目的 观察表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对脓毒症肠道损伤的作用,并探究对内质网应激(endoplasmic reticulum stress,ERS)性凋亡的途径是否存在影响.方法 选取60只雄性SD大鼠,根据随机数字表法将其分为5组:假手术组(Sham组)、盲肠结扎穿孔术致脓毒症组(cecal ligation and puncture,CLP组)、脓毒症+EGCG低剂量组(术后腹腔注射EGCG 25 mg/kg,EL组)、脓毒症+EGCG中剂量组(术后腹腔注射EGCG 50 mg/kg,EM组)、脓毒症+EGCG高剂量组(术后腹腔注射EGCG 75 mg/kg,EH组),每组均为12只.造模24 h后处死各组大鼠并收集标本.血清中炎症因子采用酶联免疫吸附试验检测;苏木精伊红染色后,光镜下观察回肠病理学改变,根据Chiu's评分进行评价;肠道组织中紧密连接蛋白-1(CLDN1,Claudin-1)、磷酸化蛋白激酶R样内质网激酶(phosphorylated PERK,p-PERK)、蛋白激酶R样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、天冬氨酸特异性半胱氨酸蛋白酶-12(cysteinyl aspartate specific protease-12,Caspase-12)、CCAAT 增强子结合蛋白同源蛋白(C/EBP-homologous protein antibody,CHOP)的蛋白表达采用蛋白质免疫印迹试验检测;肠道组织中Claudin-1、p-PERK、CHOP、Caspase-12的阳性面积采用免疫组织化学法检测.结果 对比Sham组,CLP组大鼠血清中白介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子-α水平,Chiu's评分均升高(均P＜0.05);回肠黏膜组织中Claudin-1表达减少,ERS性凋亡相关蛋白p-PERK、CHOP、Caspase-12表达量均增加(均P＜0.05).与CLP组相比,给予低、中、高剂量EGCG干预后大鼠肠道损伤均有所缓解(均P＜0.05).EL组血清炎症因子水平、Chiu's评分和小肠组织中ERS性凋亡相关蛋白p-PERK、CHOP、Caspase-12的蛋白表达水平及阳性面积均较CLP组进一步降低,且EM组较EL组、EH组较EM组均进一步降低(均P＜0.05).EL组小肠组织中Claudin-1蛋白表达水平及阳性面积均较CLP组进一步升高(均P＜0.05),且EM组较EL组、EH组较EM组均进一步升高(均P＜0.05).结论 EGCG可能通过抑制ERS性凋亡通路的活化对脓毒症大鼠肠道损伤起到一定的保护作用,且高剂量EGCG疗效更佳.
Epigallocatechin gallate attenuates intestinal injury in sepsis by inhibiting apoptosis
Objective To observe the effect of epigallocatechin gallate(EGCG)on intestinal injury in sepsis,and to investigate the effect on endoplasmic reticulum stress(ERS)apoptotic pathway.Methods Sixty male SD rats were selected and divided into five groups according to the randomized numeric table method:the sham operation group(Sham group),the cecal ligation and puncture sepsis group(CLP group),the sepsis+EGCG low-dose group(postoperative intraperitoneal injection of EGCG 25 mg/kg,EL group),the sepsis+EGCG medium-dose group(postoperative intraperitoneal injection of EGCG 50 mg/kg,EM group),and sepsis+EGCG high-dose group(postoperative intraperitoneal injection of EGCG 75 mg/kg,EH group),each group with 12 rats.The rats in each group were executed 24 h after modeling and specimens were collected.Inflammatory factors in serum were detected by enzyme-linked immunosorbent assay.Pathological changes of ileum were observed under light microscope after hematoxylin eosin staining and evaluated according to the Chiu's score.The intestinal tissues were stained for tight junction protein-1(CLDN1,Claudin-1),phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),protein kinase RNA-like endoplasmic reticulum kinase(PERK),cysteinyl aspartate specific protein-12(Caspase-12),and CCAAT enhancer-binding protein homologous protein(C/EBP-homologous protein antibody,CHOP)protein expression was detected by protein immunoblotting assay.The positive areas of Claudin-1,p-PERK,CHOP,and Caspase-12 in intestinal tissue were detected by immunohistochemistry.Results Compared with the Sham group,the serum levels of interleukin(IL)-1β,IL-6,tumor necrosis factor-α,and the Chiu's score of rats in the CLP group were increased(all P＜0.05).The ileal mucosal tissues showed reduced expression of Claudin-1,ERS apoptosis-associated protein p-PERK,CHOP,and Caspase-12 expression were increased(all P＜0.05).Compared with the CLP group,the intestinal injury in rats was alleviated after the administration of low,medium and high dose EGCG intervention(all P＜0.05).The serum inflammatory factor level,Chiu's score and the protein expression level and positive area of ERS apoptosis-related proteins,p-PERK,CHOP,and Caspase-12 in the small intestinal tissues of EL group were further reduced compared with that of the CLP group were further decreased,and EM group was further decreased than EL group,and EH group was further decreased than EM group(all P＜0.05).The protein expression level and positive area of Claudin-1 in small intestinal tissues of EL group were further increased compared with that of CLP group(both P＜0.05),and EM group was further increased compared with that of EL group and EH group was further increased compared with EM group(all P＜0.05).Conclusions EGCG may have a protective effect on intestinal injury in septic rats by inhibiting the activation of ERS-induced apoptotic pathway,and the efficacy of high-dose EGCG has a better effect.
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