Departments of Liver Diseases, Taizhou People's Hospital, Taizhou 225300, Jiangsu Province, China.
Articular Surgery, Taizhou People's Hospital, Taizhou 225300, Jiangsu Province, China.
Biomed Pharmacother. 2020 Apr;124:109930. doi: 10.1016/j.biopha.2020.109930. Epub 2020 Jan 25.
This study aims to explore the relationship between miR-195 and CD40 and its effect on Th17/Treg balance in rats with non-alcoholic fatty liver disease (NAFLD).
We established rat models of NAFLD and made seven groups, Normal group (without modeling), Model group (model rats), NC group (model rats injected with negative control vector), miR-195 OE group (model rats injected with miR-195 mimic), anti-miR-195 group (model rats injected with miR-195 inhibitor), Si-CD40 group (model rats injected with CD40 silencing vector), and anti-miR-195+Si-CD40 group (model rats injected with miR-195 inhibitor and CD40 silencing vector). Dual-luciferase reporter gene assay verified the targeting relationship between miR-195 and CD40. The mRNA and protein expression levels of miR-195, CD40 as well as Th17/Treg associated cytokines in the liver tissues were detected. The pathological changes of liver tissues were detected, and the liver lesion scoring was carried out. The liver coefficient was calculated. The levels of liver function related indices, and Th17/Treg associated cytokines and inflammatory factors in serum were determined. The proportions of Th17/Treg cells in serum were determined by flow cytometry.
Compared with Normal group, miR-195 expression level in liver tissues of rats in other six groups was significantly reduced (all P < 0.05); the serum levels of AST, ALT, GGT, IL-17, TNF-α, IL-23, IL-6, IL-8, TC, TG, HDL, and LDL, and the Th17/Treg ratio, as well as the mRNA and protein expression levels of CD40, RORyt, IL-17, TNF-α, IL-23, and IL-8 in liver tissues were significantly increased (all P < 0.05); while the mRNA and protein expression levels of Foxp3, and IL-10 level were significantly reduced (all P < 0.05). Compared with Model group, the above parameters showed an opposite trend in miR-195 OE group and Si-CD40 group were significantly reduced (all P < 0.05). Moreover, anti-miR-195 group could aggravate the imbalance of Th17/Treg cells in rats with NAFLD and promote inflammatory response. Compared with anti-miR-195 group, the combined treatment in anti-miR-195+Si-CD40 group can partially avoid the imbalance of Th17/Treg cells, and inhibit inflammatory response.
Overexpression of miR-195 can reduce the Th17/Treg ratio to maintain Th17/Treg balance by inhibiting CD40 expression in rats with NAFLD.
本研究旨在探讨 miR-195 与 CD40 之间的关系及其对非酒精性脂肪性肝病（NAFLD）大鼠 Th17/Treg 平衡的影响。
我们建立了 NAFLD 大鼠模型，并分为七组：正常组（未建模）、模型组（造模组）、NC 组（转染阴性对照载体的模型组）、miR-195 OE 组（转染 miR-195 模拟物的模型组）、抗 miR-195 组（转染 miR-195 抑制剂的模型组）、Si-CD40 组（转染 CD40 沉默载体的模型组）和抗 miR-195+Si-CD40 组（转染 miR-195 抑制剂和 CD40 沉默载体的模型组）。双荧光素酶报告基因实验验证了 miR-195 与 CD40 之间的靶向关系。检测肝组织中 miR-195、CD40 及 Th17/Treg 相关细胞因子的 mRNA 和蛋白表达水平。检测肝组织的病理变化，进行肝损伤评分。计算肝系数。检测血清中肝功能相关指标及 Th17/Treg 相关细胞因子和炎症因子水平。流式细胞术检测血清中 Th17/Treg 细胞的比例。
与正常组相比，其余六组大鼠肝组织中 miR-195 表达水平明显降低（均 P < 0.05）；血清中 AST、ALT、GGT、IL-17、TNF-α、IL-23、IL-6、IL-8、TC、TG、HDL 和 LDL 水平以及 Th17/Treg 比值，以及肝组织中 CD40、RORyt、IL-17、TNF-α、IL-23 和 IL-8 的 mRNA 和蛋白表达水平均明显升高（均 P < 0.05）；而 Foxp3 和 IL-10 水平的 mRNA 和蛋白表达水平明显降低（均 P < 0.05）。与模型组相比，miR-195 OE 组和 Si-CD40 组上述参数明显降低（均 P < 0.05）。此外，抗 miR-195 组可加重 NAFLD 大鼠 Th17/Treg 细胞失衡，并促进炎症反应。与抗 miR-195 组相比，抗 miR-195+Si-CD40 组联合治疗可部分避免 Th17/Treg 细胞失衡，抑制炎症反应。
过表达 miR-195 可通过抑制 CD40 的表达降低 NAFLD 大鼠 Th17/Treg 比值，维持 Th17/Treg 平衡。
