| Objective: H9c2 cells were used for hypoxia/reoxygenation(H/R)to construct a myocardial ischemia/reperfusion injury model in this study to observe the changes in autophagy levels of H9c2 cells in normal and H/R states,and the purpose of study was to explore the effects of TRPC6 mediated autophagy and the role of myocardial ischemia/reperfusion injury to clarify the effect of TRPC6 on myocardial cell autophagy in the H/R state.Methods: 1 Establishment and grouping of H/R model of H9c2 cardiomyocytes H9c2 cells were hypoxic for 9 hours and then reoxygenated for 6 hours to establish an H/R model to observe normal and hypoxic/reoxygenated cell morphological changes and detect changes in autophagy.After the H/R model is successfully constructed,the experiment is further divided into 6 groups,namely Control group,SAR7334 group,H/R group,H/R+SAR7334 group,H/R+3MA group and H/R+SAR7334+3MA group,among which SAR7334 is a specific inhibitor of TRPC6,and 3MA is an autophagy inhibitor.The relative cell viability was detected by CCK-8 method,and the contents of LDH,CK-MB and c Tn-T in each group were detected by microplate method.2 H9c2 cardiomyocyte HBLV-LC3B-m Cherry-e GFP-PURO virus transfection H9c2 cells were transfected with HBLV-LC3B-m Cherry-e GFP-PURO virus,screened and cultured,and verified whether the transfection was successful.After successful transfection,the autophagy changes under normal conditions and after hypoxia/reoxygenation were detected.3 Autophagy related index detection Autophagosomes were observed using Transmission electron microscope,and the aggregation of m Cherry-e GFP-LC3 fluorescent spots was observed by confocal microscope;q PCR was used to detect the transcription level of Beclin-1,LC3 and P62,etc.,the expression of LC3 fluorescence was detected by immunofluorescence,and Western blot was used to detect the expression of autophagy-related proteins.Results: 1 Establishment of H/R model of H9c2 cardiomyocytes After 9 hours of hypoxia and reoxygenation for 6 hours of H9c2 cells,the cells were shortened and rounded,with irregular shapes and poor adherence.The cell viability after H/R was significantly lower than that in the Control group(P<0.01),and the contents of LDH,CK-MB and c Tn-T in the H/R group were significantly higher than those in the Control group(P<0.01),indicating that H9c2 cells were damaged after H/R;H/R group autophagosomes,LC3 fluorescence aggregation were significantly increased compared with Control group(P<0.01),indicating that H9c2 cell autophagy flux increased after H/R.2 H9c2 cardiomyocyte HBLV-LC3B-m Cherry-e GFP-PURO virus transfection After being transfected with virus,cell culture and screening were performed.The red and green fluorescence distributions were observed under microscope.At the same time,q PCR was used to identify m Cherry-e GFP-LC3 expression in H9c2 cells,indicating that m Cherry-e GFP-LC3-transfected H9c2 cell line was successfully constructed.3 Autophagy related index detection Confocal laser scanning microscope: After using the m Cherry-e GFP-LC3 to transfect H9c2 cells H/R,the red fluorescent spots of H/R group was increased compared with control group,and the autophagy flux was increased.After H/R of H9c2 cells,autophagosomes were more in H/R group than in control group.Western blot: Beclin-1 and LC3 protein expressions in H/R group were higher than those in control group(P<0.05);Beclin-1 in H/R+SAR7334 group was lower than that in H/R group(P<0.05);after using 3MA inhibition,compared with H/R group,Beclin-1 protien levels decreased in H/R+3MA group and H/R+SAR7334+3MA group(P<0.05),and P62 increased(P<0.05).Immunofluorescence: The number of LC3 fluorescent spots in each group of H9c2 cell H/R treatment was significantly higher than that of control group(P<0.01),H/R+SAR7334 group,H/R+3MA group,H/R+SAR7334+3MA group and The H/R group had less fluorescence accumulation than LC3(P<0.01).q PCR: The transcription levels of Beclin-1 and LC3 in H/R group were higher than those in control group(P<0.01),while the P62 transcription levels in H/R group were lower than those in control group(P<0.01);H/R+SAR7334 group,compared with the H/R group,the transcription levels of Beclin-1 and LC3 in H/R+3MA group and H/R+SAR7334+3MA group were decreased(P<0.05);H/R+SAR7334 group,H/R+3MA group and the transcription level of P62 in H/R+SAR7334+3MA group was higher than that in H/R group(P<0.05).Conclusion: The autophagy flux of H9c2 cells increased under hypoxia(9 h)/reoxygenation(6 h).The TRPC6 inhibitor SAR7334 can attenuate the autophagy flux of hypoxia(9 h)/reoxygenation(6 h) H9c2 cells. |
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