛋白表达水平明显低于H-HC miR-NC组和H-HC组。结论姜黄素通过促进miR-146a表达抑制NF-κB信号途径来缓解糖尿病肾病大鼠肾脏损。

Objective To investigate the protective effects of curcumin on rat with diabetic nephropathy(DN)as well as the functional mechanism.Methods The SD rats were randomly divided into control group(N group),DN model group(DN group)and curcumin treatment group(CT group).At 4 weeks after treatment,pathological changes of renal tissues were detected and changes of nephrin protein were detected.The expression of miR-146a,pro-inflammatory factors and fibrogenic factors was detected by real-time quantitative polymerase chain reaction(RT-qPCR).HBZY-1 cells under different culture conditions were collected and the mRNA expression of miR-146a was detected.Using transfection technique,miR-146a minics was transfected into HBZY-1 cells under high-glucose culture conditions.The mRNA expression of miR-146a,Traf6,pro-inflammatory factors and fibrogenic factors as well as the change of nuclear factor kappa-B(NF-kappa B)p65 protein were detected.Results Compared with the control group,blood glucose(BG),blood urea nitrogen(BUN),serum creatinine(SCr)and urine albumin creatine ratio(UACR)were significantly increased in the DN group.After curcumin intervention treatment,the content of BG,BUN,SCr and UACR were decreased,and the difference was statistically significant.Pathological sections of kidney and Western blot results showed that compared with the control group,there was the kidney tissue damage in the DN group,which,however,was alleviated in the CT group.RT-qPCR results showed that compared with the control group,the expression of miR-146a was reduced and the expressions of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),transforming growth factor-β(TGF-β)and collagenⅠwere increased in the DN group.Compared with the DN group,the expression of miR-146a was increased,while the expression of cytokine was decreased in the CT group,suggesting significant difference.After stimulating HBZY-1 cells with curcumin at concentrations of 0,5μmol/L,15μmol/L and 25μmol/L for 24 h,the mRNA expression of miRNA-146a was gradually increased.Cells were cultured with culture medium containing 4.5 mmol/L(L-HC group),15 mmol/L(M-HC group)and 25 mmol/L(H-HC group)glucose,respectively,and the expression of miRNA-146a was gradually decreased.When miR-146a minics and negative control(NC)were transfected into HBZY-1 cells under high-glucose culture conditions,the mRNA expression of miR-146a in H-HC miR-146a group was significantly higher than that in H-HC miR-NC group,but there was no difference between the HC group and the H-HC miR-NC group.Compared with the H-HC miR-NC group,the target genes Traf6 and proinflammatory factors(TNF-αand IL-1β)and fibrosis factors(TGF-βand collagenⅠ)were reduced in the H-HC miR-146a group,and the difference was statistically significant.The expression level of p65 protein in H-HC miR-146a group was significantly lower than that in H-HC miR-NC group and H-HC group.Conclusion Curcumin alleviates kidney damage in rats with DN by promoting the expression of miR-146a and inhibiting the NF-κB signaling pathway.