【摘要】 目的研究衣霉素(Tm)诱导肠上皮细胞发生内质网应激(ERS)的过程中,ERS的不同通路调控还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶中调节蛋白P47 PHOX的表达,以评估不同通路对氧化应激(OS)的影响,探讨ERS调控NADPH氧化酶的表达在克罗恩病的作用及可能机制。方法选取克罗恩病患者手术标本10份作为实验组,再选取结肠癌患者癌旁正常肠黏膜手术标本10份作为对照组,采用免疫组织化学方法检测肠道标本中GRP78蛋白和P47 PHOX蛋白的表达情况。随后取离体培养的肠上皮细胞,观察Tm刺激细胞后,GRP78和P47 PHOX蛋白表达的情况,并考察不同浓度的4-苯基丁酸钠盐(4PBA)是否可以抑制Tm诱导的GRP78和P47 PHOX蛋白表达。再分别抑制ERS不同通路IRE1α-XBP1S、IRE1α-TRAF2-JNK、PERK-eIF2α,检测P47 PHOX蛋白表达的情况。结果与结肠癌癌旁正常肠黏膜标本比较,克罗恩病肠黏膜标本GRP78和P47 PHOX表达明显升高。在离体的细胞中Tm诱导ERS发生,GRP78蛋白、P47 PHOX蛋白表达增加,而4PBA可以抑制上述蛋白的表达。抑制IRE1α-TRAF2-JNK、PERK-eIF2α途径均可以使P47 PHOX蛋白下调,而抑制IRE1α-XBP1S使P47 PHOX蛋白表达上调。结论 ERS可能通过调控NADPH氧化酶细胞溶质中的调节蛋白P47 PHOX,从而影响OS,最终参与克罗恩病的发病过程。其中IRE1α-TRAF2-JNK、PERK-eIF2α途径可能诱导P47 PHOX蛋白表达从而促进OS的发生。而IRE1α-XBP1S可以降低P47 PHOX蛋白活性,对OS有保护作用,可增强细胞对应激的适应性。更多还原

【Abstract】 Objective To determine the effect of different pathways of endoplasmic reticulum stress(ERS) on the expression of regulatory protein P47 PHOX in NADPH oxidase in the induction of ERS by tunicamycin(Tm), and to explore the role and possible mechanism of ERS in Crohn’s disease. Methods Ten surgical specimens of Crohn’s patients were selected as the experimental group, and other 10 normal adjacent intestinal mucosa specimens of colon cancer patients were selected as the control group. The expression of GRP78 and P47 PHOX in intestinal specimens was detected by immunohistochemistry. Subsequently, the expression of GRP78 and P47 PHOX in the intestinal epithelial cells stimulated by Tm was detected by Western blot, and whether different concentrations of 4 PBA(endoplasmic reticulum stress inhibitor) inhibited the expression of GRP78 and P47 PHOX induced by Tm was determined. The expression of P47 PHOX protein induced by Tm was detected, after the IRE1α-XBP1 S, IRE1α-TRAF2-JNK, and PERK-eIF2α pathways had been inhibited respectively. Results Compared with normal intestinal mucosa adjacent to colon cancer, the expression of GRP78 and P47 PHOX in the intestinal mucosa of patient with Crohn’s disease was significantly increased. In vitro, ERS induced Tm, and the expression of GRP78 protein and P47 PHOX protein was increased, which 4 PBA inhibited. Inhibiting IRE1α-TRAF2-JNK, and PERK-eIF2α pathway down-regulated P47 PHOX protein, while inhibiting IRE1 α-XBP1 S could up-regulate the expression of P47 PHOX protein. Conclusion ERS may affect the oxidative stress by regulating the regulatory protein P47 PHOX in NADPH oxidase, and ultimately involve in the pathogenesis of Crohn’s disease. IRE1 α-TRAF2-JNK and PERKe IF2α pathways may induce the expression of P47 PHOX protein and promote the occurrence of oxidative stress and eventually cause the disease onset. IRE1 α-XBP1 S can reduce the activity of P47 PHOX protein, indicating its protective effect on oxidative stress to enhance the adaptability of cells to stress.更多还原