| Objective:1.To observe the regulation of circadian rhythm and the core clock gene Bmal1 on endothelial cells during angiogenesis after ischemic stroke;2.To explore the regulating effect of Huoxue Rongluo prescription on circadian rhythm after ischemic stroke and the mechanism of promoting angiogenesis.Methods and Results:PartⅠ Effect of environmental circadian disruption on angiogenesis after ischemic strokeMethods:(1)The rat model of environmental circadian disruption(ECD)was prepared by changing the light phase,and the rat model of middle cerebral artery occlusion(MCAO)was prepared by thread embolism;(2)TTC staining was used to detect cerebral infarction volume in each group after 24 hours of ischemia;(3)m Nss score was used to evaluate the symptoms of neurological deficit in each group;(4)Western blot was used to detect the the expression of core clock protein Bmal1 after24 hours and 10 days of ischemia among all groups,and to detect the differences in the expression of VEGF and MMP2 proteins,the key molecules of angiogenesis,after10 days of ischemia among all groups;(5)HE staining was used to detect the pathological changes in the hippocampus of each group;(6)The expression of CD34in rat brain was detected by immunofluorescence.Results:(1)ECD significantly increased infarct volume in MCAO model(P<0.01);(2)The degree of recovery of neurological impairment in MCAO model reduced by ECD group(P<0.01);(3)24 h after reperfusion,the expression of Bmal1 protein in ischemic penumbra tissue decreased significantly(P<0.05);(4)The expression level of Bmal1 on the 10th day after surgery was significantly higher than that on the 24th hour(P<0.05,P<0.001);(5)At 10 days after surgery,the protein levels of MMP-9 and VEGF,which are closely related to angiogenesis,were higher in MCAO group than in Ctrl group,and the above indexes were significantly lower in MCAO-ECD group than in MCAO group;(6)HE staining showed that the hippocampus,neurons and glial cells in the Ctrl group were arranged neatly with normal structure,while the hippocampal neurons in the MCAO group were arranged disordered,cell membranes were destroyed,cell morphology was swollen,a large number of neurons were lost and died,and part of the nuclei were dissolved,concentrated and shrank.The neurons necrosis,nuclear pyretosis and cell membrane structure destruction were aggravated in the MCAO-ECD group;(7)Microvascular density(CD34~+/Field)in cortex and hippocampus of rats in MCAO group was increased,while that in MCAO-ECD intervention group was significantly lower than that in model group(P<0.05).PartⅡ Role of circadian gene Bmal1 in angiogenesis after ischemia injuryMethods:(1)Mouse brain microvascular endothelial cells(bEnd.3 cell line)were used to establish OGD/R model;(2)Western blot was used to detect the difference of NICD and Bmal1 protein expression at 0,4,8,12,16 and 20 h after OGD injury in bEnd.3 cells;(3)The proliferative ability of bEnd.3 cells in each group was determined by CCK8 method;(4)Chip-seq technique was used to detect the genome-wide characteristics of the transcription binding sites of Bmal1 in bEnd.3cells;(5)The migration ability of cells in each group was detected by wound healing and transwell assay;(6)The angiogenesis ability of bEnd.3 cells in vitro was detected by tube forming assay;(7)The m RNA and protein expressions of VEGFR2,MMP2,MMP9,VEGF,Ang,Jag1,Notch1,DLL4,Hes1 and Hey1 were detected by RT-PCR and Western blot;(8)The expressions of Bmal1,VEGF and NICD were detected by immunofluorescence.Results:(1)After OGD injury,the protein levels of Bmal1 and NICD in bEnd.3 cells were significantly up-regulated at 12 h and 20 h,respectively(P<0.01);(2)Notch1m RNA levels began to increase at 4 h after OGD,and the oscillation rhythm was enhanced.In terms of VEGF,VEGF m RNA levels increased to varying degrees at all time points except 12 and 16 h after OGD,and the diurnal oscillation amplitude was significantly enhanced;(3)The results of Ch IP-seq suggest that Bmal1 binds to the enhancer region of Notch1,Hes1 promoter region and VEGF promoter region,and participates in various biological functions;(4)After Bmal1 knockdown,endothelial cell angiogenesis,including tube formation,transwell migration,and wound healing,was limited(P<0.05,P<0.01);(5)RT-q PCR indicate that knockdown Bmal1significantly decreased VEGF m RNA expression(P<0.05),while overexpression of Bmal1 increased the expression of angiogenesis related factors,including VEGF(P<0.001),MMP2(P<0.001),MMP9(P<0.05),ANG(P<0.05),Notch1(P<0.05),DLL4(P<0.01),Hes1(P<0.01)and Hey1(P<0.001);(6)Overexpression of Bmal1significantly increased the proliferation rate of bEnd.3 cells(P<0.05);(7)Under normal oxygen condition,overexpression of Bmal1 significantly enhanced cell migration(P<0.01),while under glucose oxygen deprivation condition,overexpression of Bmal1 significantly enhanced the migration and tubular ability of bEnd.3 cells(P<0.05,P<0.01,P<0.001);(8)Immunofluorescence detection showed that overexpression of Bmal1 further upregulated the protein expressions of VEGF and Notch1 in cytoplasm and nucleus after hypoxia injury(P<0.05);(9)Under normal oxygen conditions,overexpression of Bmal1 up-regulated the protein levels of VEGF(P<0.05)and MMP2(P<0.01),and the hypoxia-induced increase in protein expression was further enhanced by Bmal1.Including VEGF(P<0.001),VEGFR2(P<0.05),MMP-2(P<0.001),and MMP-9(P<0.05).The protein levels of NICD(P<0.05)and DLL4(P<0.05)were also up-regulated in Bmal1 overexpressed BMAL1 bEnd.3 cells.Overexpression of Bmal1 further increased the expressions of NICD(P<0.01),related receptors Jag1(P<0.01)and Dll4(P<0.05),and downstream proteins Hes1(P<0.05)and Hey1(P<0.01).PartⅢ Huoxuerongluo formula promotes angiogenesis of MCAO through Bmal1-VEGF-Notch1 signaling pathwayMethods:(1)CCK8 method was used to detect the cell survival rate of Huoxue Rongluo prescription(HXRL);(2)The effects of HXRL drug-containing serum on cell migration were detected by wound healing and transwell assay;(3)The effect of HXRL drug-containing serum on cell angiogenesis was determined by tube forming experiment;(4)Western blot analysis was performed to determine the effects of HXRL drug-containing serum on cell clock and angiogenesis related proteins in each group;(5)m Nss score was used to evaluate the effect of HXRL on neurological impairment in each group;(6)HE staining was used to detect the effect of HXRL on the pathological changes of hippocampus in each group;(7)Immunofluorescence was used to detect the expression of CD34 in cortex and hippocampus of each group of rats;(8)Western blot analysis was performed to detect the differences in the expression of Per1,Bmal1,MMP2,MMP9 and VEGF by HXRL in each group;(9)The differences in serum IL-1β,IL-18,TNF-α,Melatonin and VEGF expression of HXRL in all groups were determined by ELISA.Results:Vitro experiments:(1)OGD injury for 6h significantly decreased the survival rate of bEnd.3 cells(P<0.001),10%and 15%HXRL drug-containing serum significantly increased the survival rate of cells(P<0.01,P<0.001),and 10%Huoxue Rongluo prescription medicated serum had the best effect;(2)HXRL medicated serum could further improve the migration ability(P<0.01,P<0.001)and angiogenesis ability(P<0.05)of OGD/R injury in bEnd.3 cells.The ability of HXRL to promote migration(P<0.01,P<0.001)and angiogenesis(P<0.05)was limited;(3)Western blotting results indicated that the protein expression levels of Clock,Bmal1,NICD,DLL4,MMP2 and VEGF in bEnd.3 cells were significantly increased after OGD/R injury(P<0.05,P<0.01,P<0.001);(4)The protein expression levels of Clock,Bmal1,NICD,DLL4,MMP2 and VEGF were further increased(P<0.05,P<0.01,P<0.001)after the intervention of HXRL medicated serum.The protein expression levels of NICD,DLL4,MMP2 and VEGF were significantly decreased(P<0.05,P<0.01,P<0.001);(5)Immunofluorescence results showed that the expression levels of VEGF and NICD in bEnd.3 cells were significantly increased after OGD/R injury(P<0.001).After the intervention of HXRL drug-containing serum,the protein expression levels of VEGF and NICD were further increased(P<0.001).After Bmal1 was knocked down,the protein expression levels of VEGF and NICD were significantly decreased(P<0.01,P<0.001);Vivo experiments:(6)m Nss scores in MCAO-ECD group were higher than those in MCAO group,and scores on day 10 of administration groups were lower than those in MCAO and MCAO-ECD groups(P<0.05);(7)Cell morphology in hippocampal region of rats in MCAO-ECD and MCAO groups was damaged,showing liquefaction,vacuole-like necrosis,increased cell space,reduced volume of remaining cells,nuclear pyresis,deep staining,and unclear cell boundaries.The morphology,arrangement,nuclear clarity and interstitial staining uniformity of rat cerebral cortex cells were improved in different administration groups;(8)Compared with MCAO group,the number of CD34 positive cells was significantly decreased(P<0.05),and the number of CD34 positive cells in cortex and hippocampus was increased in each administration group(P<0.05);(9)Western blot results indicated that compared with MCAO group,the expressions of angiogenesis related factors MMP-2,MMP-9 and VEGF in Mc Ao-ecd group were significantly decreased(P<0.05).The protein expressions of MMP-2,MMP-9 and VEGF were increased in different degrees in each Chinese medicine intervention group(P<0.05 or P<0.01);(10)Compared with MCAO group,the expression levels of Bmal1 and Per1 in MCAO-ECD group were significantly decreased(P<0.01 or P<0.01),while the expression levels of Bmal1 and Per1 in each administration group were significantly increased(P<0.01 or P<0.01).Compared with the sham operation group,serum VEGF expression level in the MCAO group was significantly increased(P<0.01),melatonin was decreased(P<0.01),and IL-1β,IL-18 and TNF-αwere significantly increased compared with the sham operation group(P<0.05,P<0.01 or P<0.01).The contents of VEGF and melatonin in MCAO-ECD groups were further decreased(P<0.05 or P<0.01),while the contents of IL-1βand TNF-αin serum were further increased(P<0.05 or P<0.01),and the contents of VEGF and melatonin in serum in each administration group were significantly increased(P<0.05 or P<0.01).The contents of the above three inflammatory factors were significantly decreased(P<0.05 or P<0.01).Conclusion:1.The environmental circadian disruption delays the angiogenesis process after ischemic stroke,aggravates the inflammatory response and neurological deficit symptoms;2.Bmal1,the core gene of biological rhythm,plays an important role in endothelial cell angiogenesis,and overexpression of Bmal1 can promote ODG/R injury to the angiogenesis capacity of bEnd.3 cells;3.HXRL activates VEGF-Notch1 signaling pathway by regulating the expression of Bmal1,thus promoting angiogenesis after ischemic stroke;4.HXRL can improve the angiogenesis ability of circadian rhythm disturbance and reverse the symptoms of increased inflammation and neurological impairment. |
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