Research On The Protective Mechanism Of Blood-brain Barrier Of Acupuncture At GV20and ST36in Cerebral Ischemia Reperfusion Injury Rats

ObjectivePost-ischaemic reperfusion cause edema formation followed by blood-brain barrier (BBB) destruction thus caused cerebral ischemia reperfusion injury. This paper is to explore making stabilized rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) by using suture-occluded method, and the injury mechanism of blood-brain barrier, and to explore the protective mechanism of blood-brain barrier of acupuncture at GV20and ST36in cerebral ischemia reperfusion injury rats, and to provide the scientific basis for acupuncture treatment on ischemic cerebrovascular disease.MethodsAdult Sprague-Dawley rats were randomly divided into the following groups:the sham (S) group, the middle cerebral artery occlusion (MCAO) model groups of2h MCAO followed by1,3,5,7d of reperfusion, the electro-acupuncture (EA) groups of2h MCAO followed by1,3,5,7d of reperfusion and the edaravone (ED) groups of2h MCAO followed by1,3,5,7d of reperfusion. The MCAO model was established by placement of a suture to block the middle carotid artery, and reperfusion was triggered by suture removal in all groups except group S. The rats in EA groups received electro-acupuncture treatment by needling at GV20and left ST36. Rats in groups EA, and ED received electroacupuncture, or edaravone, respectively, every day. Neurological function was assessed using the Modified Neurological Severity Score, and infarct volume was evaluated by TTC staining, and the apoptosis cells were detected by TUNEL assay according to the post-reperfusion time point. MMP-2/AQP4/ZO-1/Occludin/Claudin-5mRNA and protein expression were evaluated by Real-time PCR, immunohistochemistry, and western blots, the levels of miRNA-29and miRNA-320in brain and serum was evaluated by Real-time PCR.Results1. TTC staining showed that the area of infarction core was in white colour compared with the common red brain tissue. Among the five slices of brain, the slice around optic chiasma area had the most stable infarction region. Electroacupuncture and edaravone both significantly decreased the volume of the MCAO-evoked infarct region (compare groups EA and ED with the MCAO groups)(P<0.05); no significant differences in infarct volume were detected between the EA groups and the ED groups(P>0.05).2. The tan dyeing was mainly expressed in the nucleus; the apoptosis percentage in the group S was lower than in the other three groups(P<0.05); the apoptosis percentage in the MCAO groups significantly increased than those in the EA group and the ED group(P<0.05); no significant differences in apoptosis percentage were detected between the EA group and the ED group(P>0.05).3. The degree of neurological deficit:the scores in the group S were0; electroacupuncture and edaravone significantly improved neurological function compaird to the MCAO group at the same time point according to the mNSS (P<0.05); and there were no significantly differences between EA groups and the ED groups at the same time point (P>0.05).4. Indicators related to BBB:①Immunofluorescence analysis results indicate that AQP4and MMP-2were both expressed in astrocyte endfeet and endothelial cell-derived blood vessels within the infarct area.②The levels of MMP-2was low in group S; the protein and mRNA levels of MMP-2in ischemia region exhibit a single-peak, the peak time in the MCAO groups, EA groups and ED groups was at5d; the expression of MMP-2in EA groups and ED groups was lower than model group obviously at the same time point (P<0.05); the protein and mRNA levels of MMP-2in the ED groups significantly decreased compared with the EA groups at1d and5d time points (P<0.05).③The mRNA and protein levels of ZO-1in MCAO groups were much lower than those in the EA groups and ED groups(P<0.05); there were no significantly differences between EA groups and the ED groups at5d and7d time point (P>0.05).④Compared with MCAO groups, the protein levels of Claudin-5in the EA groups and ED groups significantly increased at the same time point(P<0.05).⑤The mRNA and protein levels of Occludin in the EA groups and ED groups significantly increased compared with MCAO groups at Id,3d and7d, and there were no significantly differences between EA groups and the ED groups at the same time point (P>0.05).⑥The protein and mRNA levels of AQP4in ischemia region exhibit a single-peak, the peak time in the MCAO groups, EA groups and ED groups was at3d; the protein and mRNA levels of AQP4in EA groups was lower than model group obviously at5d and7d time points (P<0.05), and were lower than ED groups at7d time point (P<0.05).⑦Compared with the MCAO groups, the levels of miRNA-29c in the brain and serum in the EA groups and ED groups significantly decreased at the same time point(P<0.05); compared with the ED groups the levels of miRNA-29c in the brain and serum in the EA groups significantly increased at the1d,3d and5d.⑧Compared with the MCAO groups, the levels of miRNA-320in the brain and serum in the EA groups and ED groups significantly increased at the same time point(P<0.05); the levels of miRNA-320in the brain and serum in the EA groups were significantly decreased compared with the ED groups at the same time point.Conclusion1. The expression of the MMP-2, AQP4and miRNA-29c in the brain related to BBB disruption was increased, and the expression of the ZO-1, Occludin, Claudin-5and miRNA-320in the brain related to BBB protection was decreased in CIRI rats. 2. miRNA-29c in the serum was increased, and the miRNA-320in the brain serum was decreased in CIRI rats, and the expression trends in the serum were correspondent with the brain.3. The function of electroacupuncture at GV20and ST36may work through the intervention of the above process, the expression of the MMP-2, AQP4and miRNA-29c related to BBB disruption was decreased, and the expression of the ZO-1, Occludin, Claudin-5and miRNA-320was increased, thus reduced the blood brain barrier damage after ischemia reperfusion.4. Electroacupuncture at GV20and ST36on CIRI rats could improved neurological function, decreased infarct size and reduce the the apoptosis percentage.Innovation:CIRI model was established by using right middle cerebral artery occlusion and reperfusion, and1d,3d,5d and7d time points was chosed according to the reperfusion time. We observed the role of acupuncture at GV20and ST36on the expression of MMP-2、AQP4、ZO-1、Occludin、Claudin-5in the brain region and the expression of miRNA-29、miRNA-320in the brain and the peripheral serum, and to explore the protective mechanism of blood-brain barrier of acupuncture at GV20and ST36in cerebral ischemia reperfusion injury rats. The results showed that acupuncture treatment had the protective effects on the blood-brain barrier of cerebral ischemia reperfusion injury rats. The intervention of acupuncture at GV20and ST36on the expression of miRNA-29、miRNA-320in the brain and the peripheral serum in CIRI rats have not been reported home and abroad.