Study On The Mechanism Of Cholinergic Anti-inflammatory Pathway In The Cerebral Ischemic Tolerance Induced By Acupuncture Preconditioning

Objective:To investigate the protective effect and mechanism of the acupuncture preconditioning of"Fengchi" acupoints in cerebral infarction rats,we select cholinergic anti-inflammatory pathway as the breakthrough point,to research the action mechanism of cholinergic anti-inflammatory pathway in the cerebral ischemic tolerance induced by acupuncture preconditioning.Method:The experimental study is divided into two parts.Part one:78 adult male SD rats are randomly divided into 5 groups,including 6 rats of normal group,and 18 rats for the following groups:sham group,cerebral infarction group,ischemic preconditioning group,acupuncture preconditioning group.Each group is divided into one,three and seven days.Middle cerebral artery occlusion(MCAO)model is prepared by using the modified Zea Longa,The acupuncture preconditioning group is acupunctured�Fengchi" acupoints before the MCAO model prepared consecutive 7 days.The ischemic preconditioning group is inserted home-made occlusion through external carotid artery to block the middle cerebral artery blood flow intermittent 30 minutes before the MCAO model prepared 24 hours.We record the neurological function score in 4 hours before the rats are killed,and detect the cell morphology using the method of HE and the apoptosis using the TUNEL.Real-time PCR and Western Blot are used to detect the expression of gene and protein of a7-nAChR?TLR4?NF-?B.All results are expressed as mean�standard deviation(X�S),analyzed by the software of spss 19.0.Part two:36 adult male SD rats are randomly divided into normal group,Sham group,cerebral infarction group,acupuncture preconditioning group,acupuncture+vagotomy group and acupuncture + antagonists group,each group contains 6 rats.The method of MCAO and acupuncture preconditioning is the same as part one.The rats of acupuncture + vagotomy group are cutted off the right vagus nerve 3 days before start the acupuncture ttreatment,and then treated by acupuncture for 7 days,at length prepared the MCAO model.The rats of the acupuncture + antagonists group are acupunctured and intraperitoneal injected of MLA at the same time,and then prepared the MCAO model.The neurological function score is recorded in each group at the time of animal awake after 12 hours and death 4 hours before.The record also includes the cell morphology,the apoptosis and the expression of gene and protein of ?7-nAChRs?TLR4?NF-?B using the same method of part one.we detect the a7-nAChR expression in astrocytes and microglial cells using double immunofluorescence staining.All results are expressed as mean � standard deviation(X�S),analyzes using the software of spss 19.0.Results:Part one:the neurological function score is improved significantly compared with the cerebral infarction group in both ischemic preconditioning group and acupuncture preconditioning group,p<0.05.The cerebral infarction group has the heaviest changed of the morphology cells on 3 day,but the degree of the injury in ischemic preconditioning group and acupuncture preconditioning group is slightly compared with the cerebral infarction group at the same time point.Apoptosis test show:More TUNEL-positive cells are detected in the cerebral infarction group?ischemic preconditioning group and acupuncture preconditioning group,there are significant differences than the normal group,p<0.01.It begin to increase at 1 day,peak at 3 day and decrease at 7day in all most three groups.The numbers of TUNEL-positive cells of ischemic preconditioning group and acupuncture preconditioning group are significantly lower than that of the cerebral infarction group at the same time point.Real-time PCR result:the a7-nAChRmRNA expression of the cerebral infarction group,the ischemic preconditioning group and the acupuncture preconditioning group are decreased compared with the normal group both at 1 day and 3 day.There are higher expression in the ischemic preconditioning group and acupuncture preconditioning group than the cerebral infarction group,p<0.05.But at 7 day there are no significant differences in the three groups,p>0.05.The TLR4mRNA and NF-?B mRNA expression of sham group,cerebral infarction group,ischemic preconditioning group and acupuncture preconditioning group are increased compared with the normal group both at 1day and 3 day.There are lower expression in the ischemic preconditioning group and acupuncture preconditioning group than the cerebral infarction group,p<0.05.But the expression of TLR4mRNA and NF-?BmRNA has no significant differences in both three groups at 7 day,p>0.05.Western Blot result:the expression of a7-nAChR protein are decreased as well as the TLR4 and NF-?B protein are increased in sham group,cerebral infarction group,ischemic preconditioning group and acupuncture preconditioning group than the normal group,p<0.01.But there are higher expression of a7-nAChR protein and lower expression of TLR4 and NF-?B protein in the ischemic preconditioning group and acupuncture preconditioning group than the cerebral infarction group,p<0.01.Part two:There are something neurological function defect in both the cerebral infarction group,the acupuncture preconditioning group,the acupuncture+vagotomy group,and the acupuncture+antagonists group,and the neurological function score are improved significantly in the acupuncture preconditioning group and the acupuncture+vagotomy group compared with the cerebral infarction group,p<0.05.But the acupuncture +antagonists group is similar to the cerebral infarction group,p>0.05.HE staining results show that the cerebral infarction group has the heaviest changed of the morphology cells similar to the acupuncture+antagonists group,and there are less change in the acupuncture preconditioning group and the acupuncture+vagotomy group.Apoptosis test shows that there are more TUNEL-positive cells detected in the cerebral infarction group,the acupuncture preconditioning group,the acupuncture+vagotomy group and the acupuncture +antagonists group than the normal group,p<0.01.The numbers of TUNEL-positive cells of the acupuncture preconditioning group and the acupuncture+vagotomy group are less than the cerebral infarction group,p<0.05.But the acupuncture+antagonists group is similar to the cerebral infarction group,p>0.05.Real-time PCR test show that the expression of a7-nAChRmRNA are increased and the NF-KBmRNA are decreased in both the acupuncture preconditioning group and the acupuncture+vagotomy group than the cerebral infarction group,p<0.05.the expression of TLR4mRNA is decreased in the acupuncture preconditioning group than the cerebral infarction group p<0.01,but it is similar in the acupuncture+vagotomy group to the cerebral infarction group,p>0.05.The expression of'a7-nAChRmRNA,TLR4mRNA and NF-?B mRNA in the acupuncture+antagonists group are similar to the cerebral infarction group,p>0.05.Western Blot result:the expression of'a7-nAChR protein is increased as well as the TLR4 and NF-?B protein is decreased in both the acupuncture preconditioning group and the acupuncture+vagotomy group than the cerebral infarction group,P<0.01.But the expression of a7-nAChR,TLR4 and NF-kB protein in the acupuncture+antagonists group are similar to the cerebral infarction group,p>0.05.Immunofluorescence detection show that the protein of a7-nAChR is expressed on astrocytes and microglia in all of the test groups,there are higher expression of a7-nAChR/GFAP and a7-nAChR/CD11b in the acupuncture preconditioning group and the acupuncture+vagotomy group than the cerebral infarction group,p<0.05.But the expression in the acupuncture+antagonists group is similar to the cerebral infarction group,p>0.05.Conclusions:1.Acupuncture "Fengchi" acupoint preconditioning has the similar neuroprotective effect to the ischemic preconditioning,it can enhance the ability of brain ischemic tolerance in rats,and also improve the nervous function,reduce the injury of nerve cells,and inhibit the apoptosis.2.Acupuncture "Fengchi" acupoint preconditioning can increase the expression of the a7-nAChR mRNA and protein and decrease the expression of TLR4 and NF-kB.3.Cutting off the unilateral vagus nerve can not block the brain protective effect of acupuncture preconditioning completely,it also can not block the effect of the expression of a7-nAChR,TLR4 and NF-kB.4.MLA can block the brain protective effect of Acupuncture Preconditioning,and the the expression of a7-nAChR,TLR4 and NF-kB.5.There are important roles of cholinergic anti-inflammatory pathway in the effect of cerebral ischemic tolerance induced by acupuncture preconditioning,it is associated with the adjusted of the expression of TLR4 and NF-kB.