| Objective:To investigate the effects of effective fraction from Buyang Huanwu Decoction(EFBHD) on rats following focal cerebral ischemia and reperfusion and to explore protective effects of EFBHD on the HT22 nerve cell and the mechanism of EFBHD neuroprotections. The result suggested that EFBHD can lighten cerebral ischemia reperfusion injury in multiple ways and provide proof for clinic to facilitate work.Methods:1. Rat model of cerebral ischemia-reperfusion injury was created by the middle cerebral artery occlusion (MCAO) with modified suture method, The nervous deficit symptoms were observed and the infarct volume was measured by triphenyltetrazolium chloride (TTC) staining after the cerebral ischemia. The contents of TNF-α, IL-1β, and IL-6 in serum were measured by enzyme-linked immunosorbent assay (ELISA) methods. In addition, the activity of LDH and MDA content in serum were determined. Observing cerebral pathomorphology through HE.The expression of TNF-α,NF-κB and Nestin were detected by Immunohistochemistry technology.2. HT22 hippocampal nerve cells ischemic injury model was established with sodium dithionite in DMEM without glucose.for 24h. MTT assay and MDA,LDH releasing rate were used to detect the cell viability.Hoechst33342 staining was used to assay HT22 hippocampal nerve cells apoptotic,and apoptosis rate was measured by flow cytometer.Results:1. Neurological function deficit symptom scores and infarct volume:Compared with the sham group,The model group had seriously neurological deficit, EFBHD (200mg·kg-1)could significantly improve the grade of nerve function in comparison with the model group, decrease the cerebral infarct volume.2. Serum biochemical indicators:The activity of LDH of model rats increased, the contents of MDA, TNF-α, IL-1β, and IL-6 were increased as well.In contrast,EFBHD(100,200mg·kg-1)could significantly inhibit the activity of LDH, and reduce the contents of MDA, TNF-α, IL-1βand IL-6 in serum.which may be decreased the damage.3. HE staining: Sham group had no infarction areas and evident pathological change.In model group,the number of neurons at ischemic area decreased,nerve cells shrank and degenerated,apparent edema between tissues and vacuolization.But in EFBHD group,the number of neuron which appeared degeneration and necrosis decreased,vacuolization and inter-tissue edema have been relieved when compared with model group.4. Immunohistology:The result show that:Sham group have a few positive TNF-αand NF-κB cells;In model group, the expression of TNF-αand NF-κB seriously increased. EFBHD(100,200mg·kg-1)could inhibit the expression of TNF-αand NF-κB. Sham group have no Nestin cells, model group have a few Nestin cells. EFBHD(100,200mg·kg-1) promote the expression of Nestin cells.5. HT22 cell survival rate:In control group, HT22 hippocampal neurons present metuliform or polygon,boles and branches of synapses extended and thickening obviously,conspicuous halation,refraction strengthen,to form crowded network. Neurons damage induced by oxygen-glucose deprivation,lower outstanding refraction,parts of cell rounding,shrinkage,defluxion,flagment.EFBHD ( 100,200mg·kg-1could significantly improve the injury of hippocampal neuronal cell,it could obviously increased cell survival rate. improve and decreased the activity of LDH and the MDA content after hypoxia hypoglycemia.6. HT22 cell apoptosis rate:Hoechst33342 fluorescence staining showed that control group HT22 hippocampal neurons present orbicular-ovate,blue fluorescence was dispersed, model group appeared apoptotic body and apoptosis nuclear fragmentation,which showed half-moon or broken bits brighting blue fluorescence. EFBHD could inhibit the apoptosis of HT22 hippocampal neurons,and decreased the content of apoptotic body.FCM suggested that control group had low apoptotic peak, model group had extremely a higher apoptotic peak compared with model group. EFBHD(100,300mg·L-1)could reduce the apoptosis rate of the injured hippocampal neurons induced by hypoxia hypoglycemia. which detected by FCM.Conclusion:1. EFBHD has significant protection on cerebral ischemia-reperfusion injury in rats, which may be related to the inhibition of inflammatory cytokine secretion and expression.and inducing the inflammation in brain tissue.2. EFBHD has a significant protective effect on HT22 hippocampal nerve cell oxygen-glucose deprivation injury.The mechanism might be related to antioxidation, and inhibition of apoptosis.
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