肠道菌群代谢产物TMAO激活HMGB1/NLRP3炎症通路促进小鼠脑缺血半暗带损伤的机制研究

Mechanism of gut microbiota metabolite TMAO activating inflammatory pathway HMGB1/NLRP3 to promote cerebral ischemic penumbra damage in mice

目的探讨肠道菌群代谢产物氧化三甲胺(TMAO)通过作用于HMGB1/NLRP3对小鼠脑缺血再灌注(I/R)损伤后炎症反应的调控机制。方法TMAO和高脂饲料喂养建立小鼠动脉粥样硬化模型,采用线栓法制备小鼠MCAO模型。将小鼠随机分为假手术组,TMAO喂养2周和6周组,缺血再灌注损伤后,Western blotting检测NLRP3、HMGB1、Cle-IL-1β、ZO-1蛋白水平。TMAO喂养6周,将小鼠随机分为假手术组、I/R组、TMAO+I/R组、TMAO+DMB+I/R组。各组进行神经功能学评分,Western blotting检测NLRP3、HMGB1、Cle-IL-1β、ZO-1蛋白表达,TTC染色检测脑梗死体积,并进行脑组织含水量测定。结果与假手术组相比,随着TMAO喂养时间延长,NLRP3、HMGB1、Cle-IL-1β蛋白表达增加,ZO-1蛋白表达呈下降趋势(P<0.05);与I/R组相比,TMAO+I/R组小鼠神经评分显著下降,NLRP3、HMGB1、Cle-IL-1β蛋白表达增加,ZO-1蛋白表达显著下降(P<0.05),TTC显示脑梗死体积、脑组织含水量明显增加(P<0.05);DMB下调TMAO后,TMAO+DMB+I/R组较TMAO+I/R组小鼠神经评分明显改善,炎性蛋白NLRP3、HMGB1、Cle-IL-1β水平显著下调,ZO-1明显升高(P<0.05),而脑梗死体积、脑组织含水量显著下降(P<0.05)。结论肠道菌群代谢产物TMAO通过上调小鼠脑缺血再灌注损伤后HMGB1/NLRP3介导的炎症反应,加重血脑屏障破坏,从而加重脑组织损伤。

Objective To explore the mechanism of the gut microbiota metabolite trimethylamine-N-oxide(TMAO)acting on high mobility group box 1(HMGB1)/NOD-like receptor protein 3(NLRP3)to regulate inflammatory response after cerebral ischemia/reperfusion(I/R)injury in mice.Methods A mouse atherosclerosis model was established by feeding on TMAO and high-fat diet.A mouse model of middle cerebral artery occlusion(MCAO)was prepared using the suture method.Mice were randomly divided into sham group,2-week-TMAO-feeding group,and 6-week-TMAO-feeding group.After I/R injury,we measured the protein levels of NLRP3,HMGB1,cleaved interleukin-1β(Cle-IL-1β),and zonula occludens-1(ZO-1)by Western blotting.After six weeks of TMAO feeding,mice were randomly divided into sham group,I/R group,TMAO+I/R group,and TMAO+DMB+I/R group.For each group,we scored neurological function,determined NLRP3,HMGB1,Cle-IL-1β,and ZO-1 protein expression by Western blot,measured brain infarct volume with TTC staining,and measured the water content of brain tissue.Results Compared with those of the sham group,NLRP3,HMGB1,and Cle-IL-1β protein expression increased significantly and ZO-1 decreased significantly with the length of TMAO feeding(all P<0.05).Compared with the I/R group,the TMAO+I/R group showed a significant decline in neurological scores,significantly increased NLRP3,HMGB1,and Cle-IL-1β expression,significantly decreased ZO-1 expression,a significant increase in brain infarct volume,and a significant decrease in the water content of brain tissue(all P<0.05).With DMB lowering TMAO,the TMAO+DMB+I/R group had significantly better neurological scores,significantly lower NLRP3,HMGB1,and Cle-IL-1β levels,a significantly increased ZO-1 level,and significantly reduced brain infarct volume and brain tissue water content,as compared with the TMAO+I/R group(all P<0.05).Conclusion The gut microbiota metabolite TMAO can upregulate HMGB1/NLRP3-mediated inflammatory response in mice with cerebral I/R injury,worsening the breakdown of the blood-brain barrier to exacerbate brain tissue damage.