目的:中枢神经系统炎症反应在脓毒症相关脑病的发生和发展中起至关重要的作用.Maresin 1(MaR1)是一种抗炎和促炎症消退脂质介质,本研究拟探索MaR1对脓毒症诱导的神经炎症和认知障碍的影响.方法:将小鼠随机分为4组:假手术(sham)组(假手术+溶剂对照)、盲肠结扎穿孔(cecal ligation and puncture,CLP)组(CLP+溶剂对照)、MaR1低剂量组(CLP+1 ng MaR1)和MaR1高剂量组(CLP+10ng MaR1).从CLP手术前1h开始,隔日给予小鼠腹腔注射1次MaR1或溶剂,直到第7天.评估MaR 1对CLP小鼠7 d内存活率的影响.在术后24h,采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定小鼠血清中的炎症因子[肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)、白细胞介素(interleukin,IL)-1β 和 IL-6]水平;Morris水迷宫(Morris water maze,MWM)测试和新物体识别(novel object recognition,NOR)任务评估CLP或假手术7 d后的小鼠认知行为;实时反转录聚合酶链反应(real-time reverse transcription PCR,real-time RT-PCR)测定小鼠皮层和海马组织中 TNF-α、IL-1β、IL-6、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、IL-4、IL-10 和精氨酸酶 1(arginase 1,Arg1)的 mRNA表达水平;蛋白质印迹法测定海马组织中iNOS、Arg1、信号转导与转录激活因子6(signal transducer and activator of transcription 6,STAT6)、过氧化物酶体增殖物激活受体y(peroxisome proliferator-activated receptor gamma,PPARγ)及磷酸化STAT6(p-STAT6)的蛋白质表达水平;免疫荧光法观察小胶质细胞活化.此外,使用PPARγ拮抗剂GW9662处理小鼠,以验证该途径参与了 MaR1的作用.结果:CLP导致小鼠血清TNF-α、IL-1β和IL-6水平升高,同时体重和存活率下降(均P＜0.05).给予1 ng和10 ng的MaR1均显著降低了血清TNF-α、IL-1β和IL-6水平,并改善了小鼠的体重和存活率(均P＜0.05),2种剂量的效果差异均无统计学意义(均P＞0.05).MWM测试和NOR任务结果表明:CLP显著损害了小鼠的空间学习能力,MaR1减轻了这种损害.然而,GW9662部分抵消了 MaR1的保护作用.Real-time RT-PCR结果显示:与sham组相比,CLP术后海马组织中TNF-α、IL-1β和iNOS的mRNA表达显著增加(均P＜0.05),而IL-4、IL-10和Arg1的表达略有下降,但差异均无统计学意义(均P＞0.05).与CLP组相比,1ng和10 ng MaR1均降低了海马组织中TNF-α、IL-1β和iNOS的mRNA表达水平(均P＜0.05),并提高了IL-4、IL-10和Arg1的mRNA表达水平(均P＜0.05).免疫荧光结果显示:与sham组相比,CLP组海马区Iba-1阳性的小胶质细胞显著增多(P＜0.05);与CLP组相比,给予1和10 ng的MaR1均降低了海马区Iba-1阳性细胞的百分比面积(均P＜0.05).蛋白质印迹结果表明:与CLP组相比,1和10ng的MaR1均下调iNOS的蛋白质表达水平,上调了 Arg1、PPARγ及p-STAT-6的水平(均P＜0.05).与MaR1低剂量组相比,GW9662的加入抵消了MaR1引起的Arg1和PPARγ的上调(均P＜0.05).结论:MaR1可以抑制CLP导致的海马小胶质细胞经典活化,促进小胶质细胞选择性活化,进而减轻脓毒症引起的神经炎症,改善认知能力下降.
Maresin 1 alleviates neuroinflammation and cognitive decline in a mouse model of cecal ligation and puncture
Objective:Inflammation in the central nervous system plays a crucial role in the occurrence and development of sepsis-associated encephalopathy.This study aims to explore the effects of maresin 1(MaR1),an anti-inflammatory and pro-resolving lipid mediator,on sepsis-induced neuroinflammation and cognitive impairment.Methods:Mice were randomly assigned to 4 groups:A sham group(sham operation+vehicle),a cecal ligation and puncture(CLP)group(CLP operation+vehicle),a MaR1-LD group(CLP operation+1 ng MaR1),and a MaR1-HD group(CLP operation+10 ng MaR1).MaR1 or vehicle was intraperitoneally administered starting 1 h before CLP operation,then every other day for 7 days.Survival rates were monitored,and serum inflammatory cytokines[tumor necrosis factor alpha(TNF-α),interleukin(IL)-1β,and IL-6]were measured 24 h after operation using enzyme-linked immunosorbent assay(ELISA).Cognitive function was assessed 7 days after operation using the Morris water maze(MWM)test and novel object recognition(NOR)task.The mRNA expression of TNF-α,IL-1β,IL-6,inducible nitric oxide synthase(iNOS),IL-4,IL-10,and arginase 1(Arg1)in cortical and hippocampal tissues was determined by real-time reverse transcription PCR(RT-PCR).Western blotting was used to determine the protein expression of iNOS,Arg1,signal transducer and activator of transcription 6(STAT6),peroxisome proliferator-activated receptor gamma(PPARγ),and phosphorylated STAT6(p-STAT6)in hippocampal tissue.Microglia activation was visualized via immunofluorescence.Mice were also treated with the PPARγ antagonist GW9662 to confirm the involvement of this pathway in MaR1's effects.Results:CLP increased serum levels of TNF-α,IL-1β,and IL-6,and reduced body weight and survival rates(all P＜0.05).Both 1 ng and 10 ng doses of MaR1 significantly reduced serum TNF-α,IL-1β,and IL-6 levels,improved body weight,and increased survival rates(all P＜0.05).No significant difference in efficacy was observed between the 2 doses(all P＞0.05).MWM test and NOR task indicated that CLP impaired spatial learning,which MaR1 mitigated.However,GW9662 partially reversed MaR1's protective effects.Real-time RT-PCR results demonstrated that,compared to the sham group,mRNA expression of TNF-α,IL-1β,and iNOS significantly increased in hippocampal tissues following CLP(all P＜0.05),while IL-4,IL-10,and Arg1 showed a slight decrease,though the differences were not statistically significant(all P＞0.05).Compared to the CLP group,both 1 ng and 10 ng MaR1 decreased TNF-α,IL-1β,and iNOS mRNA expression in hippocampal tissues and increased IL-4,IL-10,and Arg1 mRNA expression(all P＜0.05).Immunofluorescence results indicated a significant increase in Iba1-positive microglia in the hippocampus after CLP compared to the sham group(P＜0.05).Administration of 1 ng and 10 ng MaR1 reduced the percentage area of Iba1-positive cells in the hippocampus compared to the CLP group(both P＜0.05).Western blotting results showed that,compared to the CLP group,both 1 ng and 10 ng MaR1 down-regulated the iNOS expression,while up-regulated the expression of Arg1,PPARγ,and p-STAT6(all P＜0.05).However,the inclusion of GW9662 counteracted the MaR1-induced upregulation of Arg1 and PPARγ compared to the MaR1-LD group(all P＜0.05).Conclusion:MaR1 inhibits the classical activation of hippocampal microglia,promotes alternative activation,reduces sepsis-induced neuroinflammation,and improves cognitive decline.
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