健脾化瘀解毒方调控PKA/NLRP3通路治疗胃癌前病变的机制

Mechanism of Jianpi Huayu Jiedu Formula on alleviating gastric precancerous lesion via regulating PKA/NLRP3 signaling pathway

目的:探讨健脾化瘀解毒方(JPHYJD)调控蛋白激酶A(PKA)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)通路治疗胃癌前病变(GPL)的作用机制。方法:动物实验一:将60只小鼠随机分为空白组,模型组,JPHYJD高、低剂量组,维酶素组,分别进行处理后,HE染色观察胃黏膜病理改变,免疫荧光染色观察NLRP3、GSDMD表达。动物实验二:将60只小鼠随机分为空白组、模型组、H89组、模型+H89组、JPHYJD组、模型+H89+JPHYJD组,Western Blot检测GSDMD-N、Caspase-1、IL-1β表达。细胞实验一:THP-1细胞分为空白组、模型组和JPHYJD组,使用LPS+ATP造模后,JPHYJD组予含药血清处理。细胞实验二:分组同动物实验二,分别予以H89及JPHYJD含药血清处理。检测焦亡相关分子表达和PI染色。结果:动物实验中,与空白组比较,模型组出现GPL病变,NLRP3、GSDMD表达显著升高(P<0.01),JPHYJD可改善胃黏膜病变,显著抑制NLRP3、GSDMD表达(P<0.01,P<0.05);与空白组比较,模型组GSDMD-N、Caspase-1、IL-1β表达显著升高(P<0.01),JPHYJD可抑制其表达(P<0.01),模型+H89+JPHYJD组表达水平显著高于JPHYJD组(P<0.05)。细胞实验中,与空白组比较,模型组NLRP3、GSDMD-N、Caspase-1、IL-1β蛋白和mRNA表达显著升高,焦亡水平显著升高(P<0.01,P<0.05),JPHYJD可显著抑制上述分子表达,减少细胞焦亡(P<0.01);与空白组比较,模型组PKA降低,NLRP3、GSDMD-N、IL-1β升高,焦亡水平升高(P<0.01),JPHYJD可上调PKA,抑制NLRP3、GSDMD-N、IL-1β,减少细胞焦亡(P<0.01,P<0.05);与JPHYJD组比较,模型+H89+JPHYJD组PKA降低,NLRP3、GSDMD-N、IL-1β表达升高,焦亡水平升高(P<0.01)。结论:JPHYJD可通过调控PKA/NLRP3通路抑制细胞焦亡从而治疗GPL。

Objective:To explore the mechanism of Jianpi Huayu Jiedu Formula(JPHYJD)in alleviating gastric precancerous lesion(GPL).Methods:Animal experiment 1:A total of 60 mice were randomly divided into control group,model group,JPHYJD high and low dose groups,and Vitacoenzyme group.After treatment,HE staining was used to observe the pathological changes of gastric mucosa,and immunofluorescence staining was used to observe the expression of NLRP3 and GSDMD:Animal experiment 2:A total of 60 mice were randomly divided into control group,model group,H89 group,model+H89 group,JPHYJD group,and model+H89+JPHYJD group.Western Blot was used to detect the expression of GSDMD-N,Caspase-1,and IL-1β.Cell experiment 1:THP-1 cells were divided into control group,model group and JPHYJD group.After modeling with LPS+ATP,the JPHYJD group was treated with drug containing serum.Cell experiment 2:THP-1 cells were divided into groups the same as animal experiment 2.After treatment with H89 and drug containing serum,the expression of pyroptosis related molecules and PI staining were detected.Results:In vivo,compared with the control group,the model group showed GPL lesions and increased expression of NLRP3 and GSDMD(P<0.01).JPHYJD could improve gastric mucosal lesions and inhibit the expression of NLRP3 and GSDMD(P<0.01,P<0.05).Compared with the control group,the model group showed increased expression of GSDMD-N,Caspase-1,and IL-1β(P<0.01),and JPHYJD could inhibit their expression(P<0.01).The expression level of the model+H89+JPHYJD group was higher than that of the JPHYJD group(P<0.05).In vitro,compared with the control group,the model group showed increased expression of NLRP3,GSDMD-N,Caspase-1,IL-1βprotein and mRNA,as well as elevated levels of pyroptosis(P<0.01,P<0.05).JPHYJD significantly inhibited the expression of these molecules and reduced cell pyroptosis(P<0.01).Compared with the control group,the model group showed a decrease in PKA,an increase in NLRP3,GSDMD-N,IL-1β,and an increase in pyroptosis levels(P<0.01).While JPHYJD upregulated PKA,inhibited NLRP3,GSDMD-N,IL-1β,and reduced cell pyroptosis(P<0.01,P<0.05).Compared with the JPHYJD group,the model+H89+JPHYJD group showed a decrease in PKA,an increase in NLRP3,GSDMD-N,IL-1βexpression,and an increase in pyroptosis(P<0.01).Conclusion:JPHYJD alleviates GPL via regulating PKA/NLRP3 pathway.