安胃汤含药血清调控NLRP3-Caspase-1-IL-1β信号轴促进MC细胞焦亡机制研究

Mechanism of Serum Containing Anwei Decoction(安胃汤)Regulating Pyroptosis of NLRP3-Caspase-1-IL-1βSignal Axis of MC Cells

目的探究安胃汤含药血清对MC(MNNG诱导GES-1恶性转化)细胞NLRP3-Caspase-1-IL-1β信号通路相关因子及细胞焦亡的影响,揭示安胃汤抗慢性萎缩性胃炎(chronic atrophic gastritis,CAG)作用机制。方法将MC细胞分为空白组、安胃汤组、安胃汤+Z-YVVD-FMK(阻断剂)组、Z-YVVD-FMK(阻断剂)组4组;CCK8分别检测12 h、24 h、48 h时间点细胞增殖情况,确定最佳检测时间点及含药血清比例;检测各组细胞上清液乳酸脱氢酶(LDH)活性;AO-EB染色法检测细胞膜的完整性及胞核形态;ELISA检测血清白介素-18(interleukin-18,IL-18)、白介素-1β(interleukin-1β,IL-1β)水平;Real-time PCR检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体(NACHT-LRR-PYD-containing proteins 3 inflammasome,NLRP3)、半胱氨酸天冬氨酸蛋白水解酶-1(cysteine-requiring aspartate protease-1,Caspase-1)、消皮素D(gasdermin D,GSDMD)、IL-18、IL-1β基因的表达;Western-blot检测NLRP3、Caspase-1、GSDMD蛋白的表达。结果选用48 h时间点、10%含药血清比例加入后续实验;与其余3组比较,安胃汤组LDH活性增高(P<0.01);给药组AO-EB染色均可见胞膜肿胀变形以及细胞核形态改变;ELISA结果提示:与空白组比较,安胃汤组IL-18、IL-1β均见升高(P<0.01),Z-YVVD-FMK组IL-18、IL-1β均见下降(P<0.01),差异具有统计学意义;Real-time PCR检测提示:与空白组比较,安胃汤组NLRP3 mRNA、Caspase-1 mRNA、GSDMD mRNA、IL-18 mRNA、IL-1βmRNA表达水平均见升高(P<0.01,P<0.05),Z-YVVD-FMK组GSDMD mRNA、IL-18 mRNA、IL-1βmRNA表达水平均见降低(P<0.01),差异具有统计学意义;Western-blot结果提示:与空白组比较,安胃汤组NLRP3/Actin、GSDMD/Actin表达上升(P<0.01),Z-YVVD-FMK组Caspase-1/Actin表达量减少(P<0.01)。结论安胃汤抗CAG可能与其通过调控NLRP3-Caspase-1-IL-1β信号转导通路诱导胃黏膜细胞焦亡逆转CAG病理状态有关。

Objective To investigate the effects of Anwei Decoction(安胃汤)on NACHT-LRR-PYD-containing proteins 3 inflammasome(NLRP3)-cysteine-requiring aspartate protease-1(Caspase-1)-interleukin-1β(IL-1β)signaling pathway related factors and pyroptosis in mast cells(MC)cells,and reveal the anti-chronic atrophic gastritis(CAG)mechanism of Anwei Decoction.Methods MC cells were divided into 4 groups:blank group,Anwei Decoction group,Anwei Decoction+Z-YVVD-FMK group and Z-YVVD-FMK group.CCK8 was used to detect cell proliferation at 12 h,24 h and 48 h and to de-termine the optimal detection time point and the proportion of drug-containing serum.The lactate dehydrogenase(LDH)activity in cell supernatant of each group was detected.AO-EB staining was used to detect the integrity of cell membrane and nuclear morphology.The levels of serum interleukin-18(IL-18)and IL-1βwere detected by ELISA.Real-time PCR was used to detect the expressions of NLRP3,Caspase-1,gasdermin D(GSDMD),IL-18 and IL-1βgenes.Western-blot was used to detect the expressions of NLRP3,Caspase-1 and GSDMD proteins.Results The 48 h time point and 10%drug-containing ser-um ratio were chosen to the follow-up experiment.Compared with that of the other three groups,the activity of LDH in the An-wei Decoction group was increased(P<001).AO-EB staining in the administration groups showed swelling and deformation of the cell membrane and changes in nuclear morphology.The results of ELISA showed that compared with those of the blank group,the levels of IL-18 and IL-1βin the Anwei Decoction group were increased(P<001),and the levels of IL-18 and IL-1βin the Z-YVVD-FMK group were decreased(P<001),and the difference was statistically significant.Real-time PCR showed that compared with those of the blank group,the expression levels of NLRP3mRNA,Caspase-1mRNA,GSDMDmR-NA,IL-18mRNA and IL-1βmRNA in the Anwei Decoction group were increased(P<001,P<0.05).The expression levels of GSDMDmRNA,IL-18mRNA and IL-1βmRNA in the blocker group were decreased(P<001),and the difference was sta-tistically significant.The results of Western-blot showed that compared with those of the blank group,the expressions of NLRP3/Actin and GSDMD/Actin in the Anwei Decoction group decreased(P<001),and the expression of Caspase-1/Actin in the Z-YVVD-FMK group decreased(P<001).Conclusion The anti-CAG effect of Anwei Decoction may be related to the re-versal of the pathological state of gastric mucosal cells induced by pyroptosis by regulating NLRP3-Caspase-1-IL-1βsignal transduction pathway.