基于miR-495/FTO通路介导的巨噬细胞极化探讨芪参汤改善胰岛素抵抗治疗2型糖尿病的分子机制

Exploration of the molecular mechanism of Qishen decoction in regulating miR⁃495/FTO pathway mediated macrophage polarization to improve insulin resistance therapy of type 2 diabetes

目的:观察芪参汤对miR‐495/FTO信号通路介导的巨噬细胞极化的影响,从而阐明芪参汤改善胰岛素抵抗治疗2型糖尿病的分子机制。方法:以佛波酯诱导THP‐1人单核细胞株分化巨噬细胞,并分为空白组、模型组、芪参汤组、miR‐495 inhibitor组和芪参汤+miR‐495 inhibitor组。除空白组外,余下各组采用30 mmol/L葡萄糖刺激构建巨噬细胞极化模型,并给与相应的药物进行干预。24 h收集各组细胞,采用酶联免疫吸附法检测炎症因子(IL‐6、IL‐1β、IL‐4和IL‐10)的含量,采用流式细胞术、qPCR和Western blot检测巨噬细胞极化标记分子、MiR‐495和FTO的表达。结果:与空白组相比,空白血清、芪参汤含药血清和转染miR‐495抑制物的巨噬细胞活性均无明显变化,差异均无统计学意义(P>0.05)。此外,与空白组相比,模型组IL‐6和IL‐1β的含量、CD68、iNOS、COX‐2和miR‐495的表达和CD68/CD206的比值均显著增加,IL‐4和IL‐10的含量、CD206、Arg‐1、YM‐1和FTO的表达均显著降低,差异均具有统计学意义(P<0.01)。与模型组相比,芪参汤组IL‐6和IL‐1β的含量、CD68、iNOS、COX‐2和miR‐495的表达和CD68/CD206的比值均显著降低,IL‐4和IL‐10的含量、CD206、Arg‐1、YM‐1和FTO的表达均显著增加,差异均具有统计学意义(P<0.01)。结论:芪参汤靶向miR‐495上调/FTO的表达促进巨噬细胞的M2型极化,从而抑制炎症达到改善胰岛素抵抗的作用。

Objective:To observe the effect of Qishen decoction on macrophage polarization mediated by miR‐495/FTO signaling pathway,and to clarify the molecular mechanism of Qishen decoction in improving insulin resistance in the treatment of type 2 diabetes.Methods:THP‐1 was induced to differentiate macrophages with phorbol ester.It was divided into the control group,the model group,the Qishen decoction group,the miR‐495 inhibitor group,and the Qishen decoction+miR‐495 inhibitor group.Except for the control group,the remaining groups were stimulated with 30 mmol/L glucose to construct a macrophage po‐larization model,and corresponding drugs were given for intervention.Cells were collected from each group for 24 hours and the content of inflammatory factors(IL‐6,IL‐1β,IL‐4,and IL‐10)were detected using enzyme‐linked immunosorbent assay.The ex‐pression of macrophage polarization marker molecules,miR‐495,and FTO were detected by flow cytometry,qPCR,and West‐ern blot to detect.Results:Compared with the control group,there was no significant change in the activity of macrophages in the control serum,Qishen decoction containing serum,and miR‐495 inhibitor transfected serum,and the difference was not statistical‐ly significant(P>0.05).In addition,compared to the control group,the content of IL‐6 and IL‐1β,the expression levels of CD68,iNOS,COX‐2,miR‐495,and the ratio of CD68/CD206,were significantly increased(P<0.01).While the content of IL‐4 and IL‐10,as well as the expression of CD206,Arg‐1,YM‐1,and FTO were significantly reduced(P<0.01).Compared with the model group,the QiShen decoction significantly reduced the contents of IL‐6 and IL‐1β,and the expression levels of CD68,iNOS,COX‐2,and miR‐495,as well as the ratio of CD68/CD206,while the content of IL‐4 and IL‐10,as well as the expression of CD206,Arg‐1,YM‐1,and FTO were significantly increased(P<0.01).Conclusion:Qishen decoction upregulate the expression of FTO to promote M2 type polarization of macrophages,thereby inhibiting inflammation and improving insulin re‐sistance by inhibiting the expression of miR‐495.