大黄素调控树突状细胞Tsc1/mTORC1通路对Th1/Th2细胞极化治疗脓毒症的影响

Efficacy of emodin for treatment of sepsis in mice and its relation with dendritic cell Tsc1/mTORC1 pathway and Th1/Th2 cell polarization

目的探讨大黄素(Emo)调控树突状细胞结节性硬化症复合体1(Tsc1)/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)通路对Th1/Th2细胞极化治疗脓毒症的影响。方法将小鼠分对照组、模型组(采用盲肠结扎和穿刺法构建小鼠脓毒症模型)、模型+Emo组;采用脂多糖(LPS)构建树突状细胞(通过小鼠股骨骨髓获取)脓毒症模型,分为对照组、LPS组、LPS+Emo组;采用Transwell构建树突状细胞与CD4^(+)T细胞(通过腹腔灌洗液获取)共培养体系,分为LPS组、LPS+Emo组、LPS+Emo+sh-NC组、LPS+Emo+sh-Tsc1组细胞;采用HE染色观察各组小鼠心、肺、肝组织损伤情况;采用流式细胞术分离CD4^(+)T细胞,以及检测Th1和Th2细胞分化情况;采用ELISA法检测小鼠血清中TNF-α和IL-1β的表达情况以及小鼠脾脏和树突状细胞培养上清液中IL-12和IL-4的表达情况;采用Western blot法检测各组小鼠脾脏和各组树突状细胞中Tsc1和mTORC1信号通路关键蛋白(S6、pS6)的表达情况。结果小鼠体内模型中,与对照组相比,模型组小鼠心、肝、肺显著损伤,脾脏中TNF-α和IL-1β表达显著升高,Th1/Th2细胞比例显著升高,Tsc1蛋白表达显著降低,S6蛋白磷酸化水平显著增高;与模型组相比,模型+Emo组小鼠心、肝、肺组织损伤部分恢复,脾脏中TNF-α和IL-1β表达显著降低,Th1/Th2细胞比例显著降低,Tsc1蛋白表达显著升高,S6蛋白磷酸化水平显著降低。体外树突状细胞模型中,与LPS组相比,LPS+Emo组细胞培养上清液中Tsc1蛋白表达升高,pS6蛋白表达降低,IL-12和IL-4表达显著降低,Th1/Th2细胞比例显著降低;抑制Tcs1表达后,与LPS+Emo+sh-NC组相比,LPS+Emo+sh-Tsc1组细胞培养上清液中IL-12和IL-4表达显著升高,Tsc1蛋白表达降低,pS6蛋白表达升高,Th1/Th2细胞比例显著升高。结论Emo对脓毒症有明显的治疗作用,其作用机制可能为通过调控树突状细胞中Tsc1/mTORC1通路,影响树突状细胞分泌T细胞极化因子IL-12和IL-4,从而影响Th1/Th2细胞极化以治疗脓毒症。

Objective To investigate the efficacy of emodin(Emo)for treatmetn of sepsis in mice and its relation with dendritic cell tuberous sclerosis complex 1(Tsc1)/mechanistic target of rapamycin complex 1(mTORC1)pathway and Th1/Th2 cell polarization.Methods The mouse sepsis model was constructed by cecal ligation and puncture,and mice were divided into control group,model group and model+Emo group.The dendritic cells(DCs)were obtained from mouse femoral bone marrow and treated with lipopolysaccharide(LPS)to construct DC sepsis model,the DCs were divided into control ra-group,LPS group and LPS+Emo group.The CD4^(+)T cells were obtained from peritoneal lavage fluid,and co-cultured with DCs,the co-cultured cells divided into LPS group,LPS+Emo+sh-NC group,LPS+Emo+sh-Tsc1 group.The pathological changes of heart,lung and liver were observed with hematoxylin-eosin staining(HE),CD4^(+)T cells were isolated and Th1/Th2 cell differentiation was detected with flow cytometry.The expressions of TNFαand IL-1βin serum and IL-12 and IL-4 in spleen and DC culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).The expression of Tsc1,anti-S6 and anti-PS6 proteins in spleen and DCs were detected with Western blot(WB).Results Compared with the control group,the heart,liver and lung in sepsis model mice showed significant damages,the expression of TNF-αand IL-1βin spleen was significantly increased,the proportion of Th1/Th2 cells was significantly increased,the expression of Tsc1 protein was significantly decreased,and the phosphorylation level of S6 protein was significantly increased.Compared with model group,the damage of heart,liver and lung tissues in model+Emo group was attenuated,the expression of TNFαand IL-1βin spleen was significantly decreased,the proportion of Th1/Th2 cells was significantly reduced,the expression of Tsc1 protein was significantly increased,and the phosphorylation of S6 protein was significantly decreased.In vitro DC model,compared with LPS group the expression of IL-12,IL-4,Tsc1 and pS6 protein,and Th1/Th2 ratio were significantly decreased in LPS+Emo group.After inhibiting the expression of Tcs1,IL 12 and IL 4 expressions were significantly increased,Tsc1 protein expression was decreased,pS6 protein expression was increased,and Th1/Th2 cell ratio was significantly increased in LPS+Emo+sh-Tsc1 group compared with LPS+Emo+sh-NC group.Conclusion Emo has significant efficacy for sepsis in mice,which may be associated with regulation of Tsc1/mTORC1 pathway in dendritic cells,affecting the secretion of IL 12 and IL-4 and rusulting to the polarization of Th1/Th2 cells.