| Objective: By establishing the model of focal cerebral ischemia-reperfusion in rats to simulate ischemic stroke,so that to observed the effects of longzhi decoction,butylphthalide and endoplasmic reticulum stress blocker on neurobehavior,brain histopathology,cerebral infarct volume,microvessel density counts,expression of GRP78,CHOP mRNA and protein.From the point of endoplasmic reticulum stress,to explore the mechanism of Longzhi Decoction in promoting angiogenesis after ischemic brain injury.Methods: (1) First Part: 24 rats were randomly divided into 4 groups:Sham-operated group(saline+sham operation),Cerebral ischemia-reperfusion24 h group(saline+surgery),Cerebral ischemia-reperfusion 3 d group(saline+surgery)and Cerebral ischemia-reperfusion 7 d group(saline+surgery),6 rats in each group.In addition to the sham-operated group,the other three groups were established with a modified Longa thread occlusion method to establish a rat model of right cerebral ischemia-reperfusion injury.(1)The neurological scores of the rats in the corresponding groups were evaluated at24 h,3d,and 7d after surgery.(2)Comparison of GRP78 and CHOP mRNA expression in each group by RT-PCR method.(2) Second Part: 108 rats were randomly divided into 6 groups: the Sham-operated group(saline+sham operation),the Model group(saline+surgery),the Longzhi Deconction group(longzhi deconction+surgery),the Butylphthalein group(butylphthalein+surgery),the 4-PBA group(endoplasmic reticulum stress blocker group,4-PBA+saline+surgery),the 4-PBA+Longzhi Deconction group(4-PBA+longzhi deconction+surgery),12 rats in each group.(1)On the 3rd day after surgery,the neurological deficits were scored on rats in each group.(2)The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.(3)Observation of pathological changes of brain tissue in hippocampus by hematoxylin-eosin(HE)staining.(4)Comparison of GRP78 and CHOP mRNA expression in each group by RT-PCR method.(5)Microvascular Density Count MVD in Cerebral Infarcted Areas was measured by immunohistochemical staining.(6)The expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP in each group was detect by Western blotting.Results: (1) First Part:(1)Compared with the sham operation group,the neurological deficit scores of the rats in each ischemia-reperfusion group were improved in varying degrees(P<0.05),and the neurological deficit scores of the rats in the cerebral ischemia-reperfusion 3d group were highest(P< 0.05).(2)Compared with the sham-operation group,the relative expression of GRP78 and chop mRNA in the brain tissues of the rest groups were significantly increased,and reached a peak value at 3d after cerebral ischemia-reperfusion.(2) Second Part:(1)Compared with the model group,the neurological deficit scores of the longzhi deconction group,the butylphthalide group,the 4-PBA group,the4-PBA + longzhi deconction group were significantly lower(P<0.05),and there was no significant difference between the longzhi deconction group and the butylphthalide group(P < 0.05),and no obvious neurological deficit symptom was observed in the sham-operation group.(2)In the sham-operated group,the brain tissue was stained with TTC dye to a uniform dark red color,while the brain tissue of the other groups showed different degrees of gray-white cerebral infarction.Compared with the model group,the percentage of cerebral infarction volume in the longzhi decoction group and the butylphthalide group was significantly reduced(P<0.05).Compared with the longzhi decoction group,the volume of cerebral infarction in the 4-PBA group was significantly larger(P<0.05),and there was no significant difference in the butylphthalide decoction group and the longzhi decoction group(P﹥0.05).(3)Under the light microscope,the morphological structure of nerve cells in the brain tissue of the sham-operation group was basically normal,while that in the other groups had varying degrees of ischemic necrosis.The nerve cells in the model group were serious ischemic necrosis,in the longzhi decoction group,the butylphthalide group,the 4-PBA+longzhi decoction group all were ischemic necrosis,and in the 4-PBA group were moderate ischemic necrosis.(4)Compared with the sham-operation group,the relative expression of GRP78 and chop mRNA in the brain tissue of the model group were significantly increased(P < 0.05).Compared with the model group,the expression level in the longzhi decoction group and the butylphthalide group were significantly decreased(P < 0.05).Compared with the longzhi decoction group,the relative expression of GRP78 and CHOP mRNA were higher in the 4-PBA group(P<0.05),and there was no significant difference between the 4-PBA+longzhi decoction group and the Longzhi Decoction group(P ﹥ 0.05).(5)Compared with the sham-operation group,the microvessel density in the brain tissue of the other groups were increased in varying degrees(P < 0.05).Compared with the model group,the microvessel density of the longzhi decoction group,the butylphthalide group,the 4-PBA+longzhi decoction group was significantly higher(P < 0.05).Compared with the longzhi decoction group,the 4-PBA group was significantly lower(P < 0.05).(6)Western blot showed that compared with sham-operation group,the expression of GRP78 and CHOP protein in the model group were significantly increased(P < 0.05).Compared with the model group,the expression level of GRP78 and CHOP protein in the longzhi decoction group,the butylphthalide group,the 4-PBA group and the 4-PBA+longzhi decoction group were significantly reduced(P<0.05).Conclusion: (1) Longzhi Decoction may decrease the expression of GRP78 and CHOP to inhibit the over endoplasmic reticulum stress response in brain tissues of rats,thereby improving the cerebral ischemia necrosis of brain tissues and exerting brain protective effects.(2) Inhibition of endoplasmic reticulum stress response of vascular endothelial cells in rats with cerebral ischemia-reperfusion may be one of its mechanisms of promoting angiogenesis. |
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