| Background Apoptosis is a highly regulated process of programmed cell death,and the degree of myocardial cell apoptosis is closely related to myocardial ischemia-reperfusion(I/R)injury.Previous studies have shown that leukocyte immunoglobulin like receptor subfamily B4(LILRB4)is involved in regulating myocardial I/R injury,but the molecular mechanism of LILRB4’s action and whether it is related to apoptosis have not been reported yet.Objective This experiment aims to investigate the effect of LILRB4 on myocardial apoptosis induced by I/R and H/R by transfecting rat myocardial tissue and H9C2 myocardial cells with recombinant adenovirus vector LILRB4,and further explore its regulatory effect on SHP-1/STAT3 signaling pathway,providing theoretical basis and new therapeutic targets for preventing and treating myocardial I/R injury.Methods(1)In vivo: 90 male SD rats were randomly divided into sham operation group(Sham group),ischemia reperfusion group(I/R group),ischemia reperfusion+up-regulated viral empty group(I/R+Ad-GFP,Ad-GFP group),ischemia reperfusion+LILRB4 up-regulated group(I/R+Ad-LILRB4,Ad-LLRB4 group),ischemia reperfusion+down-regulated viral empty group(I/R+Ad-GFP-sh RNA,Ad-GFP-sh RNA group),ischemia reperfusion+LILRB4knockdown group(I/R+Ad-LILRB4-sh RNA,Ad-LILRB4-sh RNA group).After injecting virus solution or equivalent physiological saline into the myocardium for 72 hours,a myocardial I/R injury model was established.The left anterior descending coronary artery of the rat heart was ligated for 30 minutes,and then released for 3 hours.Cardiac apex tissue was taken and fluorescence microscopy was used to observe whether the virus transfection was successful;Detection of myocardial infarction area by TTC staining method;TUNEL staining was used to detect the apoptosis index of myocardial tissue in each group;LDH content in serum was detected;Western blot was used to detect the protein expression level of LILRB4,SHP-1,STAT3,p-STAT3,cleaved Caspase-3 、 cleaved Caspase-8 、 cleaved Caspase-9、Bax、Bcl-2.(2)In vitro: H9C2 rat cardiomyocytes were cultured and randomly divided into 6 groups: blank control group(control group),hypoxia reoxygenation group(H/R group),hypoxia reoxygenation+up-regulated viral empty group(H/R+Ad-GFP,Ad-GFP group),hypoxia reoxygenation+LILRB4 up-regulated group(H/R+Ad-LILRB4,Ad-LLRB4group),hypoxia reoxygenation+down-regulated viral empty group(H/R+Ad-GFP-sh RNA,Ad-GFP-sh RNA group),and hypoxia reoxygenation+LILRB4 knockdown group(H/R+AdLILRB4-sh RNA,Ad-LILRB4-sh RNA group).After transfection of H9C2 cardiomyocytes with recombinant adenovirus vector for 48 hours,a hypoxia/reoxygenation(H/R)injury model was established,Using the Anning package system,H9C2 myocardial cells were subjected to hypoxia for 4 hours and reoxygenation for 12 hours.Adenovirus transfection was detected by fluorescence microscopy and flow cytometry;detection of cell survival by CCK-8Method;Hoechst staining was used to detect cardiomyocyte apoptosis;Western blot was used to detect the protein expression level of LILRB4,SHP-1,STAT3,p-STAT3,cleaved-Caspase-3、cleaved-Caspase-8、cleaved-Caspase-9、Bax、Bcl-2 in H9C2 cells.Results 1.In vivo:(1)Changes of LILRB4 expression in myocardial tissue after establishment of rat Myocardial I/R injury model: compared with Sham group,the expression of LILRB4 was significantly up-regulated.(2)The recombinant adenovirus vector was successfully transfected into rat cardiac tissue: Ad-GFP group,Ad-LILRB4 group,Ad-GFPsh RNA group and Ad-LILRB4-sh RNA group all showed obvious expression of green fluorescent protein.(3)LILRB4 promoted Myocardial I/R induced myocardial injury and apoptosis response: compared with Sham group,the release of serum myocardial enzyme LDH increased,myocardial tissue degeneration and necrosis,structural disorder,and myocardial infarction area significantly increased in I/R group(P<0.05).Compared with AdGFP group,the release of serum myocardial enzyme LDH in Ad-LILRB4 group increased,myocardial tissue further necrosis,myocardial infarction area further expands(P<0.05).Compared with the Ad-GFP-sh RNA group,the expression of serum myocardial enzymes in the Ad-LILRB4-sh RNA group decreased and myocardial infarction area significantly reduced(P<0.05).In addition,Tunel staining showed that myocardial apoptosis index in I/R group was significantly higher than that in Sham group(P<0.05).Compared with Ad-GFP group,the myocardial apoptosis index of Ad-LILRB4 group was significantly increased(P<0.05).Compared with Ad-GFP-sh RNA group,the myocardial apoptosis index of Ad-LILRB4-sh RNA group was significantly reduced(P<0.05).(4)LILRB4 promoted Myocardial I/R induced apoptosis cascade reaction: compared with Ad-GFP group,cleaved Caspase-3 、cleaved Caspase-8、cleaved Caspase-9、Bax in Ad-LILRB4 group increased significantly(P<0.05),Bcl-2 decreased significantly(P<0.05).Compared with Ad-GFP-sh RNA group,cleaved Caspase-3、cleaved Caspase-8、cleaved Caspase-9、Bax in Ad-LILRB4-sh RNA group decreased significantly(P<0.05),Bcl-2 increased significantly(P<0.05).(5)LILRB4regulated SHP-1/STAT3 signal transduction: compared with Ad-GFP group,SHP-1 in AdLILRB4 group increased significantly,and the phosphorylation of STAT3 decreased significantly(P<0.05).Compared with the Ad-GFP sh RNA group,the Ad-LILRB4 sh RNA group showed a significant decrease in SHP-1 and an increase in p-STAT3 levels(P<0.05).2.In vitro:(1)The recombinant adenovirus vector was successfully transfected into H9C2 cardiomyocytes: 48 hours after the recombinant adenovirus was transfected into H9C2 cells,A large amount of green fluorescence can be seen in the cytoplasm.MOI=100 was determined as the optimal multiple number of infections.(2)LILRB4 promoted the injury and apoptosis of cardiomyocytes induced by H/R: compared with the control group,the LDH level of cardiomyocytes in H/R group was significantly increased(P<0.05);Compared with Ad-GFP group,the release level of LDH in Ad-LILRB4 group was significantly increased(P<0.05);Compared with Ad-GFP-sh RNA group,the release level of LDH in Ad-LILRB4-sh RNA group was significantly lower(P<0.05).Hoechst staining showed that compared with Ad-GFP group,the number of apoptotic cells in Ad-LILRB4 group increased significantly(P<0.05);Compared with Ad-GFP-sh RNA group,the number of apoptotic cells in AdLILRB4-sh RNA group decreased significantly(P<0.05).(3)LILRB4 promoted the H/Rinduced apoptosis cascade reaction: compared with the control group,the expression of proapoptotic molecules cleaved Caspase-3、cleaved Caspase-8、cleaved Caspase-9、Bax in the myocardial cells of the H/R group increased significantly,while the expression of antiapoptotic molecules Bcl-2 decreased significantly(P<0.05).Compared with Ad-GFP group,the expression of pro-apoptotic molecules and anti-apoptotic molecules in Ad-LILRB4 group increased significantly(P<0.05);Compared with Ad-GFP-sh RNA group,The expression of the above molecules in the Ad-LILRB4-sh RNA group showed opposite effects(P<0.05).(4)LILRB4 regulated SHP-1/STAT3 signal transduction: compared with Ad-GFP group,the expression level of SHP-1 increased and the expression level of p-STAT3 decreased in AdLILRB4 group(P<0.05).Compared with Ad-GFP-sh RNA group,The Ad-LILRB4-sh RNA group showed a decrease in SHP-1 expression level and an increase in p-STAT3 expression level(P<0.05).(5)The promotion of H/R apoptosis response by LILRB4 depends on the STAT3 pathway: Compared with the Ad-GFP sh RNA group,the Ad LILRB4 sh RNA group and the Ad-LILRB4 sh RNA+AG490 group significantly reduced LILRB4 and SHP-1 levels(P<0.05);Compared with the Ad-GFP sh RNA group and the Ad-LILRB4 sh RNA+AG490group,the Ad-LILRB4-sh RNA group showed a significant increase in p-STAT3,a significant decrease in Bax,and a significant increase in Bcl-2(P<0.05).Conclusion Downregulation of LILRB4 can inhibit the expression of SHP-1,promote the phosphorylation of STAT3,thereby reducing myocardial apoptosis response,and effectively improve myocardial I/R injury. |
