| Background: Intracerebral hemorrhage(ICH)is a common neurological disease with high mortality and disability.After ICH,the hematoma compresses the brain tissue causing the direct damage.In addition,compression of the hematoma and edema,as well as hematoma products can cause endothelial cell damage,seriously damaging the microvascular system and aggravating brain damage.Angiogenesis exists in perihematoma tissue after ICH,as an important part of nerve repair.Micro R-451(mi R-451)is a kind of micro RN A that plays an important role in cell proliferation and differentiation,and is involved in the regulation of vascular endothelial function.At present,there are few studies on the role of mi R-451 after ICH,and its expression pattern and role after ICH are still unclear.Objective: This study aims to explore the expression of mi R-451 after intracerebral hemorrhage and its role and mechanism in angiogenesis after ICH.Methods: This study was divided into three parts.In the first part,the mouse caudate nucleus cerebral hemorrhage model was established by stereotactic collagenase injection,and the level of mi R-451 in the peripheral hematoma tissues at 6 time points(1,3,5,7,10 and 14 days)after cerebral hemorrhage and the brain tissues of mice after sham operation was detected by q PC R.After mi R-451 agomir was injected,angiogenesis in the peripheral hematoma tissues after intracerebral hemorrhage was detected by immunofluorescence staining and FITC perfusion.Neurological recovery was assessed by modified neurological score,forelimb placement test,and corner test.Furthermore,the mi R-451 knockout mice and wild-type mice were modeled for ICH respectively,and then angiogenesis and neurological function recovery after IC H were detected by the same methods.The second part was an in-vitro experiment,in which human microvascular endothelial cells(h BMECs)were treated with hemin to establish an in-vitro ICH model.First,h BMECs were treated with hemin a t different concentrations(5 ?M,10 ?M,20 ?M,30 ?M,40 ?M,50 ?M,60 ?M,70 ?M)to explore suitable hemin concentration.Then h BMECs were treated with the selected hemin concentration,and the level of mi R-451 in h BMECs were detected at 12 h,24 h and 48 h respectively to select the best detection time.Then h BMECs were transfected with mi R-451 mimic or mi R-451 inhibitor respectively to upregulate or down-regulate mi R-451 level,and the effect of cell transfection was verified by q PCR.After h BMECs were successfully transfected with mi R-451 mimic or mi R-451 inhibitor,the proliferation of HBMECs was detected by CCK-8 assay.The migration of h BMECs was detected by wound healing assay and transwell migration assay.The angiogenesis ability of h BMECs was detected by matrigel tube formation assay.In the third part,we explored the target genes of mi R-451 and its downstream signaling pathways in animal models.Firstly,the downstream target genes of mi R-451 were searched and predicted on target gene prediction websites such as Target Scan,mi Rand and mi RWalk.The genes with high effect intensity and related to angiogenesis were selected.In order to verify the interaction between mi R-451 and it's potential target genes,we upregulated or knocked out the expression of mi R-451 in the brain tissues of mice with ICH,and then detected the m RNA expression of target gene by q PCR.The protein expression was detected by Western blots(WB).The interaction between mi R-451 and target protein m RNA was detected by double luciferase reporter gene assay.In order to explore the downstream signaling pathway,the expression of related proteins was detected by WB.Mi R-451 knockout mice were intraperitoneally injected with inhibitor of target protein after IC H,and then WB was used to detect the the expression of related proteins.At the same time,the angiogenesis were detected by immunofluorescence staining and FITC perfusion.Results: In the first part of study,it was found that the level of mi R-451 in peri-hematoma tissue after IC H was decreased,reaching the lowest level on the 5th day,and then gradually recovered to the baseline within 14 days.Parenchymal injection of mi R-451 agomir can effectively up-regulate the level of mi R-451 in brain tissue.After up-regulation of mi R-451,less angiogenesis in peri-hematoma tissue and worse recovery of neurological function were detected as compared to mice that received negative control agomire.Mi R-451 knockout mice showed more a ngiogenesis in peri-hematoma tissue and better recovery of neurological function than wild-type mice.The second part verified the role of mi R-451 in regulating angiogenesis in vitro.CCK-8 assay was used to detect the viability of h BMECs.It was showed that the cell viability decreased by 50% after 24 hours' exposure under 60 ?M hemin.This concentration was chosen for subsequent experiments.Mi R-451 levels in hemin-treated h BMECs were detected at different time points.Mi R-451 level changed most significantly after 24 hours' hemin treatment,which was chosen to detect various indicators in subsequent experiments.QPCR results showed that the level of mi R-451 in h BMECs could be effectively upregulated or down-regulated after transfection with mi R-451 mimic or mi R-451 inhibitor,respectively.In this part of the study,it was found that up-regulation of mi R-451 in HBMECs could significantly inhibit the proliferation,migration and tube formation of h BMECs,while down-regulation of mi R-451 could significantly promote the proliferation,migration and tube formation of h BMECs.In third part,target genes of mi R-451 were retrieved through target gene prediction website.It was found that MIF is likely to be the target of mi R-451 in regulating angiogenesis.Further studies showed that MIF m RN A level did not change but protein level were decreased after up-regulation of mi R-451,while MIF m RN A level did not change but protein level were increased after knockout of mi R-451,indicating that there was an interaction between mi R-451 and MIF.Dual luciferase reporter gene assay confirmed that there was a complementary pairing sequence between mi R-451 and the 3'-untranslated region of MIF m RNA.After IC H modeling,the phosphorylation levels of ERK1/2 and Akt in mi R-451 knockout mice were higher than those in wild-type mice by WB.However,after intraperitoneal injection of MIF inhibitor in mi R-451 knockout mice with IC H,the elevated phosphorylation level was significantly reduced,and the pro-angiogenesis effect of MIF was partially offset.Conclusion: The level of mi R-451 was decreased after IC H.Mi R-451 inhibited angiogenesis and neurological recovery after intracerebral hemorrhage by negatively regulating MIF.Mi R-451 complementarily binds to the 3'-untranslated region of MIF m RNA,affecting its protein translation and thus down-regulating the level of MIF protein.MIF plays a role in regulating angiogenesis through ERK1/2 and Akt pathways. |
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