Effect Of ALDH2 Gene Overexpression On High Glucose-Induced H9C2 Cardiac Cells Pyroptosis

Background In the diabetic state,high glucose stimulation increases the production of reactive oxygen species,which is considered as an important cause of the occurrence and development of diabetic cardiomyopathy.With the prolongation of diabetes,the oxidative stress damage of myocardial tissue is increasing,and excessive reactive oxygen species stimulate the expression of various inflammatory factors and induce inflammation.Long-term inflammatory conditions can induce tissue and cell damage and induce death finally.Pyroptosis is a new form of pro-inflammatory cell death which characterized by caspase-1,accompanied by the release of a number of pro-inflammatory factors.The NLRP3 inflammatory body acts as a means of inflammatory activation and plays a key role in injury.Mitochondria is the main source of ROS production.Multiple studies have also confirmed that ROS signaling required for NLRP3 inflammatory body activation is derived from mitochondria,suggesting that mitochondria-derived ROS plays a key role in NLRP3 inflammatory body activation.Aldehyde Dehydrogenase 2(ALDH2)is a nuclear-encoded aldehyde oxidase localized to the mitochondrial matrix.Our previous study had found that ALDH2 had the protective effects on myocardium under stress conditions such as myocardial ischemia/reperfusion,alcohol and diabetes.It has been reported that NLRP3 inflammatory activation aggravates myocardial injury during myocardial ischemia/reperfusion injury in diabetes.Silencing NLRP3 gene can improve myocardial injury in type 2 diabetic rats.However,the relationship between ALDH2 and NLRP3 inflammatory body,whether the expression of ALDH2 in H9C2 cardiac cells can inhibit the occurrence of myocardial cell injury has not been reported.The purpose of this study was to investigate whether over-expression of ALDH2 gene canreduce the occurrence of high glucose-induced H9C2 cardiac cells injury and investigate the role of NLRP3 inflammatory body in cardiac cells injury.Methods: The H9C2 cardiac cells were treated with 35 mM glucose for 24 h,36h and48 h respectively.The changes of NLRP3,key factorsNLRP3,ASC and Caspase-1protein expressions were detected by Western blot.The H9C2 cardiac cells were infected with the optimal multiplicity of infection,and the infection efficiency was detected by fluorescence microscopy.H9C2 cardiac cells lentivirus negative control and ALDH2 gene over-expressing cell line were constructed,and ALDH2 mRNA and protein expressions were detected by Real-time PCR and Western blot.After verifying the successful construction of the cell line,the cells were divided into six groups:normal control(NG)group,lentivirus negative control(GFP)group,ALDH2over-expression(ALDH2-GFP)group,high glucose(HG)group,HG + GFP group and HG+ ALDH2-GFP group.The expressions of ALDH2 at mRNA and protein level were detected by Real-time PCR and western blot.The cell viability was measured by Cell Counting Kit-8 colorimetric assay.The changes of 4-HNE,IL-1? and IL-18 levels in cell cultural supernatants were measured by ELISA.The changes of mitochondrial oxidative stress were measured through Mitosox reagent,the ALDH2 activity was detected by ALDH2 kit;the protein expression levels of NLRP3,ASC and Caspase-1were assessed by western blot.Results:(1)The expression of NLRP3,ASC and caspase-1 was significantly increased in the H9C2 cardiac cells at 24 h,36h and 48 h after treatment with 35 mM glucose.The expression of NLRP3,ASC and caspase-1 was significantly higher than that of the normal group at 24h(P<0.01)and decreased gradually at 48 h.(2)The results of Western blot and Real-time PCR showed that the ALDH2 over-expressing cell line was successfully constructed.Compared with the NG group,the expression of ALDH2 protein and mRNA levels in the GFP negative control group did not change significantly.The ALDH2 protein and mRNA levels in the ALDH2-GFP group were significant.(3)The results of CCK8 cell viability showed that there was no significant changes in cardiac cell viability in the GFP group and the ALDH2-GFP groupcompared with the normal group,and the cell viability in the HG group and the HG +GFP group were decreased.Compared with the HG group,cell viability was higher,the cell viability in the HG+ALDH2-GFP group was increased(P<0.01).(4)Determination results of mitochondrial oxidative stress by Mitosox reagent: compared with the NG group,the red fluorescence intensities in HG group and HG+GFP group were significantly increased(P<0.01),suggesting that mitochondrial oxidative stress level was increased;and compared with the HG group and HG+GFP group,in the HG+ALDH2-GFP group,the red fluorescence intensity was significantly weakened(P<0.01),suggesting that the mitochondrial oxidative stress level was decreased.(5)The results of ELISA showed that compared with the NG group,the levels of 4-HNE,IL-18 and IL-1? in the supernatant of HG group and HG+ GFP group were significantly increased(P<0.01);compared with the HG group,the levels of 4-HNE,IL-18 and IL-1? in theHG+ALDH2-GFP group were significantly decreased(P<0.01).(6)Colorimetric assay results for ALDH2 activity showed that ALDH2 activity was significantly decreased in the HG group and HG +GFP group compared with the NG group(P<0.01),compared with HG group.ALDH2 activity was increased in the ALDH2-GFP group(P<0.01);(7)Western blot results showed that NLRP3,ASC,Caspase-1 protein in the HG group and the HG + GFP group were compared with the NG group.The levels of NLRP3,ASC and Caspase-1 protein in the HG+ALDH2-GFP group were significantly lower than that in the HG group(P<0.01).Conclusion: 1.In high glucose-induced cardiac cell injury,the activation of NLRP3 inflammation and pyroptosis are involved;2.ALDH2 may reduce mitochondrial ROS production,thereby inhibiting the activation of NLRP3 inflammation and improving myocardial damage induced by high glucose stimulation.