Astragaloside Ⅳ Inhibits NLRP3 Inflammasome-mediated Pyroptosis Via Activation Of Nrf-2/HO-1 Pathway And Protects Against Doxorubicin-induced Cardiac Dysfunction

Objective: Doxorubicin(DOX)is an effective antitumor drug that is used to treat a variety of cancers.However,the side effects of DOX,especially its dose-dependent cardiotoxicity,can lead to heart failure,which greatly limits its clinical application.Therefore,it is urgent to explore drugs against the cardiotoxicity of DOX.Astragaloside IV(AS-IV)is an important component of Astragalus membranaceus,which exerts cardioprotective effects through various pathways.In this experiment,we investigated the myocardial pyroptosis induced by DOX in mice,and explored the protective effect and potential molecular mechanism of ASIV to provide a theoretical basis for clinical mitigation of DOX cardiotoxicity.Methods: Forty healthy male C57BL/6 mice were divided into 4groups randomly.Myocardial injury model was established by intraperitoneal injection of DOX,and AS-IV was administered intragastric treatment.Left ventricular internal dimension at end-diastole(LVIDd)and left ventricular internal dimension at end-systole(LVIDs)were measured by echocardiography,and left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were calculated.Enzyme linked immunosorbent assay(ELISA)was used to detect creatine kinase isoenzyme(CK-MB),brain natriuretic peptide(BNP),lactate dehydrogenase(LDH)and cardiac troponin I(c Tn I),the indicators of myocardial injury.The morphological changes of myocardial tissue were observed by hematoxylin-eosin(HE)staining,wheat germ agglutinin(WGA)and masson trichrome(Masson).The arrangement of cardiomyocytes and the structure of mitochondria were observed by transmission electron microscope(TEM).Transcriptome sequencing and GO enrichment analysis were performed to screen for differential gene expression.ELISA was performed to detect the serum inflammatory factors cytokines interleukin 1β(IL-1β),IL-18 and oxidative stress index glutathione(GSH),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA.Immunohistochemistry(IHC)and Western blot(WB)were used to detect the expression of pyroptosis related protein apoptosis-associated speck-like protein containing a CARD(ASC),NOD-like receptor protein 3(NLRP3),cysteine aspartate protease 1(Caspase-1),Gasdermin D(GSDMD),Cleaved caspase-1,GSDMD-N,IL-1β,IL-18,Cleaved IL-1β,cleaved IL-18 and oxidative stress related proteins nuclear factor E2-related factor 2(Nrf-2),p Nrf-2 and heme oxygenase 1(HO-1).Results:1.Compared with the control group,cardiac dysfunctions were found post DOX injections,as evideced by increased LVIDd and LVIDs,decreased LVEF and LVFS,and increased serum levels of BNP,CK-MB,c Tn I and LDH.As compared with the model group,AS-IV significantly decreased LVIDd,LVIDs and serum levels of BNP,CK-MB,c Tn I and LDH,improved LVEF and LVFS(P<0.05).2.Compared with the control group,the heart weight/body weight(HW/BW)ratio decreased in DOX group.HE staining showed that myocardial tissue was disordered and the cytoplasmic vacuoles increased.After treatment with AS-IV,myocardial tissue was neatly arranged,cytoplasmic vacuolization decreased and HW/BW increased.Masson staining demonstrated a significant increase in myocardial fibrosis area in DOX group,and WGA showed a decrease in myocardial cross-sectional area in DOX group.After treatment with AS-IV,the degree of myocardial fibrosis and the area of cardiomyocytes were improved.TEM results revealed that a large number of mitochondria were swollen and vacuolated in DOX group,the mitochondrial ridge dissolved and disappeared,the Z line was blurred and widened,the H band disappeared,and the muscle filaments arranged disorderly.After treatment with AS-IV,the mitochondrial swelling was relieved,a small amount of mitochondrial spinal lines were dissolved,and the muscle filaments were arranged more neatly than those in the DOX group(P<0.05).3.Transcriptome sequencing indicated that DOX upregulated the gene expression of pyroptosis and oxidative stress,while AS-IV down-regulated the expression of genes related to oxidative stress response.4.Compared with the control group,the levels of serum inflammatory factors IL-1β and IL-18 in DOX group increased,and the levels of SOD and GSH decreased and MDA increased.After treatment with AS-IV,the contents of IL-1β and IL-18 decreased,while the levels of SOD and GSH increased and MDA decreased(P<0.05).5.Immunohistochemical results showed that the expression of caspase-1,GSDMD and NLRP3 in myocardial tissue of DOX group increased,and their expression decreased after AS-IV treatment(P<0.05).6.WB showed that the expression of myocardial pyroptosis-related proteins NLRP3,ASC,caspase-1,Cleaved caspase-1,GSDMD,GSDMDN,IL-1β,cleaved IL-1β,IL-18 and cleaved IL-18 in DOX group increased significantly.After AS-IV treatment,the expression of pyroptosis related proteins decreased(P<0.05).7.WB demonstrated that the expression of Nrf-2,p Nrf-2 and HO-1 in myocardium of DOX group decreased,and the expression of Nrf-2,p Nrf-2 and HO-1 increased after treatment with AS-IV(P<0.05).Conclusion:1.AS-IV alleviates DOX-induced cardiac dysfunction and myocardial injuries.2.AS-IV inhibits DOX-induced NLRP3 inflammasome-mediated pyroptosis via activation of Nrf-2/HO-1 signaling pathway.