IL-35 Protects Neonatal Cardiomyocytes Following Reoxygenation-Induced Apoptosis Via Activation Of Mitochondrial STAT3 Odontogenic Differentiation Of Human Dental Pulp Stem Cells

Background and PurposeTo explore the therapeutic potential and mechanism of new anti-inflammatory factor IL-35 in the model of hypoxia reoxygenation of primary myocardial cells of newborn mice in vitro and ischemia-reperfusion of C57BL/6J mice in vivo,so as to provide new evidence for the drug development of clinical myocardial ischemiareperfusion injury.MethodsCultured mouse neonatal cardiomyocytes were randomly divided into different groups.during the control group,neonatal cardiomyocytes were incubated with high glucose DMEM under normoxic conditions.Cardiomyocytes were subjected to 4 h hypoxia and 4 h reoxygenation in H/R group.In the IL-35 treatment group(25.50 and 100 ng/ml)IL-35 were added to the medium at the beginning of reoxygenation.The inhibitor treatment group was the same as the H/R+IL-35 group,but the cells were incubated with 5μmol/L AG490 for 4 h.Detection of cell viability by CCK-8,detection of mitochondrial reactive oxygen species by MitoSOX Red,detection of mitochondrial membrane potential by JC-1 assay,detection of apoptosis by TUNEL staining,and measurement of Bax,Bcl-2,Caspase-3,mitochondrial STAT3,mitochondrial P-STAT3 and COXIV by western blot.The in vivo model was divided into four groups(ⅰ)sham operation group;ⅱ)ischemia-reperfusion(I/R);ⅲ)IL35+I/R;(ⅳ)I/R+IL-35+AG490.Electrocardiogram to detect myocardial ischemia,HE staining to detect cardiomyocyte morphology,echocardiography to measure cardiac function,Tunnel staining to detect cardiomyocyte apoptosis.Results1.Cell viability:IL-35 at 25,50 and 100ng/ml does not affect the cell viability of cardiomyocytes under normoxic conditions;Compared with the control group,H/R significantly reduces cell viability.IL-35(25,50 And 100 ng/ml)posttreatment significantly increased cell viability in a dose-dependent manner.2.Degree of cell injury:Compared with the control group,H/R group increased the mitochondrial ROS,mitochondrial membrane potential depolarization and apoptosis rate;compared with H/R group,H/R+IL-35 group reduced the injury of cardiomyocytes.3.Protein expression:Compared with the H/R group,IL-35(50 ng/ml)decreased the up-regulation of caspase-3 and Bax/Bcl-2 expression,inhibited the release of mitochondrial cytochrome c to the cytoplasm,and promoted the expression of mitochondrial STAT3 and P-STAT3.4.AG490(a STAT3 inhibitor)abolished the protective effect of IL-35 in comparison with H/R IL-35,showing decreased cell viability,increased mitochondrial reactive oxygen species and apoptotic cell rates,increased caspase-3 and Bax/Bcl-2 expression,increased cytochrome c release,and decreased mitochondrial STAT3 and P-STAT3 expression.5.In vivo test:Electrocardiogram ST segment changes to judge myocardial ischemia-reperfusion injury in mice;HE staining showed local necrosis,blurred striation and leukocyte infiltration in myocardial infarction area,echocardiography showed a significant decrease in EF%and FS%,TUNEL staining showed a large number of myocardial apoptotic cells,the above phenomenon can be significantly alleviated after IL-35 treatment,and the use of AG490 abolished the protective effect of IL-35.ConclusionWe demonstrate that IL-35 can attenuate H/R damage by inhibiting apoptosis in primary neonatal mouse cardiomyocytes induced by mitochondrial reactive oxygen species.In addition,the cardioprotective effect of IL-35 is due in part to activation of the mitochondrial STAT3 signaling pathway.Therefore,this study provides a new direction for the clinical treatment of H/R-induced heart injury.