| Stroke is one of the major cause for neurologic disorders and limb disability. Although r-t PA is an effective treatment, only 3- 5% patients can accept and may benefit from it, duing to the relatively narrow therapeutic time window[1]. Electroacupuncture(EA) neuroprotection strategy proposed by our department is a new therapeutics strategy to prevent perioperative cerebral ischemia injury. EA can relieve neurologic disorders and reduce infarct volumes after focal cerebral ischemia[2]. Our previous researches discovered that EA could increase the production of endocannabinoid AEA, 2-AG and CB1R[3], which regulated the RISK pathway(ERKε, PKC, GSK-3β, STAT3)and inhibited neuron apoptosis[4-6]. In addition, we discovered EA could attenuate cerebral ischemic injury through regulating α7n ACh R-mediated inhibition of HMGB 1 release in rats[7].As an integration platform to trigger inflammation, NLRP3 inflammasome is composed of NLRP3, ASC and procaspase 1[8]. When all kinds of danger signals interacting with receptor TLRs on the cell membrane, it will activate the NF-κB pathway and promoted the synthesis of inflammasome proteins and pro IL-1β, pro IL-18[9]; Moreover, the intracellular DAMPs could oligomerize NLRP3 sensors and activate procaspase 1, which promoted the cleavage and release of IL-1β、IL-18. Furthermore,caspase 1 triggered a lytic mode of cell death, pyroptosis[8]. In conclusion, NLRP3 inflammasome plays an important role in initiating innate immune response, the activation of caspase 1 and neuroinflammation after cerebral ischemia[10].In this study, we aim to find out whether EA could attenuate cerebral ischemic injury through α7n ACh R-mediated inhibition of NLRP3 inflammasome caused inflammatory response in rats. Provided more experimental basis for the neuroprotection of EA, and provided a solid theoretical basis for the clinical application of EA.Experiment 1 The effect of cerebral ischemia-reperfusion on the expression of inflammasome proteins in ratsObjectivesTo observed the expression changes of inflammasome proteins at the different time points after cerebral ischemia-reperfusion injury in rat.MethodsTwenty male SD rats were randomized into five groups: Sham(n=4), MCAO6h(n=4), MCAO24h(n=4), MCAO3d(n=4), MCAO7d(n=4). Rats were subjected to MCAO for 1.5h, then withdrawn the filament and the brain blood supply was restored. After 6h, 24 h, 3d and 7d for reperfusion, the rats were sacrificed and the Western Blot were conducted to analyse the expression changes of inflammasome(NLRP3, Cl.Caspase 1, Mature IL-1β).ResultsAs compared with Sham group, the contents of NLRP3 in MCAO24 h, MCAO3 d,MCAO7d were improved(Ρ<0.01), the expression of Cl.Caspase 1 in MCAO3 d, MCAO7 d were increased(Ρ<0.01), and the levels of mature IL-1β in MCAO6 h, MCAO24 h, MCAO3 d, MCAO7 d were up-regulated(Ρ<0.01).ConclusionThe expression level of inflammasome proteins such as NLRP3, Cl.Caspase 1 and mature IL-1β were gradually increased at the different time points after cerebral ischemia-reperfusion injury and the contents were highest in MCAO3 d. So we selected MCAO3 d as the interventional time point in the further research.Experiment 2 The effect of EA to NLRP3 inflammasome-mediated inflammatory reactionObjectivesTo testify the inhibition effect of EA to the inflammatory response induced by NLRP3 inflammasome. And to probe whether EA could regulate the balance between pro-inflammatory factor and anti-inflammatory cytokine.Methods1. Whether EA could inhibit NLRP3 inflammasome associated inflammatory response.Twenty-one male SD rats divided into three groups: Sham(n=7), MCAO3d(n=7), EA+MCAO3d(n=7). The rats belonging to MCAO3 d group and EA+MCAO3d group were subjected to MCAO for 1.5h. At 3d after reperfusion, Western blot was used to detect the alternation of NLRP3, Cl.Caspase 1 and mature IL-1β. Meanwhile, immunofluorescence(IF) staining was undertook to observed the expression of NLRP3 and Caspase1.2. Whether EA regulated the balance between pro-inflammatory factor and anti-inflammatory factor.Twenty-four male SD rats were divided into 3 groups: Sham(n=8), MCAO3d(n=8), EA+MCAO3d(n=8); The levels of IL-18, TNF-α and TGF-β1, IL-10 in brain were tested using El ISA Kits.Results1. In compared with MCAO3 d group, EA decreased the expression level of NLRP3,Cl.Caspase 1 and Mature IL-1β(Ρ<0.01);Meanwhile, the results of IF staining revealed that the co-located of NLRP3 and Caspase 1 with Neu N were inhibited by EA.2. EA down-regulated the levels of IL-18 and TNF-α(Ρ<0.05), at the same time, it up-regulated the contents of TGF-β1 and IL-10(Ρ<0.01), as compared to MCAO3 d.ConclusionWe discovered that EA could not only inhibit the inflammatory response induced by NLRP3 inflammasome, but also regulate the balance between pro-inflammatory factor and anti-inflammatory cytokine.Experiment 3 Involvement of α7n ACh R in EA-induced the inhibition of NLRP3 inflammasome and neuroprotective effects in stroke ratsObjectivesTo validate α7n ACh R participated in the inhibition of NLRP3 inflammasome by EA and it possess the neuroprotective effects.MethodsOne hundred and twelve male SD rats were divided into 7 groups: Sham, MCAO, EA+MCAO, EA+α-BGT, EA+vehicle(α-BGT), PHA-543,613, vehicle(PHA-543,613)(n=16).At 3d after reperfusion, Western Blot was practiced to detect the contents of α7n ACh R in neurons and the levels of NLRP3, Cl.Caspase 1, Mature IL-1β(n=4). The neurological deficits and infarct size were evaluated at 3d after reperfusion(n=8). TUNEL staining were conducted for measurement of neuronal apoptosis(n=4).Results1. In compared with MCAO3 d, EA up-regulated the contents of α7n ACh R after cerebral ischemia in neurons(Ρ<0.01).2. When compared with EA+MCAO group, the expression of NLRP3, Cl.Caspase 1 and mature IL-1β were increased in EA+α-BGT group(Ρ<0.01); Infarct volume were increased(Ρ<0.05) and neurological score was reduced(Ρ<0.01) in EA+α-BGT group, and the celluar apoptosis was increased(Ρ<0.05).3. In compared with MCAO groups, intraperitoneal injection of PHA-543,613 could decrease infarct size(Ρ<0.01), release neurological deficits(Ρ<0.01), and reduced TUNEL positive cells(Ρ<0.01); At the same times, it could down-regulate levels of NLRP3, Cl.Caspase 1 and mature IL-1β(Ρ<0.05).Conclusion1. EA could increase the expression of α7nAChR.2. The deficiency of α7n ACh R induced by α-BGT could inverse the inhibition to NLRP3 inflammasome and neuroprotection effects of EA.3. PHA-543,613 is an agonist for α7n ACh R which could induce the similar neuroprotective effect with EA and inhibit NLRP3 inflammasome mediated inflammatory response.In conclusion, α7nAChR was involved in the inhibition of NLRP3 inflammasome by EA which have neuroprotective effects.SummaryIn current research, Stroke was induced by MCAO models in male SD rats for 1.5 hours. The result of Western-blot indicated that the contents of inflammasome proteins were gradually increased after cerebral ischemia, among which the contents were highest in MCAO3 d. Then, we selected MCAO3 d in further research. We found out that EA could inhibit the contents of NLRP3 inflammasome and regulate the balance between pro-inflammatory factor and anti-inflammatory cytokine. While the mechanism of the inhibition effect by EA is unclear. So, we used α7n ACh R antagonist(α-BGT) and agonist(PHA-543,613) to confirm that α7n ACh R plays an important role in the inhibition of NLRP3 inflammasome by EA. Our research revealed that EA provided neuroprotective effect by up-regulated α7n ACh R in neurons, which can inhibit NLRP3 inflammsome associated inflammatory response and reduce celluar apoptosis. This research provide suffient basic proof for EA in clinical translation. |
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