脓毒症诱导大鼠延髓内脏带胆碱能神经元凋亡及其对系统性炎症和行为学的影响

Apoptosis of Cholinergic Neurons in Medullary Visceral Zone Induced by Sepsis and Its Effects on Systemic Inflammation and Behavior in Rats

目的:探索脓毒症诱导的系统性炎症对延髓内脏带(MVZ)胆碱能神经元活性与凋亡、大鼠行为学的影响及胆碱能抗炎通路(CAP)干预的效应。方法:纳入无特定病原体(SPF)级Sprague Dawley(SD)大鼠64只,随机分为3组:对照组,不做任何处理。假手术组(8只),大鼠剖腹但不进行盲肠结扎穿刺法(CLP)并予哌拉西林预防感染(50 mg/kg,腹腔注射,每日3次,连续3 d)。脓毒症组(48只),制备CLP脓毒症模型并予哌拉西林预防感染(使用方法同假手术组),制备成功后随机再分为模型组、GTS-21组及甲基牛扁亭(MLA)组,各16只。模型组予生理盐水(1 mL/100 g,腹腔注射,每日3次,连续3 d);GTS-21组以α7烟碱型乙酰胆碱受体(α7nAchRs)特异性激动剂GTS-21(4 mg/kg,腹腔注射,每日1次,连续3 d)进行干预;MLA组以α7nAchRs特异性拮抗剂MLA(4.8 mg/kg腹腔注射,每日1次,连续3 d)进行干预。采用脓毒症严重程度评分(MSS)系统对大鼠每天行为进行分类评分、3 d后处死大鼠采用灌注取脑方式摘取延髓标本,采用免疫荧光双标测定延髓标本的Caspase-3/胆碱乙酰转移酶(CHAT)表达。结果:脓毒症模型组、GTS-21组及MLA组MSS评分高于对照组,且MLA组MSS评分高于GTS-21组(P<0.05),分类评分中,脓毒症3组外观、意识水平、行为表现、对刺激反应、睁眼反应、呼吸频率、呼吸质量评分均高于对照组和假手术组,3组间分类评分比较差异无统计学意义;MLA组CHAT表达较对照组降低(P<0.05),GTS-21具有升高CHAT表达的趋势(P>0.05);脓毒症各组Caspase-3表达升高(P<0.05)。结论:脓毒症诱导的系统性炎症的紊乱可能通过MVZ诱导脓毒症的病态行为,干预CAP可调控脓毒症炎症风暴和病态行为。

Objective:To explore the vitality and apoptosis of cholinergic neurons in the medullary visceral zone(MVZ)and pathological behavior induced by sepsis,and the interfering effect by cholinergic anti-inflammatory pathway(CAP).Methods:Sixty-four specific pathogen free(SPF)Sprague Dawley(SD)rats were without randomly divided into three groups.control groups(n=8),rats were normally fed.Sham group(n=8),rats were laparotomy without cecal ligation and puncture(CLP)and were administered with piperacillin(50 mg/kg,intraperitoneal injection,3 times a day for 3 consecutive days).Sepsis group(n=48),rats were prepared sepsis models with CLP and administered with piperacillin as same as sham group.After successful preparation,they were randomly divided into model group(n=16),GTS-21 group(n=16),and methyllycaconitine(MLA)group(n=16).The rats in model group were accepted normal saline(1 mL/100 g,intraperitoneal injection,3 times a day for 3 consecutive days),the rats in GTS-21 group were accepted GTS-21[α7 nicotinic acetylcholine receptor(α7nAchRs)specific agonist,4 mg/kg,intraperitoneal injection,once a day for 3 consecutive days],the rats in MLA group were accepted MLA(α7nAchRs specific antagonist,4.8 mg/kg,intraperitoneal injection,once a day for 3 consecutive days).The Murine Sepsis Score(MSS)was used to access the daily behavior of all rats.After 3 days,the rats were sacrificed to collect blood and the medulla oblongata specimens after perfusion for detection.Double immunofluorescence labeling was used to determine the expression of Caspase-3/choline acetyltransferase(CHAT)in the medulla oblongata specimens.Results:MSS scores in model group,GTS-21 group,and MLA group were higher than that in control group,MSS score in MLA group was significantly higher than that of GTS-21 group(P<0.05).For the classified scores(such as scores of the appearance,consciousness level,behavior performance,response to stimuli,eye opening,respiratory rate,and respiratory quality),rats in sepsis group were significantly higher than those of control group and sham group,there was no significant difference among three sepsis groups in these indexes.The expression of CHAT in MLA group was significantly lower than that in control group(P<0.05).GTS-21 showed a tendency to increase the expression of CHAT(P>0.05).The expression of Caspase-3 increased in three sepsis groups(P<0.05).Conclusion:Systemic inflammation disorders induced by sepsis may lead to pathological behavior through damaged MVZ.Interfering with CAP can prevent the septic inflammatory storm and the pathological behavior in sepsis.