| Objective Taking primary brain microvascular endothelial cells(BMECs)as the research object,an endothelial cell oxygen and glucose deprivation/reoxygenation and reoxygenation(OGD/R)model was established in vitro to simulate the cerebral ischemia-reperfusion process,and to explore Buyang Huanwu Decoction and its similar prescriptions(Yangyin Tongnao Granule,Buyang Huanwu Decoction and Naoxintong Capsule)protects endothelial cells.In vivo,the rat model of cerebral ischemic injury(MCAO)was established to study the effect of Buyang Huanwu Decoction on promoting cerebral microangiogenesis and explore its possible mechanism,which provides new directions and ideas for the treatment of ischemic stroke.Methods(1)Taking SD rats as the research object,the MCAO model was established.Then the rats were randomly divided into 5 groups:sham operation group,model group,Yangyin Tongnao Granule group,Buyang Huanwu Decoction group and Naoxintong capsule group.Seven days after administration,the rats were scored for neurological function.HE staining was used to observe the histopathological changes of cerebral ischemic cortex.Cerebral infarct volume was evaluated by TTC staining.Real-time quantitative PCR was used to detect the mRNA expression levels of MMP-2,HIF-1α,VEGF,Notch1 and Hes-1.Immunohistochemis--try was adopted to detect the expression of CD31 and VEGF protein in ischemic lateral brain tissue.(2)The cultivation of primary BMECs and the establishment of OGD/R model:the neonatal rats born 3 to 5 days were selected for the isolation and cultivation of BMECs.The endothelial cells were purified and identified,and the OGD/R model of the cells was established.(3)SD rats were randomly divided into normal control group,Yangyin Tongnao granule group,Buyang Huanwu decoction group and Naoxintong capsule group.They were given intragastrically for 7 days,and blood was collected from abdominal aorta to prepare drug-containing serum.The CCK-8 method was used to determine the optimal concentration of the serum containing Buyang Huanwu Decoction for acting on BMECs.(4)Taking the fourth-generation BMECs as the research object,five groups were set up,namely normal group,model group,Yangyintongnao granule group,Buyanghuanwutang group and Naoxintong capsule group.Observation of cell migration was carried out in the paddle experiment.The phalloidin staining method was used to detect the cytoskeleton.Hoechst staining was chosen to observe the apoptosis.RT-PCR was sort out to detect the change of Notch1,VEGF and Mmp-2 genes.Western blot was select to detect the expression of key protein Notch1.Results(1)Compared with the model group,the Buyang Huanwu Decoction group could alleviate cerebral ischemia-reperfusion injury to different degrees,reduce cerebral infarction volume,and reduce cerebral nerve function score.Buyang Huanwu Decoction could increase the expression of VEGF protein and reduce the mRNA expression of MMP-2,Notch1 and Hes-1 in different degrees.Among them,Naoxintong group and Buyang Huanwu Decoction had more significant regulation on VEGF.Compared with Yangyin Tongnao Granules,Buyang Huanwu Decoction and Naoxintong could increase the expression of CD31 and promote the formation of microvessels.(2)High-purity BMECs were successfully cultured.Most of the BMECs are fusiform and polygonal,with a single layer of adherent growth,and the cells are closely connected with a"swirl" distribution,which is a typical "paving stone-like".After oxygen-sugar deprivation and reperfusion,the morphology of BMECs changed significantly.The cells contracted and the ability to adhere decreased,and some cells shed or died.The cell gap become larger.The shape of the boundary was irregular,and there was a clear cavity in the cytoplasm.(3)The medicine-containing serum of Buyang Huanwu Decoction was successfully prepared.The CCK-8 method confirmed that 10%drug-containing serum was selected to act on BMECs in each administration group,and subsequent experiments were performed at the administration concentration.(4)After the OGD/R model was established,Buyang Huanwu Decoction could alleviate the damage of BMECs to different degrees,improve cell survival rate,resist apoptosis,stabilize the cytoskeleton,up-regulate the expression of Notchl,and reduce the expression of MMP-2 gene..Compared with Buyang Huanwu Decoction,the effect of Yangyin Tongnao Granule and Naoxintong Capsule on MMP-2 was more significant.Conclusion After cerebral ischemia-reperfusion injury,Buyang Huanwu Decoction may promote blood vessel recovery in the ischemic penumbra by reducing microvascularization in the cerebral tissues of the ischemic side,reducing brain injury,and accelerating the recovery of nerve function.The improvement effect of Naoxintong Capsule and Buyang Huanwu Decoction are more obvious,and its mechanism may be related to the regulation of the expression of HIF-1α/VEGF and Notch1 protein. |
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