The Role Of Leptin In Promoting Angiogenesis After Intracerebral Hemorrhage Through The Pericyte STAT3 Pathway

Objective: Angiogenesis is an important self-repair process of the nervous system after intracerebral hemorrhage.There is growing evidence that leptin promotes angiogenesis and plays a beneficial role in hemorrhagic stroke.The proangiogenic effect of leptin on cerebral hemorrhage has not been adequately studied.However,it is unclear how leptin triggers post-ICH angiogenesis via pericytes,which contributes to the formation of new blood vessels.Therefore,we intend to study the role of leptin in cerebral hemorrhage and explore its role in promoting angiogenesis after intracerebral hemorrhage,thereby providing a theoretical basis for the potential therapeutic value of leptin in cerebral hemorrhage.Methods: This study is divided into two parts,in vivo animal studies and in vitro cellular studies.In vivo animal research part:(1)C57 mice were randomly divided into normal saline control group,intracerebral hemorrhage model group,gradient concentration leptin treatment group(1,10,20 μg,intraventricular injection for 7 consecutive days).Behavioral tests(neurological scores,open field test,foot failure test,rotation test)were performed on the 7th day to evaluate the motor function of the mice,and immunofluorescence staining was performed on the blood vessels after perfusion.The number,length,area,branch and density of blood vessels were counted to screen out the optimal dose of leptin for follow-up experiments.(2)C57 mice were randomly divided into normal saline control group,intracerebral hemorrhage model group,and optimal dose of leptin treatment group(20 μg).The behavioral tests were performed at 0,1,7,and 14 days after operation,and vascular immunofluorescence staining was performed at the above time points.(3)The proliferation of endothelial cells was detected by Ed U cell proliferation assay on the 7th day after operation,and the expression levels of endothelial cell sprouting key genes(D114,ESM)and endothelial cell proliferation key gene(MYC)were detected by real-time quantitative PCR(q RT-PCR).(4)The expression levels of PDGFRβ and NG2 in pericytes were detected by immunofluorescence staining and western blot.(5)The protein and gene expression levels of vascular growth factor VEGF,VEGFA and vascular growth factor receptor VEGFR2 were detected by western blot and q TR-PCR.(6)The expression levels of Leptin R,P-JAK1,and P-STAT3 in the signaling pathway were detected by western blot.(7)The degree of cell damage and neuronal survival in the surrounding area of intracerebral hemorrhage in mice were detected by H&E staining and Nissl staining.In vitro cell research part:(1)The co-culture system of human brain microvascular pericytes(HBVPC)and human brain microvascular endothelial cells(HCMEC/D3)was established by Transwell chamber.The in vitro intracerebral hemorrhage cell injury model was constructed by Hemin,and HBVPC and HCMEC/D3 were collected.(2)The expression level of pericytes specific marker NG2 was detected by immunofluorescence staining and western blot.(3)The cell survival ability was detected by CCK8 and SYTOX methods.The cell proliferation and migration ability were detected by Ed U cell proliferation assay and scratch test.Tube formation assay was used to detect the angiogenesis in vitro.(4)western blot and q RT-PCR were used to detect the protein and gene expression levels of VEGF,VEGFA and VEGFR2 in some of the studies in vitro.(5)detection of Leptin R,P-JAK1 and P-STAT3 signaling pathway key factors expression by western blot;(6)detection of P-STAT3 protein expression level by western blot after treatment with STAT3 inhibitor BP-1-102;western blot and q TR-PCR were used to detect the protein and gene expression levels of cell proliferation(Cyclin D2 and CDK2)and migration(Rac1,Rho A and Cdc42).Results:Part of animal in vivo study:(1)On the 7th day after ICH,the number,length,area,number and density of blood vessels,modified Garcia score,the shedding time of mice in rotation test and the average speed in open field test were significantly increased,and the foot failure rate was decreased in leptin treatment group.(2)Compared with the ICH model group,the above vascular indexes and behavioral indexes were improved in the leptin treatment group on days 7 and 14 after ICH.(3)On day 7 after ICH,the number of edu-positive cells and the expression of endothelial cell proliferation(MYC)and budding markers(D114 and Esm)were increased in the leptin treatment group.(4)the expression of pericyte specific markers PDGGFR β and NG2 was up-regulated.(5)The protein and gene expressions of VEGF,VEGFA and VEGFR2 were up-regulated in the leptin treatment group.(6)Compared with the ICH model group,the protein expression levels of Leptin R and P-STAT3 were significantly increased in the leptin treatment group.(7)After intracerebral hemorrhage,the cells around the hematoma were scattered,the boundary was not clear,the number of Nissl-positive cells decreased,the number of Nissl bodies decreased,and the number of neurons decreased.Compared with the ICH model group,the morphology of Nissl bodies around the hematoma in the leptin administration group was slightly abnormal,with no significant change,and the number of issl positive cells and neurons increased.In vitro cell studies:(1)Compared with the control group,the viability of HBVPC cells in the model group decreased,the damaged cells increased,the number of edu-positive cells and the healing ability of scratch test decreased.Compared with the model group,the leptin treatment group had significant improvements in the above indicators.(2)Compared with the model group,the protein and gene expression levels of VEGF,VEGFA and VEGFR2 were significantly increased in the leptin treatment group.(3)In the co-culture system of HBVPC and HCMEC/D3,compared with the control group,the number of Ed U positive cells of HCMEC/D3 was significantly decreased,the healing ability of scratch test was significantly decreased,and the ability of tube formation in vitro was significantly weakened.Compared with the model group,the leptin treatment group had significant increases in the above indexes.(4)Compared with the model group,the protein expression levels of Leptin R and P-STAT3 were increased in the leptin treatment group,while the protein expression of P-JAK1 was not significantly changed.After treatment with BP-1-102,the expression level of P-STAT3 protein was down-regulated and restored to the level of the ICH model group,and the key proteins of cell proliferation(Cyclin D2 and CDK2)and migration(Rac1,Rho A and Cdc42)were reduced to the level of the ICH model group compared with the leptin group.Conclusion: Leptin promotes angiogenesis and improves neurological deficits after intracerebral hemorrhage through the pericellular transcriptional activator 3(STAT3)signaling pathway.