| BackgroundAccording to “2014 China Statistical Yearbook of Health and Family Planningâ€, ischemic stroke is still one of the most prevalent diseases that threat the national health. Combined with the present medical situation in our country, it has characteristics of high incidence, high prevalence, high mortality and morbidity, and brings a heavy burden to society[1]. Current clinical treatment for stroke relies mainly mainly relies on on t PA, but due to the strict treatment time window, bleeding risk, informed consent and many other reasons, most patients didcouldn’t n’t accept timely and effective treatment[2]. Therefore, researching the mechanism of brain ischemicischemic stroke and looking for the new treatments becomingbecome an urgent problem.As a family member of matrix metalloproteinases, activated MMP9 can participate the degradation process of ECM and has close relationship with the transfertransformation of tumor cells[3]. In CNS, MMP9 is considered to be one of enzymes that has the closest relation with cerebral microvascular diseases[4]. Animal experiments confirmed that matrix metalloproteinases inhibitor can reverse the degradation of TJP and relieve edema afterstroke[5], revealing the relationship between TJP and MMP9. Another study found that compared with wild type, MMP9 knock-out mice were protected against ischemic injury[6]. Furthermore, it was observed that melatonin treatment starting 1h after injury attenuated BBB disruption during focal ischemicischemia[7]. There are series of results showing that the activation of MMP9 lead to the change of BBB permeability, and cause cerebral edema and brain damage. On the contrary, taking effective measures to reduce the expression of MMP9 will reduce BBB damage after stroke.There are complex regulatory mechanisms after ischemia-reperfusion injury. Our previous investigation has already demonstrated that EA pretreatment has close relationship with the expression of MMP9 in brain[8]. But the potential mechanisms of EA pretreatment–induced neuroprotection are not clear enough, so it is significant tofurthermore explore EA preconditioning of brain protection mechanisms and will be helpful to discover new therapeutic targets.Therefore, this experiment performed the model of MCAO in rats, to observed the effects of EA pretreatment on inflammation, expression of MMP9, TJP integrity and BBB premeability of ischemic area and to investigated the internal mechanism. The aim of this research is to optimize the system of the cerebral ischemic tolerance induced by EA pretreatment and bring a new perspective in curing stroke.Experiment 1: Effects of EA pretreatment on brain ischemia and BBB permeabilityObjective: To reconfirm the cerebral protection by EA pretreatment and to observe the change of BBB permeability and brain edema.Methods:Part 1 The cerebral protection effect induced by EA pretreatment32 male SD rats were randomized to four groups(n=8): sham, EA,MCAO and EA+MCAO. The group of MCAO and EA+MCAO were suffered from occlusion MCA for 2 h, then 48 h reperfusion, the neurological scores were evaluated and infarct size was determined by TTC staining.Part 2 Effect of EA pretreatment on BBB permeability after ischemic96 male SD rats were divided in to sham group(n=24), EA group(n=24), MCAO group(n=24) and EA+MCAO group(n=24). Then each group was divided into 4 subgroups(n=6). After induction of MCAO, to detect the EB spreading in the brain parenchyma, EB permeability and brain water content, EB was injected 46 h after reperfusion by tail vein. Results:(1) EA pretreatment induced cerebral ischemic tolerance. Compared with MCAO group, EA pretreatment can reduce infarct size and improve neurological scores after MCAO(Ρ<0.05);(2) EA pretreatment significantly reduces the BBB damage and the degree of brain edema. Compared with MCAO group, EA pretreatment can reduce the EB distribution and permeability in brain(Ρ<0.05), meanwhile reduce the brain water content(Ρ<0.05).Conclusion:(1) EA pretreatment reduces infarction and improves neurological deficits.(2) EA pretreatment protects against transient MCAO-induced EB extravasation and brain edema.Experiment 2: Effects of EA pretreatment on the expression and activation of MMP9 after stroke.Objective: To testify the neuroprotective effect of EA pretreatment was related with the suppressed expression and activation of MMP9 in the ischemic brain.Methods: 72 male SD rats were randomized into 4 goups(n=18): sham, EA, MCAO and EA +MCAO. After 48 h reperfusion, both brain penumbra and contralateral tissue were collected. The expression and activation of MMP9 were detected by western blot and gelatin zymogram. At the same time, another 6 rats of each group were sacrificed for immunofluorescence staining. In order to eliminate the false positive result, a negative control group(Ab-) was set up in immunofluorescence staining.Results: Compared with MCAO group, the expression and activation of MMP9 were significant reduced in EA+MCAO group(Ρ<0.05). Meanwhile, both the expression and activation of MMP9 were found no obvious difference in contralateral brain tissues of each group.Conclusion: EA pretreatment can reduce the level of MMP9 after ischemic-reperfusion injure.Experiment 3: Effects of EA pretreatment on TJP after stroke.Objective: To investigate if TJP expression was affected by EA pretreatment through reduce the expression of MMP9 after ischemia.Methods: 30 male SD rats were randomized into 5 goups(n=6): sham, EA, MCAO, EA+MCAO and BB1101+MCAO. At 30 min before MCAO, rats were received BB1101(30mg/kg), a synthetic MMP inhibitor, via intraperitoneal injection in BB1101+MCAO group. 48 h after reperfusion, brain penumbra tissue was collected and detected the change of three main TJP by western blot.Results: Compared with Sham group, the expression of Claudin-5ã€Occludin and ZO-1 were obviously reduced in MCAO group(Ρ<0.05); Compared with MCAO group, both the cortex and striatum of infarction side, the expressions of TJPs were highly increased in EA+MCAO group(Ρ<0.05); Meanwhile EA+MCAO group and BB1101+MCAO group had no difference in the expression of Claudin-5 〠Occludin and ZO-1. Also, in contralateral brain tissues of each group no difference of TJP level was found.Conclusion: EA pretreatment attenuates MMP-9 elevation induced TJPs degradation after MCAO.Experiment 4: Effects of EA pretreatment on inflammation levels andNF-κB translocation in brain penumbra area.Objective: To investigate whether the level of inflammation factors and NF-κB nuclear translocation is related to EA pretreatment.Methods: 36 male SD rats were randomized into 4 goups: sham(n=6), EA(n=6), MCAO(n=12) and EA+MCAO(n=12). Then MCAO and EA+MCAO group was divided into 2 subgroups: The first subgroup was treated as before and detected IL-1β, IL-10 and MPO level by ELISA; The second subgroup was combined with Sham and EA group.After 48 h reperfusion, nuclear and cytosolic proteins were extracted each, and then the level of NF-κB subunit p65 was examined by western-blot.Results:(1) EA pretreatment can reduce the inflammation factors level. Compared with MCAO group, IL-1β and MPO level were declined(Ρ<0.05), while IL-10 level rose in EA+MCAO group(Ρ<0.05);(2) The result of western-blot in nuclear protein shows that compared with Sham group, MCAO treatment can increase the p65 level(Ρ<0.05). But compared with MCAO group, both cortex and striatum of infarction side, the p65 level were reduced significantly in EA+MCAO group(Ρ<0.05). On the contrary, the result of western-blot in cytosolic protein shows MCAO treatment decreases the level of p65 than Sham group(Ρ<0.05). At the same time, compared with MCAO group, both cortex and striatum had a highly increase of p65 level in EA+MCAO group(Ρ<0.05).Conclusion: EA pretreatment suppressed local inflammation factors and nuclear translocation of NF-κB in the ischemic brain.SummaryThis research found that EA pretreatment can inhibit the expression and activation of MMP9, so as to reduce the TJP degradation, and maintain the integrity and function of BBB. Also we proved that the mechanism of EA pretreatment has close relation with NF-κB translocation and local inflammatory response. |
