Combined Arsenic And Fluoride Exposure Induces H9c2 Cardiomyocyte Damage And Expression Of Autophagy-related Genes

Objectives:To explore the characteristics of injury and apoptosis of rat H9c2 cardiomyocytes induced by combined arsenic and fluoride exposure,and to elucidate the relevant mechanisms of autophagy in the process of cardiac toxicity induced by arsenic and fluoride exposure,as well as the interaction between arsenic and fluoride in this process.Methods:In this study,rat H9c2 cardiomyocytes were selected as the research object.According to the three-level factorial design of the two factors,it was divided into 9 groups:Control group（0μM NaAsO₂,0mg/L NaF）,high arsenic and high fluoride group（25μM NaAsO₂,35mg/L NaF）,high arsenic and low fluoride group（25μM NaAsO₂,10mg/L NaF）,low arsenic and high fluoride group（10μM NaAsO₂,35mg/L NaF）,low arsenic and low fluoride group（10μM NaAsO₂,10mg/L NaF）,high fluoride group（35mg/L NaF）,low fluoride group（10mg/L NaF）,high arsenic group（25μM NaAsO₂）,low arsenic group（10μM NaAsO₂）.1.CCK8 and MTT methods were used to determine the survival rate of cells exposed to arsenic and fluoride.2.Inverted microscope was used to observe the changes of cell growth state,morphology and number after arsenic and fluoride exposure.3.HE staining was used to observe the damage degree of cells after arsenic and fluoride exposure.4.The ultrastructure changes of endoplasmic reticulum,mitochondria and autophagosomes were observed by transmission electron microscopy.5.The early,late,total apoptosis rate and intracellular ROS content of the cells after arsenic-fluoride exposure were determined by flow cytometry.6.The mRNA expression levels of autophagy related genes Beclin1,LC3 and p62 were determined by qRT-PCR.7.Protein expression levels of autophagy marker molecules Beclin1,LC3-Ⅱand p62 were determined by Western Blotting.8.The expression levels of autophagy-related proteins Beclin1,LC3 and p62 were determined by immunofluorescence assay.9.Two-factor and three-level factorial analysis was used to determine the interaction between arsenic and fluoride in all the results.Result:1.Changes in cell survival rate and apoptosis rate after combined arsenic and fluoride exposure:（1）CCK8 and MTT results showed that the cell survival rate gradually decreased with the increase of arsenic and fluoride exposure dose and the time of exposure.After exposure to arsenic and fluoride in combination,the cell survival rate ranged from high to low:F₁₀>F₃₅>As₁₀>As₁₀F₁₀>As₁₀F₃₅>As₂₅>As₂₅F₁₀>As₂₅F₃₅,among which,after 24h exposure,the cell survival rate of As₂₅F₃₅ group and As₂₅F₁₀ group was significantly lower than that of the control group（p<0.001 or p<0.01）.（2）The early apoptosis rate and the total apoptosis rate were the highest in the As₂₅F₃₅ group.2.Changes in cell growth state and morphological structure after arsenic and fluoride exposure:（1）Combined with microscopic observations and HE staining results,the morphology of fluoride-exposed cells was more elongated,while the cell volume was significantly decreased after arsenic exposure.Compared with the control group,the As₂₅F₃₅group,As₂₅F₁₀ group and As₂₅ group had disordered cell arrangement,reduced number and poor cell growth.（2）Ultrastructural results showed that mitochondria cavitation occurred to different degrees in each group exposed to arsenic and fluoride alone and the endoplasmic reticulum in the As₂₅ and As₂₅F₃₅ groups was significantly enlarged.3.Results of cell oxidative damage index after combined arsenic and fluoride exposure:ROS content in the As₂₅F₃₅ group,As₂₅F₁₀ group,As₂₅ group and F₃₅ group was significantly higher than that in the control group(As₂₅F₃₅,As₂₅F₁₀,As₂₅:p<0.001,F₃₅:p<0.01),while ROS content in the As₁₀F₃₅ group,As₁₀F₁₀ group and As₁₀ group was significantly lower than that in the control group(As₁₀F₃₅:p<0.01,As₁₀F₁₀,As₁₀:p<0.001).4.Expression levels of autophagy-related molecules and changes in the ultrastructure of autophagosomes after arsenic and fluoride exposure:（1）Autophagosomes are widely distributed in the As₂₅F₃₅ group,As₂₅ group and As₁₀ group.In the As₂₅ group,autophagosomes mostly exist as monolayer autophagosomes.（2）The mRNA and protein expression levels of LC3 and p62 showed the same trend in each group.Among them,the expression levels of p62 mRNA and protein were significantly increased in the As₂₅F₃₅ group,As₂₅F₁₀ group,As₁₀F₃₅ group,As₂₅ group and As₁₀ group（p<0.01）.LC3 gene mRNA expression was significantly increased in the As₂₅F₃₅ group,As₂₅F₁₀ group and As₁₀F₃₅ group（p<0.05）,and LC3-Ⅱprotein level was also significantly increased in the As₂₅F₃₅ group,As₂₅F₁₀ group,As₁₀F₃₅ group and As₂₅ group(As₂₅F₃₅,As₂₅F₁₀:p<0.001,As₁₀F₃₅,As₂₅:p<0.05).Beclin1 protein expression level was significantly increased in the As₂₅F₃₅ group and the As₁₀ group（p<0.01）.（3）Immunofluorescence results of p62 and LC3 showed that the expression level of p62 fluorescent semi-quantitative protein was significantly increased in the As₂₅F₃₅ group,As₂₅F₁₀ group and As₂₅ group(As₂₅F₃₅:p<0.05,As₂₅F₁₀,As₂₅:p<0.01).The expression level of LC3 fluorescent semi-quantitative protein was significantly increased in the As₂₅F₃₅ group(As₂₅F₃₅:p<0.05).5.Results of factorial analysis after combined exposure to arsenic and fluoride:in the indicators of survival rate,total apoptosis rate,ROS content,p62 mRNA and protein expression level,Beclin1 protein expression level,arsenic and fluoride interacted and showed antagonism.However,no interaction was found between the two in Beclin1 mRNA expression level,p62 immunofluorescence semi-quantitative protein,LC3 mRNA,protein expression level and immunofluorescence semi-quantitative protein indicators.Conclusions:Arsenic and fluoride exposure can induce injury of rat H9c2 cardiomyocytes.When the concentration of arsenic and fluoride is at a high level,the structure of mitochondria and endoplasmic reticulum in the cell is damaged,and a large amount of ROS is produced.At the same time,increasing LC3 and p62 can induce the formation of autophagosomes,inhibit the degradation of autophagosomes,and block the autophagosome flow,contributing to the cardiotoxicity induced by arsenic-fluoride exposure.In this process,there may be antagonistic effects between arsenic and fluoride.