| Objective:The relationship between qi deficiency blood stasis pattern and AQP4,Occludin and claudin-5 was studied by observing the treatment effect of Buyang Huanwu decoction on the rats with apoplexy.The foundation of the molecular organism and the machenisim of the pathology of apoplexy would be discussed and research reference would be generated while observing the effection of the treatment of Buyang Huanwu decoction.Methods:180 male SPF(specific pathogen free animals)healthy SD rats(sprague dawley)were randomly divided into sham-operation group,cerebral ischemia model group(model group),and Buyang Huanwu decoction group,60 animals in each groups.The model of focal cerebral ischemia 120mins reperfusion in rats with qi deficiency and blood stasis pattern was made by using the middle cerebral artery occlusion(MCAO)method with controlled feeding.Buyang Huanwu decoction was applied for gavage three days before modeling.The changes of the symptoms of cerebral ischemia after modeling were observed.Score of the symptoms and signs of qi deficiency and blood stasis pattern and score of neurological function were collected.The water content of brain tissue was measured.Evans blue method was used to detect blood-brain barrier permeability.The ultrastructure of blood-brain barrier was observed by transmission electon microscope.Western Blot was used to detect the blood brain barrier closely related to the tight junction proteins Occludin and claudin-5.The expression of AQP4 in brain tissue was detected by Western Blot and immunohistochemistry.Results:(1)There were no neurological symptoms in all groups before modeling.Postoperatively,the model group appeared obvious neurological symptoms compared with the sham-operated.There was a significant difference between the two groups(P<0.001).Compared with the model group,the neurological function of the Buyang Huanwu Decoction group was significantly improved.On the 3rd day,there was a significant difference between the two groups(P<0.01).There was a difference between the two groups on the 1st and 5th days(P<0.05).(2)The symptoms of qi deficiency and blood stasis pattern in the rats of each group were obviously significant after modeling.In three days and five days,compared with the sham operation group,the qi deficiency and blood stasis patterns were more obvious in the model group,with statistical significance(P<0.01).Compared with the model group,the symptoms and signs of qi deficiency and blood stasis pattern were significantly improved in the Buyang Huanwu decoction group compared with the model group.There was a significant difference between the two groups(P<0.01).(3)In three days and five days,compared with the sham-operated group,the water content of brain tissue in the model group was significantly higher than the sham-operated group.There was a significant difference between the two groups(P<0.01).The brain tissue water content of the Buyang Huanwu decoction group was lower than the model group and there was a difference between the two groups(P<0.05).(4)In three days and five days,compared with the sham-operated group,the EB content of brain tissue in the model group was significantly higher than the sham-operated group.There was a significant difference between the two groups(P<0.01).The brain tissue EB content of the Buyang Huanwu decoction group was lower than the model group and EB content decreased with the extension of time.There was a difference between the two groups(P<0.05).(5)In three days and five days,the intact tight junction of the sham-operated group was relatively integrity,and there were a few vacuoles around the cell.Model group damage was serious and the form of the intact tight junction around the cell vacuoles degenerated.Compared with model group,the intact tight junction gap of Buyang Huanwu decoction group had widened,intermittentened and relatively formed,and there were a few vacuoles around the cell.The above phenomenon appeared a gradually reduced trend with the extension of time.(6)In three days and five days,the expression of Occludin and claudin-5 decreased and the expression of AQP4 increased in the model group compared with the sham group.There was significant statistical difference between the two groups(P<0.01).Compared with the model group,the expression of Occludin and claudin-5 increased and the expression of AQP4 decreased significantly(P<0.05).(7)The expression of AQP4 protein in brain tissue was positive with brown color,mainly distributed in astrocytes and mainly in cell membrane.In three days and five days,compared with the sham-operated group,the expression of AQP4 increased in the model group.There was significant statistical difference between the two groups(P<0.01).Compared with the model group,the expression of AQP4 decreased significantly.There was significant statistical difference between the two groups(P<0.05).Conclusion:After modeling,the rats showed obvious signs of qi deficiency and blood stasis pattern and neural function defects,indicating that the animal model had been built successfully.Buyang Huanwu decoction can obviously relieve the symptoms of rats,and it suggested that Buyang Huanwu decoction is the effective prescription for the treatment of qi deficiency and blood stasis pattern.The effection of Buyang Huanwu decoction may be regulated to rising the expression of Occludin and Claudin-5,reducing the expression of AQP4 to reduce the blood brain barrier permeability,to reduce the water content of brain tissue,thereby to relieve cerebral edema therefore have the effect of brain protection.The Occludin,claudin-5 and AQP4 expression abnormalities may be an important mechanism for the formation of qi deficiency and blood stasis pattern in apoplexy. |
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