The Effect Of Endoplasmic Reticulum Stress And Apoptosis In The Toxic Damage Of H9c2 Cardiomyocytes Induced By Arsenic-fluoride Combined

Objectives:We conducted this experiment to study the toxic effect of arsenic-fluoride combined with toxicity on H9c2 cardiomyocytes.To investigate the relationship and effect between endoplasmic reticulum stress?ERS?and apoptosis in the damage of H9c2 cardiomyocytes induced by arsenic-fluoride combined poisoning.In addition,the interaction between arsenic and fluoride in combination was analyzed.Methods:In this study,H9c2 cardiomyocyte model was used,which was divided into 9groups according to different doses of arsenic and fluoride alone and combined:control group?C group?,low arsenic group(As₁₀ group,10?M),high arsenic group(As₂₅group,25?M),low fluoride group(F₁₀ group,10 mg/L),high fluoride group(F₃₅group,35mg/L),low arsenic and low fluoride group[As₁₀F₁₀ group,10?M?As?+10mg/L?F?],low arsenic and high fluoride group[As₁₀F₃₅ group,10?M?As?+35mg/L?F?],high arsenic and low fluoride group[As₂₅F₁₀ group,25?M?As?+10 mg/L?F?],high arsenic and high fluoride group[As₂₅F₃₅ group,25?M?As?+35mg/L?F?].The survival rate of arsenic and fluoride combined exposure of H9c2 cardiomyocytes was determined by CCK-8.The rate of early cell apoptosis was detected by flow cytometry.The cell morphology,pathological morphology and ultrastructure was observed respectively by inverted microscope,light microscope and electron microscope.The activity of AST and CRE was detected.The expression of ERS related genes GRP78,PERK and CHOP in cardiomyocytes was detected by qRT-PCR.The expression of ERS and apoptosis-related protein GRP78,PERK,CHOP,and Cleaved caspase-3 was detected by Western blot.The expression of ERS marker gene GRP78 in cardiomyocytes was detected by immunofluorescence assay.Further,after intervention with ERS inhibitor 4-PBA,the survival rate of cardiomyocytes and the protein expression of ERS related genes GRP78 and CHOP was detected.SPSS22.0 was used for one-way analysis of variance and factorial analysis of experimental data.Results:1.The survival rate and apoptosis rate changes of cardiomyocytes:?1?Survival rate results showed that the cell survival rate was on the rise and reached the highest when arsenic and fluoride was exposed to different doses for 12h and 24h.After 48h,72h and 96h,the cell survival rate was time-dependent.The survival rate was significantly reduced in all the exposed group(except As₁₀ group and As₁₀F₁₀ group)after exposed 24h.?2?The apoptotic rate showed that the early apoptotic rate of exposed group was significantly higher than that of the control group.2.Morphological changes of cardiomyocytes:?1?Microscopic observations:The cells volume was contracted,the arrangement was disordered and the density was decreased in the single arsenic exposed group.The cells arranged neatly and a small number of round apoptotic cells were found in the group of F₃₅.The cells were cross-linked,the density was decreased and the round apoptotic cells were significantly increased in the combined group.?2?HE staining results:The number of cells was less,the contour was fuzzy,the chromatin in the nucleus was agglutinated and the number of necrotic cells was large in the single arsenic group.Some cells were enlarged and their contour was blurred in F₃₅ group,there was no significant change in F₁₀ group.The cells were enlarged to different degree,the cell space was widened to different degree,and chromatin in the nucleus was agglutinated in the combination group.In the As₂₅F₁₀ group and As₂₅F₃₅ group,the nuclear membrane integrity of some cells was damaged,and chromatin was agglutinated.3.Ultrastructural changes of cardiomyocytes:?1?Nucleus:No significant changes were observed in the nucleus of F₁₀ group.While the nucleus of the other exposed groups was pitted in different shades on the surface and the chromatin in the nucleus was scattered at the edge of the cells to varying degrees.The nucleus of the As₂₅ and F₃₅ group and the combined As₂₅F₃₅ group was significantly decreased.?2?ER:The endoplasmic reticulum was severely swollen and expanded into a pool in the single arsenic group.The number of dilatation and swelling of endoplasmic reticulum was significantly increased and was bubbly in the single fluoride group.In the combined group,the number of endoplasmic reticulum expansion as bubble or pool was significantly increased and endoplasmic reticulum degranulation,ribosome shedding,dissociation.4.Changes in markers of myocardial injury:Compared with the control group,AST and CRE activity was significantly increased in the arsenic,fluoride alone exposure group(except for the CRE in the F₁₀ group).In the combined exposure group,the CRE activity was increased significantly only in As₁₀F₃₅ group and As₂₅F₃₅ group,and the AST content was not statistically significant.5.Protein and mRNA expressions of ERS and apoptosis-related genes:?1?The expression of mRNA:The expression level of CHOP mRNA were significantly increased in all the exposed groups.The expression level of GRP78 mRNA in As₁₀F₃₅group and As₂₅F₁₀ group was significantly increased,and the expressions level of PERK mRNA in As₁₀F₃₅ group,As₂₅F₁₀ group and As₂₅F₃₅ group was significantly increased,but the expressions level of GRP78 and PERK mRNA only showed an upward trend in other exposed groups,but there was no statistical difference.?2?The expression of protein:The protein expression level of GRP78,PERK and CHOP was significantly increased in the exposed groups,but the protein expressions of PERK in the As₂₅ group and the protein expressions of CHOP the As₂₅F₃₅ group was no statistical difference.The protein expression level of Cleaved casapse3 was no significant change in the single arsenic group,but was significantly increased in the single fluoride group.In the combined groups,there was significantly increased only in the As₁₀F₁₀ group and As₁₀F₃₅ group.?3?Expression of immunofluorescence ERS marker protein GRP78:The mean fluorescence intensity of GRP78 was significantly enhanced only in As₂₅group,F₃₅ group,As₂₅F₁₀ group and As₁₀F₃₅ group.6.After the intervention of inhibitor 4-PBA,the cell survival rate and the expression of ERS related proteins:?1?The survival rate of cardiomyocytes:Compared with the exposed groups,the cell survival rate of exposed group(except As₁₀group and As₁₀F₃₅ group)was significantly increased after the intervention of inhibitor4-PBA.?2?Expression of ERS related proteins:Compared with the exposed group,the protein expression of GRP78 was significantly decreased after the intervention of inhibitor 4-PBA(except for group F₃₅ group,As₁₀F₃₅ group and As₂₅F₃₅ group),and the expression level of CHOP protein was decreased,but there was significantly decreased only in the F₁₀0 group and As₂₅F₁₀ group.7.The results of interaction analysis showed that there was a synergistic effect between arsenic and fluoride in the As₂₅F₁₀ group and As₂₅F₃₅ group,and an antagonistic effect between arsenic and fluoride in the As₁₀F₁₀ group and As₁₀F₃₅ group.In addition,the antagonistic effect of arsenic and fluoride on myocardial enzymes?AST and CRE?,endoplasmic reticulum stress-related mRNA?GRP78 and PERK?and proteins?GRP78,PERK and CHOP?,and apoptosis-related protein Cleaved-casapse3was found.Conclusions:1.Arsenic and fluoride combined with toxicity inhibited the survival rate of H9c2 cardiomyocytes,changed the morphology,pathology and ultrastructure of cardiomyocytes?Nucleus and ER?,activated the enzyme activity related to myocardial injury,and induced early apoptosis of cell,which eventually led to the injury of H9c2cardiomyocytes.2.After arsenic and fluoride combined poisoning,the expression levels of GRP78 and PERK were firstly activated,and then ERS early apoptotic factor CHOP was initiated,which further caused caspase-3 cascade reaction and induced apoptosis of cardiomyocytes.Therefore,we analyzed that endoplasmic reticulum apoptotic pathways may be involved in the process of myocardial cell damage caused by arsenic and fluoride combined with toxicity.3.The results of factorial analysis revealed that the interaction between arsenic and fluoride was mainly shown antagonistic effect in the process of the arsenic and fluoride combined exposed on myocardial injury,ERS and apoptosis.