鸢尾素调节PD-1/PD-L1通路增强病毒性心肌炎小鼠的抗病毒免疫水平

Iridin regulates PD-1/PD-L1 pathway and enhances antiviral immune level in mice with viral myocarditis

目的:探讨鸢尾素调节程序性死亡因子-1(PD-1)/程序性死亡因子配体-1(PD-L1)通路增强病毒性心肌炎小鼠抗病毒免疫水平的作用。方法:取50只C57BL/6小鼠经腹腔接种柯萨奇病毒B3(CVB3)构建病毒性心肌炎小鼠模型,将建模成功小鼠随机分为模型组、鸢尾素低、中、高剂量组和阳性对照组,另取10只C57BL/6小鼠记为空白对照组。阳性对照组经腹腔注射给予10000 U/g干扰素,鸢尾素低、中、高剂量组经腹腔注射给予30、60、120μg/kg鸢尾素,空白对照组和阳性对照组均给予等量生理盐水,1次/d,共2周。HE染色观察小鼠心肌组织病理改变情况;空斑形成单位(PFU)法检测小鼠心肌组织的病毒滴度;流式细胞术检测小鼠外周血T淋巴细胞亚群(CD4^(+)T、CD8^(+)T、NK细胞)比例和CD4^(+)T/CD8^(+)T;ELISA检测小鼠心肌组织中炎症细胞因子IL-6、IL-17、TNF-α和IFN-γ水平;RT-qPCR检测小鼠T淋巴细胞PD-1及心肌组织PD-L1 mRNA表达;West?ern blot检测小鼠T淋巴细胞PD-1及心肌组织PD-L1蛋白表达。结果:与空白对照组相比,模型组小鼠心肌组织结构改变明显,有大量炎症细胞浸润和成片灶性坏死,心肌组织的病理积分、病毒滴度、炎症细胞因子IL-6、IL-17、TNF-α、IFN-γ水平和PDL1 mRNA及蛋白表达升高,外周血CD8^(+)T淋巴细胞比例及T淋巴细胞PD-1 mRNA及蛋白表达升高,外周血CD4^(+)T淋巴细胞、NK细胞比例及CD4^(+)T/CD8^(+)T降低,差异均有统计学意义(P<0.05);与模型组相比,阳性对照组和鸢尾素低、中、高剂量组心肌组织病变情况均有不同程度减轻,心肌组织的病理积分、病毒滴度、炎症细胞因子IL-6、IL-17、TNF-α、IFN-γ水平和PD-L1mRNA及蛋白表达降低,外周血CD8^(+)T淋巴细胞比例及T淋巴细胞PD-1 mRNA及蛋白表达降低,外周血CD4^(+)T淋巴细胞、NK细胞比例及CD4^(+)T/CD8^(+)T升高,其中鸢尾素作用呈剂量依赖性,差异均有统计学意义(P<0.05)。结论:鸢尾素具有增强病毒性心肌炎小鼠抗病毒免疫水平作用,推测是通过抑制PD-1/PD-L1通路激活,改善小鼠外周血T淋巴细胞亚群比例失调,提高机体免疫功能实现的。

Objective:To investigate the effect of iridin on enhancing antiviral immune effect in mice with viral myocarditis by regulating programmed death factor-1(PD-1)/programmed death factor ligand-l(PD-L1)pathway.Methods:Fifty C57BL/6 mice were inoculated intraperitoneally with Coxsackie virus B3(CVB3)to construct mouse model of viral myocarditis.Successfully modeled mice were randomly divided into model group,iridin low,medium and high doses groups and positive control group.Another 10 C57BL/6 mice were recorded as blank control group.Positive control group was given 10000 U/g interferon by intraperitoneal injection,irisin low,medium and high dosed groups were given 30,60 and 120μg/kg iridin by intraperitoneal injection,and blank control group and positive control group were given the same amount of normal saline once a day for 2 weeks.HE staining was used to observe pathological changes of mouse myocardium.Virus titer in mouse myocardium was detected by plaque forming unit(PFU)method.Proportion of T lymphocyte subsets(CD4^(+)T,CD8^(+)T,NK cells)and CD4^(+)T/CD8^(+)T were detected by flow cytometry.Inflammatory cytokines IL-6,IL-17,TNF-αand IFN-γlevels in mouse myocardium were detected by ELISA.RT-qPCR was used to detect expression of PD-1 mRNA in T lymphocytes and expression of PD-L1 mRNA in myocardium of mouse.Western blot was used to detect expression of PD-1 protein in T lymphocytes and expression of PD-L1 protein in myocardium of mice.Results:Compared with blank control group,myocardial tissue structure of model group changed significantly,with a large number of inflammatory cell infiltration and focal necrosis,pathological score,virus titer,levels of inflammatory cytokines IL-6,IL-17,TNF-αand IFN-γ,and PD-L1 mRNA and protein expressions in myocardial tissue were increased,proportion of peripheral blood CD8^(+)T lymphocytes and expression of PD-1 mRNA and protein in T lymphocytes were increased,while proportion of peripheral blood CD4^(+)T lymphocytes,NK cells and CD4^(+)T/CD8^(+)T were decreased,the differences were statistically significant(P<0.05).Compared with model group,pathological changes of myocardial tissue in positive control group and low,medium and high doses groups of irisin were reduced in varying degrees.Pathological score,virus titer,levels of inflammatory cytokines IL-6,IL-17,TNF-αand IFN-γ,and PD-L1 mRNA and protein expression in myocardial tissue were decreased,the proportion of peripheral blood CD8^(+)T lymphocytes and expression of PD-1 mRNA and protein in T lymphocytes were decreased,while the proportion of peripheral blood CD4^(+)T lymphocytes,NK cells and CD4^(+)T/CD8^(+)T were increased,effect of iridin was as a dose-dependent manner,the differences were statistically significant(P<0.05).Conclusion:Iridin can enhance the antiviral immune effect of viral myocarditis mice.It is speculated that iridin can improve the proportion imbalance of T lymphocyte subsets in peripheral blood of mice and improve the immune function of the body by inhibiting the activation of PD-1/PD-L1 pathway.