萝卜硫素对猪复苏后肠黏膜损伤的保护作用研究

Study on the protective role of sulforaphane in alleviating intestinal mucosal injury after resuscitation in pigs

目的探讨萝卜硫素(sulforaphane,SFN)减轻猪复苏后肠黏膜损伤的保护效果及作用机制。方法本实验在浙江大学实验动物中心进行。24头国产健康雄性大白猪,采用随机数字表法分为假手术(Sham)组、心肺复苏(cardiopulmonary resuscitation,CPR)组、SFN组,其中Sham组6头、另两组各9头。CPR组和SFN组选择10 min心脏骤停与6 min CPR的造模参数建立猪CPR模型。SFN组在复苏后5 min时,经股静脉泵入SFN 2 mg/kg、共计10 min。在复苏后1 h、2 h、4 h和24 h时,采集静脉血标本,应用ELISA法检测肠型脂肪酸结合蛋白(intestinal fatty acid binding protein,IFABP)和二胺氧化酶(diamine oxidase,DAO)的血清水平。然后,各组选择6头猪实施安乐死,获取回肠末端组织标本,应用TUNEL法检测细胞凋亡水平,生化法检测超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性、还原型谷胱甘肽(glutathione,GSH)和丙二醛(malondialdehyde,MDA)含量,ELISA法检测过氧化物4-羟基壬烯醛(4-hydroxy-2-nonenal,4-HNE)含量,免疫荧光染色法检测活性氧物质(reactive oxygen species,ROS)荧光强度,Western blot法检测黏连蛋白-1(zonula occluden-1,ZO-1)、闭合蛋白(occludin)、核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)和血红素加氧酶-1(heme oxygenase-1,HO-1)表达水平。三组间的计量资料比较,采用单因素方差分析,进一步两两比较采用Bonferroni事后检验。结果复苏后观察期间,CPR组和SFN组肠黏膜损伤标志物IFABP和DAO的血清水平均高于Sham组(均P<0.05)。然而,SFN组IFABP在复苏后2 h、4 h和24 h时、DAO在复苏后1 h、2 h、4 h和24 h时均低于CPR组(均P<0.05)。复苏后24 h时,CPR组和SFN组细胞凋亡指数高于Sham组,SOD和CAT活性、GSH含量均降低,MDA和4-HNE含量、ROS产物均升高,ZO-1和occludin表达下调、Nrf2和HO-1表达上调(均P<0.05)。但是,SFN组细胞凋亡指数低于CPR组,SOD和CAT活性、GSH含量均升高,MDA和4-HNE含量、ROS产物均降低,ZO-1、occludin、Nrf2和HO-1表达均上调(均P<0.05)。结论SFN具有积极减轻猪复苏后肠黏膜损伤的保护作用,其机制可能与激活Nrf2/HO-1通路后抑制组织氧化应激与细胞凋亡有关。

Objective To explore the protective efficacy of sulforaphane(SFN)in alleviating intestinal mucosal injury after resuscitation in pigs and its possible mechanism.Methods This experiment was performed in the laboratory animal center,Zhejiang university.Using a random number table,twenty-four domestic healthy male white pigs were randomly divided into the Sham group,cardiopulmonary resuscitation(CPR)group,and SFN group,in which the Sham group had 6 pigs,and the other two groups had 9 pigs,respectively.The experimental parameters of 10 min of cardiac arrest and 6 min of CPR were chosen to establish the porcine model of CPR in the CPR and SFN groups.At 5 min after resuscitation,a dose of 2 mg/kg of SFN was infused via the femoral vein within 10 min in the SFN group.At 1 h,2 h,4 h,and 24 h after resuscitation,vein samples were collected,and then the levels of intestinal fatty acid binding protein(IFABP)and diamine oxidase(DAO)in serum were measured by ELISA.Subsequently,6 pigs were chosen to be euthanized in each group,and then tissue samples were harvested from distal ileum to measure the level of cell apoptosis by TUNEL,the activities of superoxide dismutase(SOD)and catalase(CAT)and the contents of glutathione(GSH)and malondialdehyde(MDA)by biochemical method,the contents of 4-hydroxy-2-nonenal(4-HNE)by ELISA,the fluorescence intensity of reactive oxygen species(ROS)by immunofluorescence staining,and the expression levels of zonula occluden-1(ZO-1),occludin,nuclear factor E2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)by Western blot.Continuous variables were compared with one way analysis of variance among the three groups,and Bonferroni test was used for further pairwise comparison.Results During the observation period after resuscitation,the serum levels of biomarkers of intestinal mucosal injury including IFABP and DAO were significantly higher in the CPR and SFN groups than in the Sham group(all P<0.05).However,the serum levels of IFABP at 2 h,4 h,and 24 h after resuscitation and the serum levels of DAO at 1 h,2 h,4 h,and 24 h after resuscitation were significantly lower in the SFN group than in the CPR group(all P<0.05).At 24 h after resuscitation,apoptotic index was significantly increased,SOD and CAT activities and GSH contents were significantly decreased,MDA and 4-HNE contents and ROS production were significantly increased,ZO-1 and occludin expression were significantly down-regulated,and Nrf2 and HO-1 expression were significantly up-regulated in the CPR and SFN groups when compared with the Sham group(all P<0.05).However,apoptotic index was significantly decreased,SOD and CAT activities and GSH contents were significantly increased,MDA and 4-HNE contents and ROS production were significantly decreased,and ZO-1,occludin,Nrf2,and HO-1 expression were significantly up-regulated in the SFN group when compared to the CPR group(all P<0.05).Conclusion SFN could effectively protect against intestinal mucosal injury after resuscitation in pigs,and its mechanism was possibly related to the inhibition of oxidative stress and cell apoptosis via the activation of Nrf2/HO-1 pathway.