Intervention Of Oxidative Stress Mediated Myocardial Pyroptosis By TSA Via FoxO3a

Acute myocardial infarction is the disease that seriously impact human life and health.Though thrombolytic therapy or interventional therapy can restore the infarcted myocardium to reperfusion,cause myocardial ischemia reperfusion injury similar to inflammation,the structure and function of myocardium get to hurt more seriously.How to alleviate reperfusion injury after myocardial ischemia has become a hotspot of current research.The mechanisms of myocardial ischemia reperfusion injury are very complex,and many results of animal experiments show that the mechanism of myocardial ischemia reperfusion injury relates to calcium overload,neutrophil infiltration,angiotensin,oxygen free radicals,and nitric oxide production,etc.In another word,myocardial ischemia reperfusion injury is a chain reaction caused by a series of energy metabolism abnormal,and the production of ROS is one of the main reasons induce of the early injury of the cardiomyocytes.The redundant ROS may react with biological macromolecules as proteins,lipids and nucleic acids,and then induce the macromolecular degeneration,biofilm damage and lipid peroxidation,which result in the damage of myocardial cell structure and function,ultimately cause apoptosis.It is demonstrated that if the ROS derived from mitochondria over express,it will induce NLRP3 to recruit ASC,and then activate Caspase-1,which plays an important role in the progress of the inflammation induced apoptosis.Trichostatin A is known to be one of the broad spectrum histone deacetylase inhibitor.It can increase the level of histone acetylation,and it is widely used as an experimental study of antitumor so far.Our previous experiments showed that TSA ameliorated myocardial ischemia/reperfusion injury,but the underlying mechanisms remain to be elucidated.FoxO,a member of the fork protein family,plays a very important role in the progress of the metabolism,cell survive and anti-oxidative stress.It has been shown that FoxO3 a,a member of the FoxO proteins,relieved myocardial injury by increasing oxidative stress resistance in mice,but there is no relative report in rat.Recently,it has been reported that mir-30 d directly repressed FoxO3 a expression and its downstream protein,apoptosis repressor with caspase recruitment domain,upregulating caspase-1 and inducing pyroptosis in diabetic cardiomyopathy.Endogenous HDAC inhibitor β-OHB can alleviate oxidative stress injury by inhibiting the activity of HDAC1,increasing histone acetylation at the FoxO3 a promoters and promoting FOXO3 a transcription.Thus,we hypothesized that FoxO3 a may play an important role in TSA alleviating myocardial ischemia/reperfusion injury.Moreover,FoxO3 a may be involved in the regulation of pyroptosis during this process.In order to verify our speculation,we conducted the following experiment.1.Myocardial ischemia reperfusion injury is accompanied by the formation of pyroptosisThe myocardial ischemia-reperfusion injury model was established by ligating the left anterior descending coronary artery.The expression of Caspase-1,IL-1β and IL-18 were detected by immunohistochemical method in the myocardium of rats.The results have shown that the I/R group show that the expression of Caspase-1,IL-1β and IL-18 in inflammatory tissue of myocardium was significantly increased compared to the Sham group,indicating that I/R injury may induce pyroptosis in rat myocardium cells.At the same time,we examined the expression of ROS in myocardium of rats with reperfusion injury,the results showed that the level of ROS in myocardium of I/R group was significantly increased compared with Sham group,and it was confirmed that rat I/R could induce pyroptosis by increasing ROS in myocardium.Following that we selected H9c2 cell line,the use of H2O2 to establish oxidative stress injury model for a further observation whether increased ROS induced by oxidative stress injury can lead to myocardial pyroptosis.In order to detect the survival rate of H9c2 we used CCK-8 kit to screen out the best working concentration of H2O2.ROS expression at different time points was measured by ROS kit.The expression of pyroptosis protein Caspase-1,IL-1β and IL-18 was detected by immunofluorescence assay.It was found that the expression of Caspase-1,IL-1β and IL-18 in H2O2-treated group was significantly higher than that in Control group,suggesting that oxidative stress could induce pyroptosis in H9c2 cells.2.TSA attenuates pyroptosis induced by oxidative stress in ratsIn the first part,we examined the myocardial ischemia-reperfusion injury in vitro and in vivo,which may lead to pyroptosis by increasing ROS.We have shown that TSA protects the rat myocardium from apoptosis induced by endoplasmic reticulum stress,so as to protect the rat myocardium from I/R injury.In order to clarify the protective effect of TSA on the myocardium after reperfusion whether related to pyroptosis induced by its oxidative stress was induced by I/R damage in rats.And we carried out the following experiment.(1)The effect of TSA on myocardial injury induced by I/R injury in rats: To further validate the effect of TSA on rat I/R injury,we used TTC to determine the myocardial infarct size of the rats after I / R injury.And the activity of myocardial enzymes in rat myocardium was detected by the enzymes kits,HE staining was used to observe the morphology of myocardium.The results have shown that TSA could decrease the myocardial infarct size,decrease the activity of myocardial enzymes and improve the morphology of myocardium in rats,thus reducing the injury of rats.(2)Effects of TSA on oxidative stress in rats with I/R injury: We used flow cytometry to detect the expression of ROS in rat cardiac tissue,the results have shown that the expression of ROS in myocardium of TSA-treated I/R rats was significantly decreased.SOD and MDA kits were used to detect the expression of MDA and SOD in myocardium of rats.The results have shown that the expression of MDA in myocardial tissue of TSA-treated I/R rats decreased,but the activity of SOD increased.(3)Whether TSA can affect rat pyroptosis induced by I/R injury: First,we used flow cytometry to observe the effect of TSA on cardiomyocyte apoptosis in I/R-induced rats,the results showed that the apoptotic rate of cardiomyocytes in TSA group was significantly lower than that in I/R group.The effects of TSA on the I/R-induced expression of Caspase-1,IL-1β and IL-18 were observed by immunohistochemistry and Western blot.The results show that TSA can effectively reduce the expression of Caspase-1,IL-1β and IL-18 of cardiomyocytes in I/R-induced rats.(4)The effect of TSA on serum inflammatory related factors in I/R-injured rats: A large number of proinflammatory cytokine release in the development of pyroptosis process.In order to test whether TSA affects the expresion of inflammatory factors during pyroptosis.We used the ELISA method to detect the expression of inflammatory factors in serum.The results show that the expression of inflammatory factors TNF-α、IL-6、IL-18 and IL-1β in serum of TSA intervention group was significantly lower than that of I/R group.3.FoxO3 a is a key molecule in the process of intervention of oxidative stress mediated myocardial pyroptosis by TSAResearches has been shown that FoxO3 a relieved myocardial injury by increasing oxidative stress resistance in mice,and endogenous HDAC inhibitor,β-hydroxy butyric acid can increase FoxO3 a promoter region acetylated against oxidative stress reaction of HEK293 cells.In order to clarify improving myocardial injury of TSA whether associated with FoxO3 a,so we adopt the following research was conducted on the experiments in vivo and in vitro.(1)The effect of TSA on FoxO3 a expression in oxidative stress injury rats: First,the expression of FoxO3 a in I/R rats and H2O2 induced H9c2 cells in different time points was detected by Western blot method,the results showed the expression of FoxO3 a decreased in a time-dependent manner during reperfusion 6h,12 h and 24 h,and is especially significant the reperfusion 24 h,FoxO3 a expression also gradually decline in H2O2 induced H9c2 cells in different time point,and decreased significantly after 400μM H2O2 induced 2h.we adopt Western blot method to detect the effect of TSA on the expression of FoxO3 a at reperfusion 24 h,the results showed that 0.2 mg·kg-1 TSA can obviously increase the expression of FoxO3 a in I/R rats.Immunofluorescence and Western blot results show that the TSA can increase the expression of the FoxO3 a in H9c2 cells induced by H2O2.(2)The effect of TSA on oxidative stress damage by H2O2 induced H9c2 cells: The expression of ROS was measured by DCFH-DA as a fluorescent probe,the dye JC-1 was used as a fluorescent probe to detect mitochondrial membrane potential,the expression of the SOD2 and Catalase was detected by Western blot method.The results show that ROS and mitochondrial membrane potential decreased in TSA treatment group,the expression of SOD2 and Catalase significantly increased,prompting that TSA may reduce oxidative stress injury.(3)The effect of TSA on H2O2 induced rat myocardial H9c2 cells pyroptosis: To preliminary explore the effect of TSA on H2O2 induced rat myocardial H9c2 cells pyroptosis,we adopt Annexin V-FITC/PI method to detect the H9c2 cell apoptosis rate,using Western blot method to observe the expression of Caspase-1,IL-1β and IL-18 which are the pivotal protein of pyroptosis,the results showed that rats myocardial H9c2 cells apoptosis rate significantly decreased in TSA intervention group,the expression of Caspase-1,IL-1β and IL-18 also significantly reduced,prompting that TSA may reduce H2O2 induced rat myocardial H9c2 cells pyroptosis.(4)The effect of TSA on H2O2 induced rat myocardial H9c2 cells inflammatory related factors: To test whether the TSA would affect the expression of inflammatory factors in the process of pyroptosis,the expression of inflammatory related factors was detected in rat myocardial H9c2 cell culture medium,the results show that the expression of inflammatory related factors TNF-α,IL-6,IL-18 and IL-1β in TSA intervention group was lower than that in H2O2 group,prompting that TSA may reduce the release of inflammatory cytokines.(5)TSA based on FoxO3 a intervention of oxidative stress mediated rat myocardial pyroptosis: we use interference technology to knock down the FoxO3 a in rats myocardial H9c2 cells,on the premise of ensure the transfection efficiency,FoxO3 a si RNA can effectively restrain FoxO3 a gene m RNA and protein levels,and FoxO3 a si RNA can restrain FoxO3 a gene m RNA and protein levels,meanwhile,Catalase and SOD2 in H2O2 and TSA treatment group was obviously suppressed;the expression of pyroptosis related proteins Caspase-1,IL-1β and IL-18 were significantly increased in H2O2 and TSA treatment group,prompting that TSA may be based on FoxO3 a intervention of oxidative stress mediated rat myocardial pyroptosis.4.The mechanism of TSA regulating FoxO3 a expressionIn the third part of experiment,we preliminary confirm that TSA may base on FoxO3 a intervention oxidative stress mediated rat myocardial pyroptosis,but how does TSA regulate FoxO3 a,we made the following experiment.(1)classⅠHDACs(HDAC1,HDAC2,HDAC3,HDAC8)is closely related to the development of cardiovascular diseases,so we adopt HDAC activity kits using colorimetric method for testing the activities of HDACs,the results show that compared with the control group,classⅠHDACs activity increased significantly in H2O2 group cells,classⅠHDACs activity was obviously reduced in TSA intervention group.Prompting that oxidative stress damage may result in activities raised of classⅠHDACs in H9c2 cells,TSA can effectively reduce the activities of classⅠHDACs in H2O2 induced H9c2 cells.(2)Effect of TSA on the acetylation levels of histone H3 and H4 in the promoter region of FoxO3a:To investigate whether TSA affects the levels of histone acetylation of the FoxO3 a gene promoter and then FoxO3 a gene expression,we adopt Ch IP method to detect the acetylation levels of histone H3 and H4 in the promoter region of FoxO3 a in TSA pretreatment rat myocardial H9c2 cells,the results show that TSA elicited a significant increase in H4 acetylation of the FoxO3 a promoter region compared with that of the H2O2 group.The level of H3 acetylation of FoxO3 a was not significantly different.The above results show that TSA changed the H4 acetylation of the FoxO3 a promoter region,thus affecting the expression of FoxO3 a.Through the above experiment,we get the following conclusion:1.Oxidative stress injury can induce pyroptosis in rat myocardium.2.Acetylation attenuates pyroptosis induced by oxidative stress in rats.3.FoxO3 a is a key molecule in the process of intervention of oxidative stress mediated myocardial pyroptosis by TSA.4.TSA may affect FoxO3 a expression by changing FoxO3 a gene promoter region acetylation of histone H4 levels.In summary,intervention of oxidative stress mediated myocardial pyroptosis by TSA via FoxO3a.