| BackgroundWith the deepening of population aging process,cerebrovascular disease has become a common and frequently-occurring disease that endangers the health of middle-aged and old people.Because of its high incidence,high disablement rate and high recurrence rate,regeneration and repair of nerve after cerebral ischemia is a hot and difficult topic in neuroscience.More and more studies show that the axons of injured neurons can regenerate if placed in the appropriate microenvironment.Tongluo Huatan Capsule is optimized on the basis of "Huatan Tongluo Decoction"and has been used in clinical treatment of cerebral infarction.On the basis of previous research,we used cell biology and molecular biology to study the effect of Tongluo Huatan Capsule on Nogo-A,GAP-43 protein expression and glial scar formation,explore its effect on axonal regeneration,and to provide theoretical basis for the application of traumatic hemorrhagic shock,traumatic diffuse brain swelling and subarachnoid hemorrhage in cerebral ischemia injury and other diseases.Objective1 To study the effect of Tongluo Huatan Capsule on the expression of Nogo-A protein,GAP-43 protein and glial scar formation after cerebral ischemia injury in MCAO rats.2 To study the effect of serum containing Tongluo Huatang with different concentration on the proliferation of glial cells and the expression of neurocan,which is highly correlated with glial cell proliferation,and to explore its effect on the regeneration of axons.Methods1 48 rats were randomly divided into normal group,model group,low-dose group of Tongluo Huatan capsule and high-dose group of Tongluo Huatan capsule.Model of middle cerebral artery ischemia in rats was made with reference to Koizumi method in experimental group(Middle Cerebral Artery Occlusion,MCAO).The anterior median incision was made in front of the neck in rats,and the common carotid artery,external carotid artery and internal carotid artery were carefully dissociated,The right common carotid artery and the external carotid artery and the internal carotid artery were ligated and only the internal carotid artery was retained in the skull trunk.4-0 nylon thread was inserted at the bifurcation of the common carotid artery,Push 17-18mm along the internal carotid artery,showing that the plug head had passed through the beginning of the middle cerebral artery to block the middle cerebral artery,tighten the distal end of the carotid artery,and leave 1cm outside the nylon thread,Suture the skin,after 1 hour of ischemia,gently pull the remaining thread to the resistance,suggesting that the end of the nylon thread has reached the incision of the common carotid artery,realizing the middle cerebral artery reperfusion,and the molding is completed.After successful molding,low and high doses of Tongluo Huatan capsule solution were given 324mg/kg,648mg/kg per day,The model group was given normal saline and the blank group was kept for 7 days without any treatment.Six rats from each group were killed and their brains were harvested at two time points on the 7th and 14th day respectively,the expression of Nogo-A and GAP-43 was detected by Immunohistochemical Method.2 Rats were randomly divided into normal group and Tongluo Huatan capsule group.Tongluo Huatan Capsule group was given Tongluo Huatan Solution 648mg/kg every day for 7 consecutive days.On the 7th day,blood was taken from femoral artery for 2 hours.Leave at 4℃,2000r/min centrifugally for 10mins,and then take the supernatant,56℃,30mins inactivation,finally kept at-20℃.The rat primary cell glial cells cultured in serum containing different concentrations were cultured in vitro.The effect of different concentration of serum containing drug on the migration of glial cells was observed by scratch test.The expression of neurocan was measured by western blot technique.Statistical methods:SPSS 23.0 statistical software was used to complete the statistical analysis.The data were expressed in the form of mean±standard deviation(x±s).The statistical method was One-way ANOVA,and P<0.05 was statistically significant.Result1 After the establishment of cerebral ischemia reperfusion model in rats,Tongluo Huatan capsule solution was given by gastric perfusion.The expression of Nogo-A was decreased in both high and low dose groups of Tongluo Huatan capsules(P<0.05),and the expression of GAP-43 protein was up-regulated(P<0.05).But there was no significant difference between the high and low doses of Tongluo Huatang group and the above-mentioned two indexes.2 According to the results of scratch test,although the drug-containing serum did not exert a complete inhibitory effect on the injured rat primary glial cells,when the concentration gradient increased,the glial cells showed a certain growth inhibition trend.Neurocan was detected in primary glial cells of rats cultured in vitro for 21 days.The results showed that the content of Neurocan in the cell culture of the highest drug containing serum decreased to some extent.ConclusionTongluo Huatan Capsule can up-regulate the expression of GAP-43 protein in cerebral ischemia area,and decrease the expression of Nogo-A protein at the same time,and it can inhibit glial scar formation to some extent.Thus,Tong luo Hua tan Capsule can probably promote axonal regeneration in cerebral ischemia area. |
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