金汁调控自噬与凋亡修复脓毒症小鼠肠黏膜受损的实验研究

Experimental Study on the Regulation of Autophagy and Apoptosis by Jinzhi in Repairing Intestinal Mucosal Damage in Septic Mice

目的探讨金汁对脓毒症小鼠肠黏膜的保护作用及其机制。方法50只健康的SPF级BALB/c小鼠进行适应性饲养3 d,随后分为对照组(10只)和模型组(40只),模型组采用盲肠结扎穿孔(CLP)法制备脓毒症小鼠模型,制备成功后再随机分为CLP组、CLP+生理盐水(NS)组、CLP+金汁(Jinzhi)组、CLP+金汁(Jinzhi)+NF-κB抑制剂(PDTC)组。CLP+NS组的小鼠每次给予生理盐水灌胃(0.01 mL·g^(-1)),CLP+Jinzhi组每次给予金汁灌胃(0.01 mL·g^(-1)),CLP+Jinzhi+PDTC组每次给予金汁(0.01 mL·g^(-1))灌胃并在造模前2 h腹腔注射抑制剂PDTC(120 mg·g^(-1))1次。模型组小鼠造模结束后分别在12、24、36 h给予上述相关操作。于48 h取各组小鼠小肠组织和血清。HE染色法观察小鼠小肠组织形态学变化;ELISA法检测小鼠血清中的TNF-α、IL-6以及IL-1β的表达水平;RT-qPCR法和Western blot法分别检测小鼠小肠组织中LC3Ⅰ、LC3Ⅱ、Caspase 3以及Caspase 8的mRNA和蛋白的表达情况。结果与对照组比较,CLP组小鼠肠上皮细胞均出现不同程度的损伤,肠黏膜绒毛出现不同程度的肿胀,并可见炎性细胞浸润,血清中TNF-α、IL-6以及IL-1β的表达升高(P均<0.05),小肠组织中LC3Ⅰ、LC3Ⅱ、Caspase 3、Caspase 8的mRNA和蛋白表达增加(P均<0.05)。与CLP组比较,CLP+NS组上述指标差异不大,CLP+Jinzhi组、CLP+Jinzhi+PDTC组上述指标均得到不同程度改善,且CLP+Jinzhi+PDTC组效果更佳。结论金汁可改善脓毒症小鼠肠黏膜损伤,其机制可能是通过抑制自噬相关蛋白LC3Ⅰ、LC3Ⅱ及凋亡相关蛋白Caspase 3、Caspase 8的表达,降低小鼠体内TNF-α、IL-1β、IL-6的水平,抑制机体炎性反应起到治疗作用。

Objective To investigate the protective effect and mechanism of Jinzhi on intestinal mucosa of septic mice by regulating autophagy and apoptosis.Methods Fifty healthy SPF BALB/c mice were adaptively fed for 3 days,and then divided into control group(n=10)and model group(n=40).The sepsis model was established by cecal ligation and puncture(CLP)method in model group.After successful preparation,the mice were randomly divided into CLP group,CLP+normal saline(NS)group,CLP+Jinzhi group,and CLP+Jinzhi+NF-κB inhibitor(PDTC)group.The mice in the CLP+NS group were given normal saline(0.01 mL·g^(-1))by gavage,and the mice in the CLP+Jinzhi group were given Jinzhi(0.01 mL·g^(-1))by gavage.The CLP+Jinzhi+PDTC group was treated with Jinzhi(0.01 mL·g^(-1))and injected with PDTC(120 mg·kg-1).Intragastric administration was performed at 12 h,24 h and 36 h after grouping.Intestinal tissue and serum were collected at 48 h.HE staining was used to observe the morphological changes of the intestine.ELISA was used to measure the levels of TNF-α,IL-6 and IL-1βin serum.The mRNA and protein expressions of LC3Ⅰ,LC3Ⅱ,Caspase 3 and Caspase 8 in the small intestine were detected by RT-qPCR and Western blot,respectively.Results In contrast to the control group,the mice in CLP group had a high number of necrotic intestinal mucosal epithelial cells,varied degrees of intestinal mucosal villi swelling,and a large number of inflammatory cells infiltrating the submucosa.The expression levels of TNF-α,IL-6,and IL-1βin serum(P all<0.05)and the protein and mRNA expression levels of LC3Ⅰ、LC3Ⅱ,Caspase 3,and Caspase 8 in small intestine were significantly increased(P all<0.05).Compared with the CLP group,there was no significant difference in the above indicators in the CLP+NS group,but the above indicators in the CLP+Jinzhi group and the CLP+Jinzhi+PDTC group were significantly improved,and the CLP+Jinzhi+PDTC group had better effects.Conclusion Jinzhi could improve intestinal mucosal injury in septic mice through inhibiting the expression of autophagy-related proteins LC3Ⅰ,LC3Ⅱand apoptosis-related proteins Caspase 3 and Caspase 8,decreasing the levels of TNF-α,IL-1β,and IL-6,and inhibiting the inflammatory response of the organism,thus playing a therapeutic role.