二氢硫辛酸琥珀酰转移酶在小鼠肾间质纤维化中的作用

Role of dihydro lipoamidesuccinyl transferase in mice renal interstitial fibrosis

目的:探讨二氢硫辛酸琥珀酰转移酶(DLST)在小鼠肾间质纤维化中的作用。方法:采用结扎小鼠单侧输尿管建立单侧输尿管梗阻(UUO)模型和HK2细胞缺氧模型,60只C57b/l小鼠随机分为假手术组(Sham,n=15)和UUO模型组(n=45),UUO模型组又随机分为1周(n=15)、2周(n=15)和3周组(n=15);HK2细胞实验分为对照组、缺氧组、缺氧加DLST高表达组和DLST干扰组。HE和Masson染色观察各组小鼠肾脏病理性变化,免疫组化观察在体实验不同时间点DLST的表达,Western-blot检测DLST、I型胶原蛋白(Col-Ⅰ)和α-SMA的蛋白变化,细胞实验中采用质粒高表达和shRNA干扰DLST。结果:HE和Masson染色显示,与Sham组相比,UUO模型小鼠均出现肾小管损伤,肾小管上皮细胞变性,肾间质有大量炎性细胞浸润,损伤程度逐渐加重。Western Blot结果表明,与Sham组相比,UUO模型小鼠肾脏Col-Ⅰ和α-SMA的表达显著增加(P<0.05),而DLST的表达明显减少(P<0.05),均呈现时间依赖性。免疫组化结果表明,与Sham组相比,UUO组DLST的表达逐渐减少。细胞实验结果显示,正常条件下干扰DLST后,纤维化蛋白Col-Ⅰ表达明显增加(P<0.01),而缺氧条件下,高表达DLST后可明显减少Col-Ⅰ蛋白的表达(P<0.01)。结论:小鼠肾脏DLST蛋白表达的减少是导致肾间质纤维化的重要原因,可能通过引起能量代谢障碍参与肾间质纤维化的发生过程。

Objective： To invetigate the role of dihydrolipoamidesuccinyltransferase（ DLST） in mice renal interstitial fibrosis. Methodology：Unilateral ureteral obstruction（ UUO） was established by ligation of unilateral ureter in mice.Sixty C57 b/l mice were randomly divided into sham operation group（ n = 15）,UUO model group（ n = 45）,and the UUO model group was randomly divided into 1 week（ n = 15）,2 weeks（ n = 15） and 3 weeks group（ n = 15）. HK2 cells were divided into control group,hypoxia group（ HY）,HY plus DLST overexpression group（ HY ＋ DLST OE） and DLST interference group（ DLST＋ shRNA）. The pathological changes of kidney in each group were observed by HE and Masson staining,the expression of DLST was observed by immunohistochemistry,and Western blot was used to measure the expressions of DLST,collage type Ⅰ（ Col-Ⅰ） and α-SMA protein expression. Results： HE and Masson staining examination showed renal tubules injury at each time point in the UUO group. The renal tubular epithelial cells were degenerated. There was a large number inflammatory cells infiltration in the interstitium and the interstitial fibrosis degree gradually increased. Western-blot results showed that the expression of Col-Ⅰ and α-SMA in UUO group increased significantly（ P〈0. 05） and displayed a time-dependent tendency. The immunohistochemistry showed that the expression of DLST in UUO was gradually decreased over time. Cell experiments showed that the expression of Col-Ⅰ was significantly increased after DLST shRNA under normal conditions（ P〈0. 01）,while the expression of Col-Ⅰwas significantly decreased after DLST overexpression（ P〈0. 01）. Conclusion： The expression of DLST was decreased in mice renal interstitial fibrosis,which may be involved in the process of renal interstitial fibrosis by causing energy metabolism disorders.