| Objective To determine the role of miR-199a-5p in neurogenesis of cerebral ischemia rats and explore the mechanism of action from the target gene hypoxia-inducible factor 1α(HIF-1α),caveolin-1(Cav-1),vascular endothelia growth factor(VEGF)and brain-derived neurotrophic factor(BDNF);then observe whether Buyang Huanwu Decoction(BYHWD)promote neurogenesis in rats with cerebral ischemia by regulating miR-199a-5p.Methods(1)The middle cerebral artery occlusion(MCAO)model was induced by the method of suture and the reperfusion was performed 90 min after ischemia.(2)After 24 hours of ischemia,the first part was divided into model group(MCAO),MCAO+miR-199a-5p agomir(199a-5p agomir)group,MCAO+miR-199a-5p agomir NC(199a-5p agomir NC)group,MCAO+miR-199a-5p antagomir(199a-5p antagomir)group,MCAO+miR-199a-5p antagomir NC(199a-5p antagomir NC)group,5 d after ischemia,intracerebroventricular administration of miR-199a-5p agomir/NC 2.5 nmol and miR-199a-5p antagomir/NC 10 nmol were performed respectively;The second part of the experiment was divided into sham group,model group(MCAO),Buyang Huanwu Decoction(BYHWD)group,Buyang Huanwu Decoction+miR-199a-5p antagomir(BYHWD+199a-5p antagomir)group,Buyang Huanwu Decoction+miR-199a-5p antagomir NC(BYHWD+199a-5p antagomir NC)group.After 24 h of ischemia,Buyang Huanwu Decoction(1 ml/100g)was intragastrically administered and Brd U 50 mg/kg was intraperitoneally injected.(3)On the 1st,3rd,7th,and 14th day after ischemia,neurological function was evaluated by modified nerve injury severity score(m NSS)and corner test.On the14th day after ischemia,toluidine blue staining was used to measure the volume of cerebral infarction.(4)On the 14th day after ischemia,Brd U/Nestin,Brd U/DCX,Brd U/Neu N and Brd U/GFAP immunofluorescence double markers were used to detect neurogenesis in cerebral ischemia rats.(5)On the 14th day after ischemia,qRT-PCR was used to detect the expression of miR-199a-5p,HIF-1αand Cav-1 mRNA,and Western Blot was used to detect the expression of HIF-1α,Cav-1,VEGF and BDNF.Results The first part of the experiment:(1)Compared with the sham-operated group,the expression of miR-199a-5p in the model group increased significantly 14days after ischemia(P<0.01);Compared with the model group,the expression of miR-199a-5p in miR-199a-5p agomir group was significantly up-regulated(P<0.01),the expression of miR-199a-5p in miR-199a-5p antagomir group was significantly decreased(P<0.01);(2)Compared with the model group,miR-199a-5p agomir significantly promoted neurological recovery(P<0.05)and decreased infarct volume(P<0.05),and increased Brd U~+/Nestin~+,Brd U~+/DCX~+,Brd U~+/Neu N~+positive cells in cerebral ischemia rats.(P<0.05),down-regulated the expression of HIF-1αand Cav-1mRNA and protein(P<0.05)and up-regulated the expression of VEGF and BDNF protein(P<0.05).The effect of miR-199a-5p antagomir was the opposite of the above;The second part of the experiment:(1)Compared with the model group,the expression of miR-199a-5p in the Buyang Huanwu Decoction group was significantly increased 14 days after ischemia(P<0.05).After injecting miR-199a-5p antagomir into the lateral ventricle,miR-199a-5p expression was significantly inhibited(P<0.05);(2)Compared with the model group,Buyang Huanwu Decoction significantly promoted neurological recovery(P<0.05)and infarct volume(P<0.05),and increased the number of Brd U~+/Nestin~+,Brd U~+/DCX~+,Brd U~+/Neu N~+positive cells in cerebral ischemia rats(P<0.05),down-regulated the expression of HIF-1α,Cav-1 mRNA and protein(P<0.05)and up-regulated VEGF and BDNF protein expression(P<0.05).The miR-199a-5p antagomir reverses these effects.Conclusion miR-199a-5p promotes neurogenesis in cerebral ischemic rats.Buyang Huanwu Decoction promotes neurogenesis in cerebral ischemic rats by upregulating miR-199a-5p,its mechanism is related to regulating VEGF,BDNF,HIF-1,Cav-1 expression. |
PDF Full Text Request