积雪草酸抑制TXNIP/NLRP3信号通路改善小鼠糖尿病肾病的机制研究

Mechanism of Asiatic Acid in Improving Diabetic Nephropathy in Mice Through Inhibition of TXNIP/NLRP3 Signaling Pathway

目的 基于TXNIP/NLRP3信号通路探讨积雪草酸(ASA)对糖尿病肾病小鼠的作用及机制。方法 将60只雄性C57BL/6小鼠随机分为空白组、模型组及积雪草酸低、中、高剂量组(10、20、40 mg·kg^(-1));采用高脂饲料喂养结合腹腔注射链脲佐菌素(STZ)建立糖尿病肾病小鼠模型;造模成功后,按设定剂量灌胃给药,每日1次,连续8周。检测小鼠血糖、糖化血清蛋白水平,以及肾脏指数、24 h尿量、血清肌酐及尿素氮等肾功能指标;采用HE、Masson染色法观察肾脏组织病理改变;ELISA法测定小鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-18的水平;免疫组织化学检测肾脏组织中α平滑肌肌动蛋白(α-SMA)、E-cadherin、NLRP3的表达;RT-qPCR法检测肾脏组织TNF-α、IL-1β、IL-18、α-SMA、E-cadherin mRNA表达水平;Western Blot法检测肾脏组织TXNIP、NLRP3、Cleaved Caspase-1蛋白表达水平。结果 与模型组比较,积雪草酸高剂量组小鼠的血糖水平显著降低(P<0.01),但积雪草酸对小鼠的糖化血清蛋白水平影响不明显(P>0.05);积雪草酸中、高剂量组小鼠的肾脏指数、24 h尿量、血清肌酐及尿素氮水平明显降低(P<0.05,P<0.01);积雪草酸中、高剂量组小鼠的肾小管损伤评分及胶原纤维阳性区域平均光密度值显著降低(P<0.01),肾脏组织病理损伤有明显改善;积雪草酸中剂量组小鼠血清TNF-α、IL-1β、IL-18水平明显降低(P<0.05,P<0.01),肾脏组织中的E-cadherin蛋白表达水平显著升高(P<0.01),α-SMA、NLRP3蛋白表达水平显著降低(P<0.01),TNF-α、IL-1β、IL-18、α-SMA mRNA表达水平显著降低(P<0.01),E-cadherin m RNA表达水平显著升高(P<0.01),TXNIP、NLRP3、Cleaved Caspase-1蛋白表达水平显著降低(P<0.01)。结论 积雪草酸对糖尿病肾病小鼠的肾脏具有保护作用,可能与其抑制TXNIP/NLRP3信号通路所介导的炎症反应,并抑制α-SMA的表达改善肾脏的纤维化有关。

Objective To explore the effect and mechanism of asiatic acid(ASA)in mice with diabetic nephropathy based on the TXNIP/NLRP3 signaling pathway. Methods Sixty male C57BL/6 mice were randomly divided into the blank group,the model group and the low-,medium-and high-doses of ASA(10,20 and 40 mg·kg^(-1))groups;a mouse model of diabetic nephropathy was established by high-fat diet combined with intraperitoneal injection of streptozotocin(STZ). After successful modeling,the drug was administered by gavage at the set dose once daily for consecutive 8 weeks. The levels of blood glucose,glycated serum protein and renal function indexes such as renal index,24-hour urine volume,serum creatinine and urea nitrogen were detected. The pathological changes of renal tissue were observed by HE and Masson staining. The serum levels of tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β)and IL-18 were determined by ELISA. The expressions of α smooth muscle actin(α-SMA),E-cadherin and NLRP3 in kidney tissue were detected by immunohistochemistry. The mRNA expression levels of TNF-α,IL-1β,IL-18,α-SMA and E-cadherin in kidney tissue were detected by RT-qPCR. The protein expression levels of TXNIP,NLRP3 and Cleaved Caspase-1 in kidney tissue were detected by Western Blot. Results Compared with the model group,the blood glucose level in mice was significantly decreased in the high-dose group(P<0.01),but the effect of ASA on the glycated serum protein level was not significant(P>0.05);the renal index,24-hour urine volume, serum creatinine and urea nitrogen levels were significantly decreased in the mice in the medium-and high-dose groups(P<0.05,P<0.01);the renal tubular injury score and the mean optical density of the collagen fiber-positive region in mice were significantly decreased in the medium-and high-dose of ASA groups(P<0.01).The mean optical density values of renal tubular injury score and collagen fiber positive area were significantly decresaed(P<0.01),and there was significant improvement in renal histopathological injury;levels of serum TNF-α,IL-1β and IL-18 were significantly decreased in mice in the medium-dose of ASA group(P<0.05,P<0.01),and the protein expression level of E-cadherinin in renal tissue was significantly increased(P<0.01),the protein expression levels of α-SMA and NLRP3 were significantly decreased(P<0.01),the mRNA expression levels of TNF-α, IL-1β, IL-18 and α-SMA were significantly decreased(P<0.01), the mRNA expression level of E-cadherin was significantly increased(P<0.01),and the protein expression levels of TXNIP,NLRP3 and Cleaved Caspase-1 were significantly reduced(P<0.01). Conclusion The protective effect of ASA on the kidneys of mice with diabetic nephropathy may be related to its inhibition of the inflammatory response mediated by the TXNIP/NLRP3 signaling pathway and its inhibition of α-SMA expression to improve renal fibrosis.