摘要目的 观察胆碱酯酶抑制剂石杉碱甲A(HupA)对脓毒症大鼠脑皮质胆碱乙酰转移酶(ChAT)及毒蕈碱型乙酰胆碱受体M1 (CHRM1)表达的影响,探讨胆碱能抗炎通路是否介导了HupA对脓毒症相关性脑病(SAE)的保护作用.方法 8周龄雄性Wistar大鼠按随机数字表法分为对照组、脓毒症模型组和HupA干预组,每组18只.采用腹腔注射脂多糖(LPS)10 mg/kg(1 mL)诱导大鼠脓毒症模型,对照组给予等量生理盐水.HupA干预组于术前30 min腹腔注射HupA 0.04 mg/kg(1 mL),对照组及模型组给予等量生理盐水.观察大鼠一般情况;每组分别于术后3、12、24 h处死6只大鼠取大脑皮质组织,采用酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α (TNF-α)和白细胞介素-1β (IL-1 β)等炎性介质水平;采用天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)和神经元核抗原(NeuN)免疫双标染色实验观察神经元细胞凋亡情况;采用实时定量反转录-聚合酶链反应(RT-qPCR)及免疫荧光法检测ChAT和CHRM1的mRNA表达和阳性细胞.结果 模型组大鼠给药后3h即出现精神萎靡、毛发竖立及静止懒动等脓毒症症状,12h表现最为明显;并伴随大脑皮质TNF-α和IL-1β过表达,神经元凋亡细胞明显增多,ChAT和CHRM1阳性表达明显减少,ChAT和CHRM1的mRNA表达均明显降低,与对照组比较差异均有统计学意义[12h的TNF-α(ng/L):84.97±31.84比40.31±10.37,12h的IL-1 β(ng/L):1 095.98±127.09比622.62±117.25,12h的ChAT mRNA (2-△△Ct):1.34(0.67,1.86)比1.92(1.12,2.87),12h的CHRM1 mRNA(2-△△Ct):0.65±0.12比1.16±0.42,均P＜ 0.05].HupA干预后可明显缓解大鼠脓毒症症状;抑制大脑皮质TNF-α和IL-1β过表达及神经元细胞凋亡,同时可明显上调ChAT和CHRM1阳性表达及mRNA表达,与模型组比较差异均有统计学意义[12 h的TNF-α(ng/L):48.38±12.62比84.97±31.84,12h的IL-1 β (ng/L):718.13±163.33比1095.98±127.09,12h的ChAT mRNA (2-△△Ct):18.04(17.22,19.23)比1.34(0.67,1.86),12h的CHRM1 mRNA (2-△△Ct):1.46±0.69比0.65±0.12,均P＜0.05];24 h上述症状和数值基本恢复.结论 大脑皮质胆碱能神经功能异常及炎症反应均参与了SAE的发病过程;HupA可通过恢复胆碱能神经功能及胆碱能抗炎通路对SAE起到一定的保护作用.

abstractsObjective To explore whether the cholinergic anti-inflammatory pathway is involved in the neuroprotective effect of acetylcholinesterase inhibitor huperzine A (HupA) on sepsis-associated encephalopathy (SAE) by observing the effect of HupA on the expressions of choline acetyltransferase (CHAT) and cholinergic muscarinic receptor M1 (CHRM1) of sepsis rats.Methods Fifty-four male Wistar rats,8 weeks old,were divided into three experimental groups according to random number table:control group,sepsis group,and HupA group,with 18 rats in each group.The rat model of sepsis was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS,1 mL),and the rats in control group were given the same volume of normal saline.The rats in HupA group were intraperitoneally administered with HupA 0.04 mg/kg (1 mL) at 30 minutes before model reproduction,while the rats in control group and sepsis group were treated with the same volume of saline instead.At 3,12,and 24 hours after model reproduction,6 rats in each group were sacrificed after clinical manifestation observation,and cerebral cortex tissue was collected.Pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1 β) in cerebral cortex were determined with enzyme linked immunosorbent assay (ELISA),along with the measurement of neuronal apoptosis by caspase-3 and neuronal nuclear antigen (NeuN) immune double standard staining.The mRNA expressions and positive expressions of ChAT and CHRM1 were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunofluorescence methods.Results Clinical manifestations of sepsis rats were present at 3 hours and reached a peak at 12 hours,including lethargy,vertical hair and lazy to move.Over-expression of pro-inflammatory cytokines TNF-α and IL-1β was found in sepsis group,and apoptotic neurons marked by two fluorescences were significantly increased in sepsis rats,in comparison with deficient ChAT and CHRM1 proteins marked by red fluorescence,and low-expressed ChAT and CHRM1 mRNA as well.The differences between sepsis group and control group were statistically significant [12-hour TNF-α (ng/L):84.97±31.84 vs.40.31 ± 10.37,12-hour IL-1 β (ng/L):1 095.98± 127.09 vs.622.62± 117.25,12-hour ChAT mRNA (2-△△Ct):1.34 (0.67,1.86) vs.1.92 (1.12,2.87),12-hour CHRM1 mRNA (2-△△Ct):0.65±0.12 vs.1.16±0.42,all P ＜ 0.05].The septic symptoms were relieved after HupA administration,as well as the reduction of pro-inflammatory cytokines and the neuronal apoptosis,which might attribute to the increased expressions of ChAT and CHRM1.The differences between HupA group and sepsis group were statistically significant [12-hour TNF-α (ng/L):48.38 ± 12.62 vs.84.97 ± 31.84,12-hour IL-1 β (ng/L):718.13 ± 163.33 vs.1 095.98 ± 127.09,12-hour ChAT mRNA (2-△△Ct):18.04 (17.22,19.23) vs.1.34 (0.67,1.86),12-hour CHRM1 mRNA (2-△ △Ct):1.46 ± 0.69 vs 0.65 ± 0.12,all P ＜ 0.05].The clinical manifestations and neuroinflammation mainly recovered at 24 hours.Concltsions The cortical cholinergic neurons dysfunction and the abnormal inflammatory response are involved in the onset of SAE process.HupA plays a protective role in SAE through recovering the function of cholinergic neurons and cholinergic anti-inflammatory pathway.