氧化苦参碱减轻糖尿病肾病小鼠肾组织炎症及纤维化反应的机制研究

Mechanism underlying roles of oxymatrine in alleviating inflammation and fibrosis in renal tissues of mice with diabetic kidney disease

摘要:

目的 通过观察氧化苦参碱（OMT）对同源结构域相互作用蛋白激酶2（HIPK2）、Toll样受体4（TLR4）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、上皮钙黏素（E-cadherin）、纤维连接蛋白（Fibronectin）、白细胞介素-1β（IL-1β）和IL-18表达的影响，初步探讨OMT在糖尿病肾病（DKD）中抗炎抗纤维的可能保护机制。方法 健康6周龄的db/db小鼠适应性喂养2周，随机分为糖尿病组（DM组）和OMT组，每组10只。OMT组给予腹腔注射氧化苦参碱［120 mg/（kg·d）］，持续8周，并设同月龄同背景db/m小鼠为正常对照组（NC组）。处死小鼠前收集血液和尿液，检测各项生化指标，HE和Masson染色观察小鼠肾组织病理形态学改变，Western blotting检测、免疫组织化学染色观察各组小鼠肾皮质TLR4、HIPK2、NLRP3、E-cadherin、Fibronectin的表达水平及部位；酶联免疫吸附试验检测各组小鼠血清IL-1β、IL-18水平。采用Pearson法对HIPK2与TLR4、NLRP3蛋白表达进行相关性分析。结果 DM组体重、血糖、24 h尿蛋白量、总胆固醇、甘油三酯水平较NC组均升高（P <0.05），OMT组24 h尿蛋白量、总胆固醇、甘油三酯水平较DM组降低（P <0.05）。病理染色显示，DM组可见肾小球系膜区节段性增生，基底膜无明显增厚，肾小管管腔明显扩张，肾小管上皮细胞空泡样变性，间质区炎症细胞浸润和蓝色胶原纤维沉积；经OMT治疗后，相较于DM组，OMT组肾小球系膜区增生程度减轻，肾小管病变有所改善，间质区炎症细胞浸润减少。DM组TLR4、HIPK2、NLRP3、Fibronectin蛋白相对表达量较NC组升高（P <0.05），E-cadherin蛋白相对表达量较NC组降低（P <0.05）；OMT组小鼠TLR4、HIPK2、NLRP3、Fibronectin蛋白相对表达量较DM组降低（P <0.05），E-cadherin蛋白相对表达量较DM组升高（P <0.05）。DM组血清中IL-1β、IL-18相对表达量较NC组升高（P <0.05），OMT组较DM组降低（P <0.05）。Pearson相关相关性分析显示，DM组肾组织中HIPK2蛋白与TLR4、NLRP3蛋白表达均呈正相关（r =0.881和0.774，均P <0.05）。OMT组HIPK2蛋白与TLR4、NLRP3蛋白表达均呈正相关（r =0.814、0.871，均P <0.05）。结论 OMT抑制DM小鼠肾组织的炎症反应和纤维化程度，可能与抑制HIPK2及炎症信号通路的活化有关。

Abstract:

Methods Healthy 6-week-old db/db mice were accustomed to feeding for 2 weeks, and were then randomly divided into the diabetes mellitus (DM) group and the OMT group with 10 mice in each group. The OMT group was given intraperitoneal injection of OMT [120 mg/ (kg·d)] for 8 weeks, and db/m mice of the same month of age and background were set as the control group (NC group). The blood and urine samples were collected to detect the changes of biochemical indicators before the mice were sacrificed. H&E and Masson staining were used to observe the histopathological changes of renal tissues. Western blotting and immunohistochemical staining were performed to observe the expressions and distribution of TLR4, HIPK2, NLRP3, E-cadherin and fibronectin in the renal cortex of mice from each group, and the serum levels of IL-1β and IL-18 of mice in each group were detected by the enzyme-linked immunosorbent assay (ELISA). Pearson's correlation test was performed to analyze the correlation between the protein expression of HIPK2 and that of TLR4 and NLRP3.Results The levels of fasting blood glucose, 24-hour urine protein, total cholesterol, and triglycerides in the DM group were significantly higher than those in the NC group (P < 0.05), while levels of 24-hour urine protein, total cholesterol, and triglycerides in the OMT group were lower than those in the DM group (P < 0.05). The pathological staining exhibited mesangial segmental hyperplasia, no obvious glomerular basement membrane thickening, significantly dilated renal tubular lumen, vacuolar degeneration in the renal tubular epithelial cells, and inflammatory cell infiltration and collagen fiber deposition in the interstitial area in the DM group. After OMT treatment, the OMT group showed that the mesangial hyperplasia, the renal tubular lesions, and the infiltration of inflammatory cells in the interstitial area were alleviated compared with those in the DM group. The protein expressions of TLR4, HIPK2, NLRP3, and fibronectin in the DM group were higher than those in the NC group, and the protein expression of E-cadherin in the DM group was lower than that in the NC group (P < 0.05). The protein expressions of TLR4, HIPK2, NLRP3, and fibronectin in the OMT group were lower than those in the DM group, while the protein expression of E-cadherin in the OMT group was higher than that in the DM group (P < 0.05). The serum levels of IL-1β and IL-18 in the DM group were higher than those in the NC group (P < 0.05), and those in the OMT group were lower relative to those in the DM group (P < 0.05). Pearson's correlation test demonstrated that the protein expression of HIPK2 in renal tissues was positively correlated with the protein expressions of TLR4 and NLRP3 in renal tissues in the DM group (r = 0.881 and 0.774, both P < 0.05). In addition, the protein expression of HIPK2 in renal tissues was also positively correlated with the protein expressions of TLR4 and NLRP3 in renal tissues in the OMT group (r = 0.814 and 0.871, both P < 0.05).Conclusions OMT inhibits the inflammation and fibrosis in the renal tissues of DM mice, which may be achieved by suppressing the activation of HIPK2 and the inflammatory signaling pathway.Odjective To preliminarily investigate the possible mechanism underlying roles of oxymatrine (OMT) in protecting against inflammation and fibrosis in diabetic kidney disease (DKD) by observing the effects of OMT on expressions of homeodomain interacting protein kinase 2 (HIPK2), Toll-like receptor 4 (TLR4), Nod-like receptor family pyrin domain containing 3 (NLRP3), E-cadherin, fibronectin, interleukin (IL)-1β and IL-18.