基于ROS介导NLRP3炎症小体通路探讨蠲痛饮对子宫内膜异位症大鼠自噬的作用机制

Exploring the mechanism of action of Juantong Drink on autophagy in rats with endome-triosis based on ROS-mediated NLRP3 inflammatory vesicle pathway

目的基于ROS介导NLRP3炎性小体通路探讨蠲痛饮对子宫内膜异位症自噬的作用机制。方法建立EMs大鼠模型,随机将72只SD大鼠分为假手术组、模型组、蠲痛饮(高、中、低)剂量组、地诺孕素组、氧化应激组,分别给予蠲痛饮高、中、低剂量(25.4,12.7,6.35g·kg^(-1))灌胃,地诺孕素组(0.21mg/kg)灌胃,氧化应激组,模型组和假手术组给予生理盐水(1ml/100g)灌胃,于取材前八天,氧化应激组通过腹腔注射10mg/kg BW Diquat建立氧化应激模型,对照组则注射等量生理盐水,之后饲养给药如常。各组大鼠连续用药28d。对各组大鼠光镜下异位内膜病理改变进行检测,DCFH-DA荧光探针检测内膜组织活性氧(ROS)水平,电镜观察内膜组织自噬小体数量;荧光定量PCR、免疫组化、蛋白免疫印迹法(Western blot)检测NLRP3、Caspase-1、LC3II、Beclin-1的mRNA及蛋白表达;酶联免疫吸附法(ELISA)检测血清中TNF-α的表达情况。结果与假手术组比较,模型组异位内膜ROS水平、NLRP3、Caspase-1蛋白及mRNA表达显著上调,Beclin-1、LC3B表达与自噬小体数量显著降低(P<0.05),血清TNF-α含量显著升高(P<0.05);与模型组对比,蠲痛饮高剂量组异位内膜ROS含量显著下调(P<0.05),自噬小体数量增加(P<0.05),蠲痛饮(高、中剂量组)、地诺孕素组NLRP3、Caspase-1蛋白及mRNA表达显著下调(P<0.05),血清TNF-α含量显著下调(P<0.05),蠲痛饮(高、中剂量组)、地诺孕素组LC3B、Beclin-1蛋白及mRNA表达均升高(P<0.05),其中蠲痛饮高剂量组升高最为显著(P<0.05),异位内膜组织病理形态明显改善。结论蠲痛饮能抑制异位内膜组织氧化应激反应,调控NLRP3炎性小体通路,激活自噬水平,促使异位内膜细胞凋亡,达到治疗EMs的目的。

Objective To investigate the mechanism of Juantong Drink on autophagy in endometriosis rats based on ROS-media-ted NLRP3 inflammatory vesicle pathway.Methods Establishment of the EMS rat model,and 72 SD rats were randomly divided into the sham operation group,model group,Juantong Drink(high,medium and low)dose group,Dinorelgestrol group,oxida-tive stress group and oxidative stress+high dose group,and the oxidative stress and oxidative stress+high dose groups were injec-ted intraperitoneally with 10mg/kg body The oxidative stress and oxidative stress+high dose groups were injected intraperitoneal-ly with 10mg/kg body weight Diquat to establish the oxidative stress model,while the control group was injected with an equal a-mount of saline,and then fed as usual.Each group was administered the drug continuously for 28 d.The pathological changes of ectopic endothelium in each group were examined by light microscopy,DCFH-DA fluorescent probe to detect the level of reactive oxygen species(ROS)in endothelial tissues;Fluorescent PCR,immunohistochemistry,and protein immunoblotting(Western blot)to detect the proteins and proteins of NLRP3,Caspase-1,and LC3II,Beclin-1 protein and mRNA expression;enzyme-linked immunosorbent assay(ELISA)to detect the expression of TNF-αin serum.Results Compared with the sham-operated group,the ectopic endothelial ROS level,NLRP3,Caspase-1 protein and mRNA expression were significantly up-regulated in the model group,and the expression of Beclin-1,LC3Bwere significantly reduced(P<0.05),and serum TNF-αcontent was significantly increased(P<0.05);compared with the model group,ectopic endothelial ROS content was significantly down-regulated in the Juantong Drink high-dose group and high-dose+oxidative stress group(P<0.05),NLRP3,Caspase-1 protein and mRNA expression was significantly down-regulated in the dienogest group(P<0.05),serum TNF-αcontent was significantly down-regulated(P<0.05),and the expression of LC3B,Beclin-1 protein and mRNA was elevated in the Juan-tong Drink(high and medium dose groups),dienogest group(The expression of LC3B,Beclin-1 protein and mRNA were all increased in Juantong Drink(high and medium dose groups)and Dinorelgestrol group(P<0.05),and the histopathological morphology of ectopic endothelium was significantly improved in Juantong Drink(high dose group).Conclusion Juantong Drink can inhibit oxidative stress in ectopic endothelial tissue,regulate NLRP3 inflammatory vesicle pathway,activate autophagy level,and promote apoptosis of ectopic endothelial cells to achieve the purpose of treating EMs.