Salvianolate Inhibits AFB1-induced H9C2 Cell Injury Through The NLRP3 Inflammasome Signaling Pathway

Objective:On the basis of the previous work,H9C2 cells were selected as the research object,and the model of AFB1-induced oxidative damage of H9C2 cells was established to evaluate the inhibitory effect of salvianolate on AFB1-induced cardiotoxicity in vitro and explored its molecular mechanism.Method:1.The establishment of oxidative damage model:the cck-8 method was used to determine t best inductive conditions of AFB1 so as to establish the cell damage model.2.The design of experimental group:H9C2 cells were randomly divided into 5 groups:control group,AFB1 injury group and AFB1+low,medium and high concentration Sal intervention group.3.The testing indicators of experiments:Observation of cell morphology was completed under inverted microscope;After DCFH-DA staining,intracellular ROS fluorescence was observed under an inverted fluorescence microscope;After JC-1 method staining,the fluorescence of mitochondrial membrane potential was observed using confocal microscope;The levels of antioxidant related proteins such as superoxide dismutase（SOD）and catalase（CAT）,as well as the expression levels of pyroptosis-related proteins such as NLRP3,cleaved-Caspase-1（p20）,GSDMD-FL and GSDMD-N were detected by Western blot.Results:1.The establishment of oxidative damage model:After 36h treatment with AFB1,the relative survival rate of H9C2 cells decreased gradually with the increase of AFB1concentration.After 36 h treatment with 128?mol.L^-1 AFB1,the relative survival rate of H9C2cells was 56.83%.And the cell survival rate was close to 50%,which was suitable for the establishment of AFB1 injury model.2.Determination of salvianolate concentration:According to the results of cck-8experiment,the safe use concentration range of salvianolate was 5-60 mg·L^-1,and the low,medium and high intervention concentrations of salvianolate were 5,20 and 40 mg·L^-1,respectively.3.The cell morphology of each group:H9C2 cells in the control group grew in good condition,with fusiform cells,clearly visible boundaries and firm adherent to the wall.H9C2cells in the AFB1 group grew at a lower density,with large cells exfoliated,and some cells shrank and became round and irregular in shape,with cell debris visible.The growth morphology and state of H9C2 cells were improved in different doses of salvianolate protection group.4.The changes of cell survival rate in each group:The H9C2 cell viability of AFB1 group decreased significantly（p<0.01）compared with that of the control group.After the intervention of high concentration salvianolate,the cell viability of the H9C2 cells was higher than that of the injured group（p<0.05）.5.The level of ROS in H9C2 cells:The AFB1 intervention on H9C2 cells could obviously increase the level of internal ROS（p<0.01）,and the level of intracellular ROS could be decreased by high concentration of salvianolate intervention（p<0.05）.6.Mitochondrial membrane potentials in H9C2 cells of different groups:The ratio of red fluorescence to green fluorescence decreased obviously（p<0.01）in the AFB1 group,indicating that the mitochondrial membrane potential was in a decreasing state.Compared with the AFB1 group,high concentration of salvianolate intervention increased the intracellular red-green fluorescence ratio to a certain extent（p<0.05）,representing an increase in mitochondrial membrane potential.7.Western blot results:The expression of SOD2,CAT protein in AFB1 group was significantly decreased（p<0.01）compared with that in the control group（all p<0.01）.After the intervention of high concentration of salvianolate,CAT protein was obviously up-regulated（p<0.01）,and SOD2 protein expression was also increased to some extent（p<0.05）.8.Western blot results:The expression levels of intracellular NLRP3,p20,GSDMD-FL and GSDMD-N protein were significantly upregulated after AFB1 treatment compared with control group（all p<0.01）.The protein expressions of GSDMD-N and p20 in high concentration salvianolate treatment group were down-regulated（all p<0.05）,and the protein expression of NLRP3 was obviously decreased（p<0.01）.Conclusion:Salvianolate can attenuate AFB1-induced injury in H9C2 cells byimproving mitochondrial dysfunction and reducing ROS production,with mechanisms that may be associated with inhibiting NLRP3-ASC-Casepase-1 inflammasome activation and downstream cell pyrotosis.