Research On The Relationship Of Autophagy And Apoptosis Induced By Silica Nanoparticles In H9c2 Cell

【Objective】By detecting the toxic effects of silica nanoparticles on cardiomyocytes,we explored the autophagy and apoptosis induced by silica nanoparticles on rat H9c2 cardiomyocytes,and the relationship between them.The application and protection provide a theoretical basis.【Methods】Rat H9c2 cardiomyocytes were selected as the research object,and the normal cultured cell group,which not stained with silica nanoparticles,was used as the control group to prepare different concentrations of silica nanoparticles suspension(12.5mg/m L、25mg/m L,50mg/m L,100mg/m L,150mg/m L、200mg/m L).CCK-8 method was used to detect the cell viability at different time points(6,12,24,28 h)after different concentrations of cells to determine the most stable exposure time and concentration.Transmission electron microscopy was used to detect autophagosomes induced by silica nanoparticles entering rat cardiomyocytes.Flow cytometry was used to detect the level of intracellular reactive oxygen species and apoptosis rate.Immunofluorescence was used to locate and semi-quantitatively detect the number of autophagy-associated protein LC3.Hoechst 33342 staining was performed by labeling the apoptotic nuclei to observe cells.Western Blot assay for autophagy and apoptosis-related protein expression detection.The autophagy inhibitor 3-MA+100 m g/m L silica nanoparticles group and the apoptosis inhibitor Z-VAD-FMK+100 m g/m L silica nanoparticles group were established at the same time,the normal control group and 100mg/m L silica nanoparticles were used as control.Flow cytometry was used to detect the level of intracellular reactive oxygen species and apoptosis rate.The autophagy-associated protein LC3 was detected by immunofluorescence,and the expression of autophagy and apoptosis-related protein was detected by Western Blot.【Results】1.Characterization of silica nanoparticlesThe results of the characterization of silica nanoparticles showed that the 60 nm silica nanoparticles we used had good stability and monodispersity in both distilled water and medium.2.General toxicity of silica nanoparticles to H9c2 cardiomyocytesThe results of CCK-8 showed that silica nanoparticles could effectively inhibit cell growth,and the toxic effect on cells increased with the increase of the concentration.In this experiment,the maximum toxicity was achieved at the concentration of 200mg/m L,and the cell survival rate was obvious decrease(P<0.05).Compared with the control group,the survival rate of the silica nanoparticles group at 6,12,24,and 48 hours both decreased significantly(P<0.05),and the longer the time,the more obvious the decrease.,and the difference was statistically significant.Silica nanoparticles can change the state of H9c2 cardiomyocytes.After 24 hours of exposure,the cytoplasm was shrunk and the nucleus was broken in the high concentration group under the inverted microscope.Silica nanoparticles induce an increase in the level of reactive oxygen species in H9c2 cardiomyocytes.After 24 h exposure of silica nanoparticles,the level of intracellular reactive oxygen species increased significantly with concentration compared with the control group,and the difference was statistically significant(P<0.05,P<0.01).3.Silica nanoparticles induce autophagy in H9c2 cardiomyocytes.Immunofluorescence and semi-quantitative results showed that the number of LC3 detectable in the cytoplasm of the cells increased with the increasing concentration,and the difference was statistically significant(P<0.05).Silica nanoparticles induced changes in the expression of LC3 in H9c2 cardiomyocytes.As the concentration increased,LC3 I changed to LC3 II,and the difference was statistically significant(P<0.05,P<0.01).4.Silica nanoparticles induce apoptosis of H9c2 cardiomyocytes.Compared with the control group,the apoptotic rate induced by silica nanoparticles increased with the increase of concentration dose,and it was statistically significant(p<0.05).Hoechst33342 staining showed significant nuclear fragmentation.Silica nanoparticles induced changes in the expression of Bax,Bcl-2 and Caspase-3 in H9c2 cardiomyocytes.As the concentration increased Bax and Caspase-3 proteins.The expression of Bcl-2 protein decreased with the increase of expression,and the difference was statistically significant(P<0.05,P<0.01).5.The relationship between silica nanoparticles induced autophagy and apoptosis in H9c2cardiomyocytesThe effect of inhibitors on apoptosis rate and reactive oxygen species was detected by flow cytometry.Compared with the control group and 100mg/m L SNPs group,the apoptosis rate and the level of reactive oxygen specie of autophagy inhibitor 3-MA+100 m g/m L silica nanoparticles group is higher than that of the apoptosis inhibitor Z-VAD-FMK+100m g/m L silica nanoparticles group,which apoptotic rate and the level of reactive oxygen species decreased(P<0.05,P<0.01).Immunofluorescence shows that a large amount of LC3 protein was produced in the cytoplasm of the cells after exposure,and the autophagy inhibitor 3-MA+100 mg/m L silica nanoparticles group had low fluorescence intensity compared with both control group and100m g/m L silica nanoparticles group.The fluorescence intensity of the apoptosis inhibitor Z-VAD-FMK+100 m g/m L silica nanoparticles group was enhanced,and it was statistically significant after semi-quantitative analysis(P<0.05).Under the action of inhibitors,the expression of LC3,Bax,Bcl-2 and Caspase-3 in rat H9c2 cardiomyocytes was changed by silica nanoparticles,which was compared with the control group and 100mg/m L silica nanoparticles group.Compared with autophagy inhibitor3-MA,it can inhibit the transformation of LC3 I to LC3 II and promote the protein expression of Bax and Caspase-3.The inhibitor of apoptosis Z-VAD-FMK can inhibit the protein expression of Bax and Caspase-3 and promote LC3 I to LC3 II.The anti-apoptotic protein Bcl-2 was significantly increased,and the difference was statistically significant(P<0.05,P<0.01).【Conclusion】1.Silica nanoparticles can induce cytotoxicity in rat H9c2 cardiomyocytes.2.Silica nanoparticles can induce autophagy in rat H9c2 cardiomyocytes.3.Silica nanoparticles can induce apoptosis in rat H9c2 cardiomyocytes.4.There is a mutual inhibition relationship between autophagy and apoptosis induced by silica nanoparticles.