Effect Of Gsk-3? On HIF-1? Against Myocardial Ischemia/reperfusion Injury

Acute myocardial infarction is currently the leading cause of high morbidity and mortality worldwide.Thrombolytic and percutaneous coronary intervention(ie,early reperfusion)are the most effective and important treatments for current acute myocardial infarction.However,myocardial damage induced by reperfusion therapy to a certain extent can not be ignored,and there is no effective treatment.Recent studies have found that hypoxia inducible factor-1?(HIF-1?)induced by hypoxia in tissue cells has anti-ischemic/reperfusion injury effects.Under the condition of normal oxygen content,the proline residue P564 of HIF-1? was dehydroxylated by hydroxylation of proline hydroxylase,and only under hypoxic conditions,hydroxylation was inhibited by HIF-1?.Therefore,we used the proline hydroxylase(PHD)inhibitor dimethyloxalyl glycine(DMOG)to stabilize the expression of HIF-1?.Studies have shown that the expression level of p-GSK3? protein is increased after HIF-1? overexpression in oral squamous carcinoma cell line(OSCC).GSK-3? is a very conserved serine/threonine kinase that is ubiquitous in mammalian eukaryotic cells and,in addition to modulating the activity of glycogen synthase(GS),it phosphorylates a variety of substrates.Including metabolism and signaling,structural proteins and transcription factors,regulating cell differentiation,proliferation,survival and apoptosis.Whether HIF-1? protects against myocardial ischemia/reperfusion injury is related to GSK3? has not been reported.Therefore,the aim of this study was to explore this issue based on the use of DMOG to stabilize HIF-1?expression.Recently,GSK-3? signaling has been shown to inhibit mitochondrial permeability transition pore(m PTP)opening and control cell fate.Some researchers have found thatGSK3? is inactivated by phosphorylating the GSK3? serine site(Ser9),which can protect against myocardial ischemia-reperfusion injury.In normal myocardium,p-GSK3?(Ser9)is mainly located in the cytosol,but under stress conditions such as ischemia/reperfusion,such as 30 minutes after ischemia and reperfusion in rats,p-GSK3?(Ser9)may be transferred to In the mitochondria.Therefore,whether GSK-3?mediates the protective effect of HIF-1? on myocardial ischemia and reperfusion through mitochondria still needs further confirmation.Objective:1 To observe the effect of GSK-3? on HIF-1? protection of myocardial ischemia/reperfusion injury in rats.2 To explore the possible mechanism of GSK-3?-mediated HIF-1? in alleviating myocardial ischemia/reperfusion injury in rats.Methods:Eight-week-old healthy male clean SD rats weighing 200-250 g were randomly divided into 5 groups: 1 sham operation group(Sham);2 myocardial ischemia/reperfusion group(I/R);3HIF-1 Stabilizer DMOG+ myocardial ischemia/reperfusion group(DMOG+I/R);4 HIF-1? inhibitor YC-1+I/R group(YC-1+I/R);5DMOG+GSK-3? inhibitor SB216763+ Myocardial ischemia/reperfusion group(DMOG+SB216763+I/R).According to the classic ligation of the left anterior descending coronary artery,myocardial ischemia for 45 min,followed by reperfusion for3 h,left the left ventricular anterior wall tissue.After 3 hours of reperfusion,the left ventricular apical myocardial tissue was taken.The expression of HIF-1?,total GSK3?and p-GSK3?(Ser9)and UCP3 protein in myocardial tissue was detected by Western blot.Real-The m RNA levels of VEGF,HO-1 and Bcl2 were detected by time PCR;the myocardial infarct size was detected by Evens blue/TTC staining;the myocardial damage was observed by HE staining;the inflammatory cell infiltration was detected by immunohistochemistry;the myocardial cells were detected by TUNEL staining.Results:1 DMOG pretreatment can up-regulate the protein expression of HIF-1? in I/R myocardial tissue and the m RNA expression of downstream factors VEGF,HO-1and Bcl2 Compared with the Sham group,the protein expression of HIF-1? was increased in the I/R group;compared with the I/R group,the protein expression level of HIF-1? was significantly increased after DMOG pretreatment,and HIF-was given.1? After the inhibitor YC-1,the protein expression level of HIF-1? was significantly decreased(P<0.05),indicating that DMOG can stabilize the protein expression of HIF-1?.Compared with the I/R group,the m RNA expression levels of the three were significantly increased after the stable HIF-1?,while the m RNA expression levels of the three were decreased after the HIF-1? inhibitor YC-1(P<0.05).2 HIF-1? significantly up-regulated the expression level of p-GSK3? protein in I/R myocardial tissue There was no significant difference in total GSK3? protein expression between the groups.Compared with the I/R group,the expression level of p-GSK3? protein in the DMOG pretreatment group was significantly increased,while the expression of p-GSK3? protein was expressed after the HIF-1? inhibitor YC-1.The level was significantly down-regulated(P<0.05).This result indicates that HIF-1? can significantly up-regulate the expression level of p-GSK3? protein in I/R myocardial tissue.3 HIF-1? Reduces myocardial damage and inflammatory cell infiltration through GSK3?The results of HE staining showed that the myocardial structure of the Sham group was normal,the fiber bundles were neatly arranged,and the coloration was uniform.The myocardial tissue structure of the I/R group was significantly impaired,the muscle fibers were arranged disorderly,and the texture was blurred.After DMOG intervention,themyocardial tissue structure is relatively complete and arranged neatly.However,after administration of the GSK3? inhibitor SB216763,the myocardial tissue structure was broken and the muscle fibers were disordered(P<0.05).The results of CD45+immunohistochemistry showed that the number of CD45+ leukocytes increased after the expression of HIF-1? was stabilized compared with the I/R group,and the number of CD45+ leukocytes decreased after administration of the GSK3? inhibitor SB216763(P<0.05).These results indicate that GSK3? mediates HIF-1? attenuates myocardial damage and inflammatory cell infiltration after myocardial I/R in rats.4 HIF-1? reduces cardiomyocyte apoptosis through GSK3?TUNEL staining showed that compared with Sham group,the apoptotic rate of cardiomyocytes in I/R group increased significantly,and the apoptosis rate of cardiomyocytes decreased after DMOG stabilized HIF-1?,while the apoptosis rate of cardiomyocytes increased after administration of GSK3? inhibitor SB216763..See Figure 5.The results indicated that GSK3? mediates HIF-1? attenuates cardiomyocyte apoptosis after myocardial I/R in rats(P<0.05).5 The protective mechanism of GSK3?-mediated HIF-1? on the heart may be related to the mitochondrial structural protein UCP3 Compared with the I/R group,the expression of UCP3 protein in DMOG pretreatment group was significantly increased,while the expression of UCP3 protein was significantly down-regulated after administration of GSK3? inhibitor SB216763(P<0.05).To further verify the above results,m RNA expression of UCP3 was also detected,and the results were consistent with the above(P < 0.05).Conclusion:HIF-1? can up-regulate the protein expression of p-GSK3?,while GSK3? inhibitor reverses the inhibitory effect of HIF-1? on inflammatory cell infiltration and cardiomyocyte apoptosis in rat I/R injury,and GSK3-? mediates HIF-1? Upregulates the protein and m RNA expression of mitochondrial inner membrane protein UCP3,indicating that GSK3? mediates the protective effect of HIF-1? on the heart and may be related to the mitochondrial structural protein UCP3.