Mechanism Of Mitochondrial Apoptosis Induced By ROS By Recombinant Protein Of Trichinella Spiralis HSP70 In Rat Cardiomyocytes

Trichinella spiralis is a zoonotic pathogen that spreads widely among carnivorous mammals,T.spiralis infection can result in death from myocarditis,encephalitis and pneumonia,among which myocarditis is one of the most common causes.The myocarditis caused by T.spiralis infection has two possibilities,one is the direct myocarditis caused by the transient migration of T.spiralis larvae from the myocardium,and the other is the indirect myocarditis caused by the substances secreted by T.spiralis.Heat shock protein 70(Hsp70)is an important secreted protein of T.spiralis.It was found that Toxoplasma gondii Hsp70 could reduce the body’s antioxidant capacity and induce hepatocyte apoptosis,but the study of the role of T.spiralis Hsp70 in apoptosis has not been reported.Thus,in the present study,H9c2 cells were stimulated by recombinant protein Hsp70(Ts-Hsp70)in vitro to investigate whether Ts-Hsp70 could induce apoptosis in H9c2 cells through the mitochondrial pathway and to resolve the relationship between T.spiralis infection and host cardiomyocyte injury.In this study,the optimal concentration of Ts-Hsp70 stimulation for H9c2 cells was screened by using CCK-8 kit,the results showed the optimal concentration was 60 μg/mL.After stimulating H9c2 cells with 60 μg/mL of Ts-Hsp70 for 6 h,12 h and 18 h,the cell culture medium was collected,and detected.The results showed that H9c2 cells stimulated by Ts-Hsp70 released large amounts of LDH and CK,and the trend of LDH and CK contents increased with the extension of stimulation time,indicating that the cell membrane permeability of H9c2 cells was changed and cell damage occurred after Ts-Hsp70 stimulation.In the flow cytometric assay,it was found that Ts-Hsp70 stimulation led to an increase in the apoptosis rate of H9c2 cells,and the results of immunofluorescence and spectrophotometry showed that caspase-3 activity was increasing with the extension of the stimulation time.These results preliminarily confirmed that Ts-Hsp70 could induce apoptosis of H9c2 cells.Mitochondria are the main component of cardiomyocytes,and it was suggested that oxidative stress aggravates the autoimmune process in myocarditis.This experiment used a fluorescent probe DCFH-DA labeling method to detect the content of ROS in cells,and the results showed that H9c2 cells could release a large amount of ROS after Ts-Hsp70 stimulation,Meanwhile,the GSH content in H9c2 cells was significantly reduced after Ts-Hsp70 stimulation,indicating the occurrence of oxidative stress in cardiac myocytes.qPCR and Western Blot showed that the gene transcription level and protein expression level of Bax were significantly decreased,while the gene transcription level and protein expression level of Bcl-2 were significantly increased.The decrease of membrane potential is one of the important signs of apoptosis through the mitochondrial pathway,and in JC-10 staining,it was found that Ts-Hsp70 stimulation could cause the decrease of mitochondrial membrane potential of H9c2 cells.Therefore,the present study continued to find that Ts-Hsp70 stimulation could lead to the release of Cytochrome C and the activation of Caspase-9 and Caspase-3 in mitochondria by Western-blot assay.The above experimental results suggest that Ts-Hsp70 regulates H9c2 cell apoptosis through the mitochondrial pathway.To verify that ROS overexpression is related to mitochondrial apoptosis in H9c2 cells,the cells were treated with NAC inhibitor,and the fluorescence intensity of ROS was detected by fluorescence probe DCFH-DA.Compared with the Hsp70 group,the fluorescence intensity of ROS was significantly reduced in the NAC pretreatment group,and the apoptosis rate was found to be significantly reduced by flow cytometry.qPCR and Western-Blot method revealed that the gene transcription level and protein expression level of Bax were significantly decreased,while the gene transcription level and protein expression level of Bcl-2 were significantly increased,and caspase-3 activity was significantly decreased and cellular activity was significantly increased.The above results suggest that Ts-Hsp70 can activate ROS overexpression,and mediate apoptosis regulated by the mitochondrial pathway.JNK signaling pathway can be activated to promote apoptosis,and it has been found that the increase of endogenous ROS is an important trigger for activation of the JNK pathway.Based on this,the present study used Western-blot to detect the levels of JNK and p38 protein phosphorylation,and the results showed that the overall protein phosphorylation level of JNK increased with the extension of stimulation time,while the phosphorylated p38 bands did not change significantly.H9c2 cells treated with NAC revealed a significant decrease in JNK protein phosphorylation level by Western-blot assay,indicating that ROS can activate the JNK pathway.After adding JNK inhibitor(sp600125),western-blot assay revealed a significant decrease in JNK protein phosphorylation level.qPCR and Western-Blot assay revealed a significant decrease in gene transcription level and protein expression level of Bax,and a significant increase in gene transcription level and protein expression level of Bcl-2.Immunofluorescence and Western-blot assays revealed a significant decrease in Caspase-3 shear activation level and a significant increase in cellular activity.Together,the above findings demonstrated that Ts-Hsp70 stimulated H9c2 cells can cause ROS overexpression to activate the JNK pathway,and transduce apoptotic signals downstream,resulting in the activation of the downstream mitochondrial apoptotic pathway leading to apoptosis in H9c2 cells.In this study,we demonstrated that Ts-Hsp70 stimulation of H9c2 cells triggered ROS overexpression and activated the JNK pathway to transmit the apoptotic signal downstream,which led to the activation of the downstream mitochondrial apoptotic pathway,resulting in changes in the levels of Bcl-2 and Bax that enhanced mitochondrial membrane permeability and released Cytochrome C,then activated Caspase-9 and Caspase-3 and eventually led to apoptosis.The results of this study are important for investigating the pathogenesis of T.spiralis and providing methods for control.