红萸饮对溃疡性结肠炎小鼠NLRP3/Caspase-1/ASC细胞焦亡通路的影响

Effects of Hongyu Decoction on pyroptosis pathway of NLRP3/Caspase-1/ASC cells in mice with ulcerative colitis

目的:探析红萸饮对溃疡性结肠炎(UC)小鼠细胞焦亡通路的调控机制。方法:将40只C57/BL6小鼠随机分为正常组、模型组、红萸饮组(1.5g/kg)和美沙拉秦组(0.50g/kg),采用自由饮用3%葡聚糖硫酸钠溶液1周的方法造模。造模后正常组和模型组予蒸馏水,红萸饮组和美沙拉秦组予相应药物灌胃7d,期间观察小鼠体质量、粪便等一般情况及疾病活动指数评分(DAI);HE染色观察结肠黏膜变化;微板法测定结肠组织中乳酸脱氢酶(LDH)水平;ELISA法检测血清中IL-18和IL-1β的含量;TUNEL荧光法检测结肠组织细胞凋亡水平;免疫组化法检测通路蛋白(ASC、Caspase-1、NLRP3)变化;Western blot法检测焦亡关键蛋白(IL-1β、IL-18、GSDMD)的表达。结果:与正常组比较,造模后动物体质量降低,DAI评分上升,结肠黏膜损伤严重,结肠组织蛋白上清中LDH含量显著升高(P<0.01),血清炎症因子(IL-1β、IL-18)含量均显著增加(P<0.01),TUNEL染色阳性明显,结肠组织焦亡通路蛋白(ASC、Caspase-1、NLRP3)表达量均显著增加(P<0.01),Westernblot法焦亡关键蛋白(IL-1β、IL-18、GSDMD)表达量均显著增加(P<0.01)。与模型组比较,给药组小鼠体质量增加,DAI评分降低,结肠黏膜损伤较轻,结肠组织蛋白上清中LDH含量表达显著降低(P<0.01),血清炎症因子(IL-1β、IL-18)含量表达均显著降低(P<0.01),TUNEL染色阳性减弱,结肠组织焦亡通路蛋白(ASC、Caspase-1、NLRP3)表达量均显著下降(P<0.05,P<0.01),Westernblot法焦亡关键蛋白(IL-18、GSDMD)表达量均下降(P<0.01,P<0.05)。结论:红萸饮对UC小鼠细胞焦亡有抑制作用,其作用机制可能是通过调控NLRP3/Caspase-1/ASC通路实现的。

Objective:To explore the regulatory mechanism of Hongyu Decoction on the scorchification pathway of ulcerative colitis(UC)mice.Methods:Forty C57/BL6 black rats were randomly divided into normal group,model group,Hongyu Decoction group(1.5 g/kg)and Mesalazine group(0.50 g/kg).The rats were modeled by free drinking of 3%sodium glucan sulfate solution for 1 week.After modeling,normal group and model group were given distilled water,and Hongyu Decoction group and Mesalazine group were given corresponding drug intragastric administration for 7 d.Body mass,feces and other general conditions of mice and disease activity index score(DAI)were observed between periods.The changes of colonic mucosa were observed by HE staining.Lactate dehydrogenase(LDH)levels in colon tissues were determined by microplate method.The contents of IL-18 and IL-1βin serum were determined by ELISA.Colon tissue was detected by TUNEL fluorescence assay cell apoptosis level;The changes of pathway proteins(ASC,Caspase-1,NLRP3)were detected by immunohistochemistry.The expressions of key proteins(IL-1β,IL-18,GSDMD)were detected by Western blot.Results:Compared with the normal group,the body weight of the animals was decreased,DAI score was increased,colon mucosa was seriously damaged,LDH content in colon protein superneant was increased(P<0.01),serum inflammatory cytokines(IL-1β,IL-18)were increased(P<0.01),and TUNEL staining was significantly positive.The expressions of focal death pathway proteins(ASC,Caspase-1,NLRP3)in colon tissues were increased(P<0.01),and the expressions of key focal death proteins(IL-1β,IL-18,GSDMD)were increased by Western blot(P<0.01).Compared with model group,the body weight of mice in administration group was increased,DAI score was decreased,colon mucosa was less damaged,LDH content in colon tissue superneant was decreased significantly(P<0.01),serum inflammatory cytokines(IL-1β,IL-18)were decreased(P<0.01),TUNEL staining was decreased.The expressions of focal death pathway proteins(ASC,Caspase-1,NLRP3)in colon tissues were decreased(P<0.05,P<0.01),and the expressions of key focal death proteins(IL-18,GSDMD)were decreased by Western blot(P<0.01,P<0.05).Conclusion:Hongyu Decoction can inhibit the scorch death of UC mice cells,and the mechanism may be realized by regulating the NLRP3/Caspase-1/ASC pathway.