过表达miR-449a对溃疡性结肠炎大鼠肠黏膜的保护作用及机制研究

Protective effect and mechanism of overexpression of miR-449a on intestinal mucosa of rats with ulcerative colitis

目的探究过表达miR-449a对溃疡性结肠炎大鼠肠黏膜的保护作用及机制。方法选取40只SD健康雄性大鼠,将其中10只作为对照组,其余30只建立溃疡性结肠炎大鼠模型后分为模型组、抑制miR-449a组、过表达miR-449a组。抑制miR-449a组大鼠尾部静脉注射30 mg/kg antagomiR-449a进行干预,过表达miR-449a组大鼠尾部静脉注射30 mg/kg agomiR-449a进行干预,对照组及模型组大鼠给予等量生理盐水干预。采用酶联免疫吸附法检测白细胞介素-8(IL-8)、白细胞介素-10(IL-10)、白细胞介素-17(IL-17)、肿瘤坏死因子(TNF-α);免疫透射比浊法检测免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白G(IgG)、环氧化酶-2(COX-2);采用免疫组织化学染色法检测紧密连接蛋白Claudin-1、Toll样受体4(TLR4);采用Western blot法检测p-p38、过氧化酶体增殖物激活受体γ(PPARγ)、核因子κB(NF-κB)蛋白表达。结果与对照组相比,模型组大鼠IL-8、IL-17、IgA、IgM、IgG、TNF-α、COX-2、TLR4较高,IL-10、Claudin-1较低,差异具有统计学意义(P<0.05);与模型组相比,抑制miR-449a组大鼠IL-8、IL-17、TNF-α、COX-2、TLR4下降,IL-10、IgA、IgM、IgG、Claudin-1上升,差异具有统计学意义(P<0.05);与抑制miR-449a组相比,过表达miR-449a组大鼠IL-8、IL-17、IgA、IgM、IgG、TNF-α、COX-2、TLR4较低,IL-10、Claudin-1较高,差异具有统计学意义(P<0.05)。与对照组相比,模型组大鼠PPARγ下降,p-p38、NF-κB上升,差异具有统计学意义(P<0.05);与模型组相比,抑制miR-449a组大鼠PPARγ上升,p-p38、NF-κB下降,差异具有统计学意义(P<0.05);与抑制miR-449a组相比,过表达miR-449a组大鼠PPARγ上升,p-p38、NF-κB下降,差异具有统计学意义(P<0.05)。结论采用过表达miR-449a对溃疡性结肠炎大鼠肠黏膜的损伤进行修补,能够有效改善其肠黏膜的破损,激活PPARγ,抑制p38/NF-κB通路,效果显著。

Objective To explore the protective effect and mechanism of overexpression of miR-449a on intestinal mucosa in rats with ulcerative colitis.Methods Forty healthy male SD rats were selected,10 of which were used as the control group,and the other 30 rats were divided into the model group,miR-449a inhibition group,and miR-449a overexpression group after establishing ulcerative colitis rat model.Rats in the miR-449a inhibition group were injected with 30 mg/kg antagomiR-449a in the tail vein for intervention,rats in the miR-449a overexpression group were injected with 30 mg/kg agomiR-449a for intervention,and rats in the control group and the model group were given the same amount of normal saline for intervention.Interleukin-8(IL-8),interleukin-10(IL-10),interleukin-17(IL-17)and tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay.Immunoglobulin A(IgA),immunoglobulin M(IgM),immunoglobulin G(IgG)and cyclooxygenase-2(COX-2)were detected by immunity transmission turbidity.Tight junction protein Claudin-1 and toll-like receptor 4(TLR4)were detected by immunohistochemical staining.The expressions of p-p38,peroxisome proliferator activated receptorγ(PPARγ)and nuclear factorκB(NF-κB)protein were detected by Western blot.Results Compared with the control group,the levels of IL-8,IL-17,IgA,IgM,IgG,TNF-α,COX-2 and TLR4 in the model group were higher,while the levels of IL-10 and Claudin-1 were lower,with statistically significant differences(P<0.05).Compared with the model group,the levels of IL-8,IL-17,TNF-α,COX-2 and TLR4 in the miR-449a inhibition group were decreased,while the levels of IL-10,IgA,IgM,IgG and Claudin-1 were increased,with statistically significant differences(P<0.05).Compared with the miR-449a inhibition group,the levels of IL-8,IL-17,IgA,IgM,IgG,TNF-α,COX-2 and TLR4 in the miR-449a overexpression group were lower,and the levels of IL-10 and Claudin-1 were higher,with statistically significant differences(P<0.05).Compared with the control group,the level of PPARγin the model group was decreased,while the levels of p-p38 and NF-κB were increased,with statistically significant differences(P<0.05).Compared with the model group,the level of PPARγwas increased and the levels of p-p38 and NF-κB were decreased in the miR-449a inhibition group,with statistically significant differences(P<0.05).Compared with the miR-449a inhibition group,the level of PPARγwas increased and the levels of p-p38 and NF-κB were decreased in the miR-449a overexpression group,with statistically significant differences(P<0.05).Conclusion Overexpression of miR-449a can repair the damage of intestinal mucosa in rats with ulcerative colitis,activate PPARγand inhibit p38/NF-κB pathway with significant effects.