黄芪甲苷对脓毒症小鼠肠屏障功能障碍的影响

Effect of astragaloside IV on intestinal barrier dysfunction in septic mice

目的评价黄芪甲苷对脓毒症小鼠肠屏障功能障碍的影响。方法清洁级健康雄性C57BL/6J小鼠120只,6~8周龄,体重20~25 g,采用随机数字表法分为4组(n=30):假手术组(SH组)、假手术+黄芪甲苷组(SH+A组)、脓毒症组(S组)、脓毒症+黄芪甲苷组(S+A组)。采用盲肠结扎穿孔(CLP)法制备小鼠脓毒症模型。SH+A组和S+A组分别于CLP术后1 h时尾静脉注射黄芪甲苷3 mg/kg,SH组和S组分别注射等体积二甲基亚砜。记录术后7 d各组小鼠的生存情况;于CLP术后24 h时心尖穿刺采集血样,采用ELISA法测定血清IL-1β和IL-18浓度;随后处死小鼠取小肠组织,采用ELISA法测定IL-1β和IL-18含量;采用HE染色法观察小肠组织病理学损伤并行Chiu评分;采用尤斯灌流仪检测小肠黏膜跨上皮电阻值(TER);分别采用Western blot法和RT-PCR法测定小肠组织NOD样受体家族3(NLRP3)、caspase-1及其mRNA的表达。结果与SH组比较,S组和S+A组小鼠7 d生存率和TER降低,血清IL-1β和IL-18浓度、小肠组织Chiu评分、IL-1β和IL-18含量升高,NLRP3、caspase-1及其mRNA表达上调(P<0.05);与S组比较,S+A组小鼠7 d生存率和TER升高,血清IL-1β和IL-18浓度、小肠组织Chiu评分、IL-1β和IL-18含量降低,NLRP3、caspase-1及其mRNA表达下调(P<0.05);SH组与SH+A组比较,上述指标差异均无统计学意义(P>0.05)。结论黄芪甲苷可改善脓毒症小鼠肠屏障功能障碍,其机制可能与抑制NLRP3/caspase-1信号通路的激活,从而减轻炎症反应有关。

Objective To evaluate the effect of astragaloside IV(AS-IV)on intestinal barrier dysfunction in septic mice.Method One hundred and twenty clean-grade healthy male C57BL/6J mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=30 each)using a random number table method:sham operation group(group SH),sham operation plus AS-IV group(group SH+A),sepsis group(group S),and sepsis plus AS-IV group(group S+A).The model of sepsis was established by cecal ligation and puncture(CLP)in anesthetized mice.AS-IV 3 mg/kg was injected via the tail vein at 1 h after CLP in group SH+A and group S+A.The equal volume of dimethyl sulfoxide was injected instead in group SH and group S.The survival rate of mice in each group was recorded at 7 days after operation.Blood samples were collected from the cardiac apex at 24 h after CLP operation to measure concentrations of interleukin-1beta(IL-1β)and IL-18 in serum by enzyme-linked immunosorbent assay.Mice were then sacrificed,and intestinal tissues were removed to determine contents of IL-1βand IL-18(by enzyme-linked immunosorbent assay),transepithelial electrical resistance(TER)(by Ussing chamber system),expression of NOD-like receptor family pyrin domain containing 3(NLRP3)and caspase-1 protein and mRNA(by Western blot or real-time polymerase chain reaction)and to examine the pathological changes(using HE staining).Intestinal damage was assessed and scored according to Chiu.Results Compared with group SH,the 7-day survival rate and TER were significantly decreased,the concentrations of IL-1βand IL-18 in serum,Chiu′s score,contents of IL-1βand IL-18 were increased,and the expression of NLRP3 and caspase-1 protein and mRNA was up-regulated in group S and group S+A(P<0.05).Compared with group S,the 7-day survival rate and TER were significantly increased,the concentrations of IL-1βand IL-18 in serum,Chiu′s score,contents of IL-1βand IL-18 were decreased,and the expression of NLRP3 and caspase-1 protein and mRNA was down-regulated in group S+A(P<0.05).There was no significant difference in the above indicators between group SH+A and group SH(P>0.05).Conclusion AS-IV can improve sepsis-induced intestinal barrier dysfunction in mice,and the mechanism may be related to inhibiting the activation of NLRP3/caspase-1 signaling pathway and thus reducing inflammatory responses.