基于分子对接技术的藤茶总黄酮对高尿酸血症肾功能损伤保护机制研究

目的探讨藤茶总黄酮(total flavonoids from Ampelopsis grossedentata,AGTF)对高尿酸血症(hyperuricemia,HUA)肾功能损伤的保护机制。方法利用分子对接技术,将藤茶总黄酮主要活性成分与HUA相关靶点蛋白尿酸重吸收转运体1(uric acid reabsorption transporter 1,URAT1)、葡萄糖转运蛋白9(glucose transporter 9,GLUT9)、三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate binding cassette transporter G2,ABCG2)、黄嘌呤氧化酶(xanthine oxidase,XOD)和腺苷脱氨酶(adenosine deaminase,ADA)进行分子对接。采用腺嘌呤联合乙胺丁醇复合方法制备HUA肾功能损伤大鼠模型,以AGTF进行干预,检测大鼠血清中尿酸代谢酶XOD和ADA活性、尿酸以及肾功能生化指标肌酐和尿素氮的水平;苏木素-伊红(HE)染色观察大鼠肾脏组织病理变化;Western blotting法检测大鼠肾组织中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)和转化生长因子-β(transforming growth factor-β,TGF-β)蛋白的表达;免疫组化法检测大鼠肾组织中URAT1、GLUT9和ABCG2蛋白的表达。结果分子对接结果表明,AGTF主要活性成分二氢杨梅素、杨梅素、槲皮素和藤茶素与疾病相关靶点对接得分均较高,提示AGTF可能通过干预URAT1、GLUT9、ABCG2、ADA和XOD发挥抗HUA肾功能损伤的作用。AGTF能显著降低HUA肾功能损伤模型大鼠血清中XOD和ADA的活性以及尿酸、肌酐、尿素氮的水平(P<0.05、0.01)。HE染色结果显示,AGTF能缓解模型大鼠肾小球基底膜增厚,减轻肾小管排列紊乱及坏死,抑制病变区域内纤维组织增生。Westernblotting和免疫组化结果显示,AGTF能显著降低大鼠肾组织中炎症因子TNF-α、IL-1β、IL-6、TGF-β蛋白和尿酸盐重吸收相关蛋白URAT1、GLUT9的表达(P<0.05、0.01),显著升高尿酸盐排泄相关蛋白ABCG2的表达(P<0.05、0.01)。结论 AGTF对HUA肾功能损伤大鼠具有较好的保护作用,其作用机制可能与降低TNF-α、IL-1β、IL-6和TGF-β表达,抑制炎症反应,并调节尿酸盐转运蛋白URAT1、GLUT9和ABCG2的表达,促进尿酸代谢有关,与分子对接预测结果一致。

Protective effect and mechanism of total flavonoids from Ampelopsis grossedentata on renal function injury of hyperuricemia based on molecular docking technology

Objective To explore the protective mechanism of the total flavonoids from Ampelopsis grossedentata(AGTF) on renal function injury of hyperuricemia(HUA). Methods Main active components of AGTF were connected with HUA related target proteins uric acid reabsorption transporter 1(URAT1), glucose transporter 9(GLUT9), adenosine triphosphate binding cassette transporter G2(ABCG2), xanthine oxidase(XOD), and adenosine deaminase(ADA) by using molecular docking technology. Renal injury rats model of HUA was prepared by adenine combined with ethambutol, and AGTF was used for intervention. Activities of urate metabolizing enzyme such as XOD and ADA, levels of uric acid, renal function biochemical indicators such as creatinine and urea nitrogen in serum were detected. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat kidney. Expressions of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), and transforming growth factor-β(TGF-β) in renal tissues were determined by Western blotting. Expression of URAT1, GLUT9, and ABCG2 were detected by immunohistochemistry. Results Molecular docking results showed that the main components of AGTF of dihydromyricetin, myricetin, quercetin, and grossedentatasin all had higher docking scores with disease-related targets, suggesting that AGTF may prevent and treat HUA renal function injury by acting on URAT1, GLUT9, ABCG2, ADA and XOD. AGTF significantly reduced the activities of XOD and ADA, levels of uric acid, creatinine and urea nitrogen in serum of HUA renal injury rats(P < 0.05, 0.01). AGTF alleviated the thickening of glomerular basement membrane in model rats, reduced the disorder and necrosis of renal tubular arrangement, and inhibited the proliferation of fibrous tissue in lesion area. Western blotting and immunohistochemistry results showed that AGTF significantly reduced the expressions of inflammatory factors such as TNF-α, IL-1β, IL-6, TGF-β, and urate reabsorption related proteins such as URAT1 and GLUT9(P < 0.05, 0.01), significantly increased the expression of urate excretion related protein such as ABCG2 in renal tissue(P < 0.05, 0.01). Conclusion AGTF has a good protective effect on renal function injury of HUA. AGTF could inhibit the expressions of TNF-α, IL-1β, IL-6, and TGF-β to alleviate inflammatory pathway reactions, and regulate the expressions of urate transporter related proteins such as URAT1, GLUT9, and ABCG2 to promote metabolism of uric acid, which are consistence with predicted results of molecular docking.