消退素D2对CVB3诱导的病毒性心肌炎小鼠炎症反应和内质网应激损伤的保护作用及机制研究

Protective effect of resolvin D2 on inflammatory response and endoplasmic reticulum stress injury of CVB3-induced viral myocarditis mice and its mechanism

目的明确消退素D2(resolvin D2,RvD2)在病毒性心肌炎小鼠体内发挥的抗炎和抗内质网应激作用并初步探究其作用机制。方法采购50只雄性BALB/c小鼠并给予相应编号,通过随机数表法抽取40只雄性BALB/c小鼠,每组各10只。分别设为正常对照组、RvD2对照组、病毒性心肌炎组和RvD2治疗组。RvD2对照组小鼠给予连续腹腔注射RvD2治疗7 d;病毒性心肌炎组小鼠给予腹腔注射柯萨奇B3病毒(Coxsackievirus B3,CVB3)构建病毒性心肌炎动物模型;RvD2治疗组小鼠在构建病毒性心肌炎动物模型后给予连续腹腔注射RvD2治疗7 d。7 d后处死各组小鼠并留取心脏组织及血清样本。ELISA检测各组小鼠血清中炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达水平;HE染色检测各组小鼠心肌组织内炎症细胞浸润情况;Western blot检测各组小鼠心肌组织内炎症相关蛋白IL-1β、TNF-α以及内质网应激相关蛋白质葡萄糖调节蛋白78(glucose regulated protein 78 kD,GRP78)、C/EBP同源蛋白(C/EBP-homologous protein,Chop)的表达水平。剩余10只BALB/c小鼠在构建病毒性心肌炎动物模型后给予腹腔注射RvD2和G蛋白偶联受体18(G protein-coupled receptor 18,GPR18)蛋白抑制剂处理,Western blot检测各组小鼠心肌组织内GPR18蛋白、炎症相关蛋白IL-1β、TNF-α以及内质网应激相关蛋白GRP78、Chop的表达水平。结果与正常对照组相比,病毒性心肌炎组小鼠血清中炎症因子IL-1β、TNF-α表达水平显著上调;心肌组织内炎症细胞浸润程度显著增加;心肌组织内炎症相关蛋白IL-1β、TNF-α以及内质网应激相关蛋白GRP78、Chop表达水平显著增加。与病毒性心肌炎组相比,RvD2治疗组小鼠血清炎症因子IL-1β、TNF-α表达水平显著降低;心肌组织内炎症细胞浸润程度显著降低;心肌组织内炎症相关蛋白IL-1β、TNF-α以及内质网应激相关蛋白GRP78、Chop表达水平显著降低。与RvD2治疗组相比,给予病毒性心肌炎小鼠腹腔注射RvD2和GPR18抑制剂处理后小鼠心肌组织内炎症相关蛋白IL-1β、TNF-α以及内质网应激相关蛋白GRP78、Chop表达水平显著增加,而GPR18蛋白表达水平显著降低。结论RvD2可通过结合膜蛋白受体GPR18抑制病毒性心肌炎小鼠炎症反应并改善心肌组织所受的内质网应激损伤,从而在病毒性心肌炎小鼠体内发挥心脏保护作用。

Objective To clarify the anti-inflammatory effects and anti-endoplasmic reticulum stress effects of resolvin D2(RvD2)in viral myocarditis mice and to explore its possible mechanism.Methods Fifty male BALB/c mice were collected and assigned corresponding numbers.Then 40 male BALB/c mice were selected randomly with 10 mice in each group.They were set as normal control group,RvD2 control group,viral myocarditis group and RvD2 treatment group.Afterwards,mice in the RvD2 control group received continuous intraperitoneal injection of RvD2 for 7 days,while mice in the viral myocarditis group received intraperitoneal injection of Coxsackievirus B3 virus(CVB3)in the purpose of constructing an animal model of viral myocarditis.Then,mice in the RvD2 treatment group were given continuous intraperitoneal injection of RvD2 for 7 days.After these 7 days,the mice of each group were sacrificed and their cardiac tissue and serum samples were taken.The expression levels of serum inflammatory factors including IL-1βand TNF-αwere detected by ELISA in each group of mice,and HE staining were used to detect the inflammatory cell infiltration in myocardial tissue of each group.Meanwhile,the expression levels of inflammation-related proteins IL-1β,TNF-αas well as endoplasmic reticulum stress-related proteins like GRP78 and Chop in the myocardial tissue in each group of mice were detected by Western blot experiment.The remaining 10 BALB/c mice were treated with intraperitoneal injection of RvD2 as well as GPR18 protein inhibitors after constructing the animal model of viral myocarditis mentioned above.In the end,the expression levels of GPR18 protein,inflammation-related proteins including IL-1βand TNF-αas well as endoplasmic reticulum stress-related proteins like GRP78 and Chop in the myocardial tissue of each group were detected by Western blot experiments.Results Compared with the normal control group,the expression levels of inflammatory factors IL-1βand TNF-αin the serum of mice with viral myocarditis were significantly increased,and the degree of infiltration of inflammatory cells in myocardial tissue was also significantly increased.Besides,the expression levels of the inflammation-related proteins IL-1β,TNF-αas well as endoplasmic reticulum stress-related proteins including GRP78 and Chop increased largely.While compared with the viral myocarditis group,the expression levels of serum inflammatory factors IL-1βand TNF-αin the mice of the RvD2 treatment group were significantly reduced and the degree of infiltration of inflammatory cells in the cardiac tissue was significantly reduced.Also,the expression levels of inflammation-related proteins IL-1βand TNF-αas well as endoplasmic reticulum stress-related proteins GRP78 and Chop were significantly reduced.After intraperitoneal injection of RvD2 and GPR18 inhibitor,in the mice treated with viral myocarditis,the expression levels of IL-1β,TNF-αand endoplasmic reticulum stress-related proteins like GRP78 and Chop in myocardial tissue of these mice significantly increased when it came to compare with the RvD2 treatment group,while the expression levels of GPR18 protein were significantly reduced.Conclusions RvD2 can inhibit the inflammatory response and endoplasmic reticulum stress injury in mice with viral myocarditis by binding to the membrane protein receptor GPR18,thus exerting a protective effect on heart.