Kuanxiong Aerosol Regulates TLR4/MyD88/NLRP3/Caspase-1 Pathway to Reduce Cardiomyocyte Injury in Myocardial Infarc-tion Rats / 浙江中医药大学学报

ABSTRACT

[Objective]To investigate the effects of Kuanxiong aerosol(KXA)on pyroptosis and inflammatory response in isoproterenol(ISO)-induced myocardial infarction(MI)rats,and its effect on the key pathway of pyroptosis Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/NOD-like receptor pyrin domain containing 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1).[Methods]Thirty male Wistar rats were randomly divided into five groups,6 rats in each group,as control group(0.9％sodium chloride solution),model group(ISO 120 mg·kg-1),isosorbide mononitrate(IMSN)group(ISO 120 mg·kg-1+IMSN 5 mg/kg·d),KXA low dose group(ISO 120 mg·kg-1+KXA 0.1 mL/kg·d),and KXA high dose group(ISO 120 mg·kg-1+KXA 0.3 mL/kg·d),gave continuous intragastric administration for 14 days,and intraperitoneal injection of ISO on the 13th and 14th day.After the last intervention,collected heart tissues and blood under anesthesia.Enzyme-linked immunosorbent assay(ELISA)was performed to investigate the expression of creatine kinase-MB(CK-MB)and cardiac troponin T(cTnT),as well as serum inflammatory indicators such as interleukin-1β(IL-1β),interleukin-18(IL-18),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α).The histopathological changes in heart tissue were evaluated using hematoxylin-eosin(HE)staining,and RNA-sequencing was used to detect the differential expression genes among groups.And the expression of the pyroptosis relevant protein was detected by Western blot.[Results]The results of the ELISA showed that CK-MB and cTnT expression in model group were significantly higher than those in control group(P＜0.01),which meant successful model construction.Pathological staining results showed disordered and fractured muscle fibers were significantly improved after KXA and IMSN intervention.RNA-seq results showed there were 2 646 different genes between model group and control group,while 714 other genes were in KXA and model group.After analyzing these two compared groups,it found that there were 130 up-regulated genes and 7 down-regulated genes;among them,inflammation related TLR4 pathway was significantly enriched.Furthermore,compared with model group,the expression of inflammatory factors IL-1β,IL-18,IL-6 and TNF-α decreased significantly in KXA groups and IMSN group(P＜0.01,P＜0.05),and Western blot showed that the protein expression of TLR4,MyD88,phospho-nuclear factor-KB(p-NF-KB)p65,NLRP3,cleaved cysteinyl aspartate specific proteinase-1(cleaved caspase-1)and Gasdermin D-N(GSDMD-N)increased significantly in model group while significantly down-regulated in KXA groups and IMSN group(P＜0.05,P＜0.01).[Conclusion]KXA can improve myocardial ischemia,reduce cardiac damage,and inhibit cardiomyocyte pyroptosis and inflammatory response,the mechanism may be related to regulating the TLR4/MyD88/NLRP3/caspase-1 signaling pathway.