二黄新伤止痛软膏治疗急性软组织损伤的作用机制研究

Role of NLRP3 Inflammatory Vesicle Activation-Mediated Cell Pyroptosis in Acute Soft Tissue Injury and the Antagonistic Efficacy of Erhuangxinshang Analgesic Ointment

目的:探究二黄新伤止痛软膏治疗急性软组织损伤对NOD样受体蛋白3(NLRP3)炎性小体活化介导的细胞焦亡的抑制作用。方法:将SD大鼠随机分为正常组、模型组、软膏治疗组及Caspase-1抑制剂处理组,除正常组以外建立急性软组织损伤模型。各组大鼠在治疗期间进行软组织损伤评分记录,苏木精-伊红(HE)染色观察大鼠软组织损伤,TUNEL染色观察大鼠软组织细胞焦亡情况,扫描电子显微镜观察大鼠损伤组织超微结构,Western Blot法检测细胞焦亡相关因子蛋白表达水平,ELISA法检测大鼠血清中IL-1β和IL-18的含量。结果:与模型组对比,经二黄新伤止痛软膏治疗或Caspase-1抑制剂处理的大鼠,软组织损伤评分逐日降低,肌肉组织病理变化得到改善,软组织细胞焦亡率显著降低,差异有统计学意义(P<0.05)。软组织内肌纤维排列由紊乱溶解趋于整齐清晰,NLRP3及GSDMD-N蛋白表达水平显著降低,Caspase-1及Cleaved Caspase-1蛋白表达有一定减少,差异有统计学意义(P<0.05)。血清中IL-1β及IL-18含量显著减少,差异有统计学意义(P<0.05)。结论:二黄新伤止痛软膏可以抑制NLRP3炎性小体活化介导的细胞焦亡,从而起到对急性软组织损伤的治疗作用。

Objective:To investigate the inhibitory efficacy of Erhuangxinshang analgesic ointment on NOD-like receptor family,Pyrin domain containing 3(NLRP3)inflammatory vesicle activation-mediated inflammatory response and pyroptosis on the treatment of acute soft tissue injury.Methods:SD rats were randomly divided into normal group,model group,ointment treatment group and Caspase-1 inhibitor treatment group,and acute soft tissue injury model was established in addition to normal group.The rats in each group were scored for soft tissue injury during the treatment.Rat soft tissues were stained with Hematoxylin-eosin(HE)to observe pathological changes.Rat soft tissue cells were stained with TUNEL staining to observe pyroptosis.The ultrastructure of injured tissues was observed by scanning electron microscope(SEM).Western Blot was used to detect the protein expression level of cell death related factors.The content of IL-1β and IL-18 in rat serum was detected by ELISA.Results:In comparison with the model group,the soft tissue injury scores decreased day by day in rats treated with Erhuangxinshang analgesic ointment or Caspase-1 inhibitor.The pathological changes of muscle tissue were improved.The pyroptosis rate of soft tissue cells was significantly reduced.The arrangement of the muscle fibres in the soft tissue tends to dissolve from disorganised to neat and clear.There was a significant decrease in the expression levels of NLRP3 and GSDMD-N proteins,and the expression of Caspase-1 and Cleaved Caspase-1 protein decreased to some extent,signifi-cant reduction in serum levels of IL-1β and IL-18.Conclusion:Erhuangxinshang analgesic ointment can inhibit the inflammatory reaction and pyroptosis mediated by the activation of NLRP3 inflammatory bodies,thus achieving the antagonistic efficacy on acute soft tissue injury.