Astragalosides Ⅳ Alleviates Doxorubicin-Induced Cardiomyocytes Damages Via Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis

Objective: Doxorubicin(DOX)is an effective and widely used antitumor drug,but its severe cardiotoxicity may cause cardiac dysfunction in patients,which is the limitation of the application of this drug.Astragalosides IV(ASIV)is the main active ingredient of Astragalus membranaceus,and ASIV can exert cardioprotective effects through various mechanisms.In this research,we established a DOX myocardial injury model at the cytological level to investigate the role of ASIV on DOX-induced myocardial damage in pyroptosis and the molecular mechanismMethods:(1)Different concentrations of DOX and ASIV were used to intervene in H9c2 cells,and cell counting kit 8 was used to detect cardiomyocyte viability and select the appropriate concentration and time of drug intervention.(2)There are four groups in this experiment: control group,ASIV group(100 μmol/L ASIV),DOX group(1 μmol/L DOX)and DOX+ASIV group(1 μmol/L DOX+100 μmol/L ASIV).Cell morphology was examined under light microscope,lactate dehydrogenase(LDH)was measured by enzyme linked immunosorbent assay(ELISA),reactive oxygen species(ROS)fluorescence intensity was observed by fluorescence inverted microscope,malondialdehyde(MDA)level was measured;cell sub-microscopic structure was studied under electron microscope;adenosine-triphosphate(ATP)concentration was measured;interleukin-1β(IL-1β)and interleukin-18(IL-18)levels were measured by ELISA;real-time fluorescence quantitative polymerase chain reaction(RT-q PCR)was used to determine the m RNA expression of NOD-like receptor heat protein domain-associated protein 3(NLRP3),apoptosisassociated spot-like protein(ASC),cysteine aspartate protein hydrolase 1(caspase-1),and Gasdermin D(GSDMD);immuno-fluorescence analysis was used to observe the fluorescence expression of NLRP3 and GSDMD.The expression levels of proteins(NLRP3,ASC,cleaved caspase-1,GSDMD-N,cleaved IL-1β and cleaved IL-18)were detected by western blotting(WB).(3)The NLRP3 agonist Nigericin was administered.The groups were: Control group,DOX group,DOX+ASIV group and DOX+ASIV+Nigericin group.IL-1β and IL-18 levels were measured by ELISA and the expression levels of proteins related to the classical pathway of pyroptosis were measured by WB.Results:(1)DOX decreased the relative activity of H9c2cardiomyocytes(P<0.05),and ASIV had no significant effect on the relative activity of H9c2 cardiomyocytes(P>0.05).DOX 1μmol/L,24 h,ASIV 100μmol/L was selected as the intervention condition.(2)Compared with the control group,cell damage occurred in H9c2 cells in the DOX group,which could be observed under light microscopy,and LDH levels were significantly increased(P<0.01),and ASIV could inhibit the increase in LDH levels(P<0.05).(3)DOX significantly increased ROS and MDA levels in H9c2 cells(P<0.05),and ASIV significantly decreased ROS and MDA levels in cardiomyocytes compared with the DOX group(P<0.05).(4)DOX inhibited energy metabolism in cardiomyocytes,and energy metabolism was restored in the DOX+ASIV group.Mitochondrial swelling and partial loss of mitochondrial cristae were observed in H9c2 cells under electron microscopy in the DOX group,and mitochondrial morphology was better in the DOX+ASIV group compared to the DOX group.DOX caused a decrease in cardiomyocyte ATP levels compared to the control group(P<0.01),and ASIV restored cardiomyocyte ATP levels after DOX intervention(P<0.05).(5)The levels of IL-1β and IL-18 were increased by ELISA(P<0.01)and significantly decreased after ASIV treatment(P<0.05).The partial disappearance of cell membrane and detachment of cytoplasm from cytosol in DOX group could be observed under electron microscope,and ASIV restored the above changes to some extent.Compared with the control group,the levels of m RNA(NLRP3,ASC,caspase-1,GSDMD),NLRP3,GSDMD fluorescence intensity and(NLRP3,ASC,cleaved caspase-1,GSDMD-N,cleaved IL-1β,cleaved IL-18 protein levels)were increased in the DOX group(P<0.05),and the above changes were suppressed after ASIV treatment(P<0.05).(6)Compared to the treatment group,NLRP3 agonist attenuated the protective effect of ASIV on DOX-induced H9c2 myocardial injury,and increased IL-1β and IL-18 levels(P<0.05)and expression of proteins related to the classical pathway of pyroptosis(P<0.05).Conclusion:(1)DOX induces cardiomyocyte damages via the NLRP3 Inflammasome-mediated pyroptosis classical pathway.(2)ASIV inhibition of NLRP3 Inflammasome-mediated pyroptosis alleviates DOX-induced cardiomyocyte damages.