机构地区:[1]大连大学附属中山医院急诊科,辽宁大连116622 [2]山西省大同市第五人民医院急诊科,山西大同037056
出 处:《中华卫生应急电子杂志》2024年第6期359-367,共9页Chinese Journal of Hygiene Rescue(Electronic Edition)
基 金:国家自然科学基金资助项目(81673801,81473512)。
摘 要:目的基于TLR4/NF-κB信号通路介导的自噬研究高脂饲喂小鼠肠黏膜屏障应激能力的变化情况并分析其可能机制。方法40只SPF级小鼠随机分为普通对照组(NCD组)、高脂饮食对照组(HFD组)、普通-重症急性胰腺炎组(NSAP组)和高脂饮食-重症急性胰腺炎组(HSAP组),每组10只。NCD组予以普通饲料喂养,HFD组予以高脂饲料喂养,共8周。NSAP组、HSAP组腹腔注射20%L-精氨酸(L-Arg)(3.5 g/kg间隔1 h,共2次)作为应激刺激,NCD组、HFD组腹腔注射等量等渗盐水。ELISA法检测血清TC、TG、AMY、LPS、IL-6、TNF-α的含量;HE染色观察胰腺、结肠和肺组织病理变化;免疫组化法观察结肠上皮紧密连接蛋白Claudin、ZO-1和Occludin及自噬相关蛋白LC3Ⅱ及通路相关蛋白TLR4和NF-kB的分布;蛋白免疫印迹法(Western Blot)检测结肠Claudin、ZO-1、Occludin、TLR4、NF-kB及LC3Ⅱ的表达水平。结果与NCD组相比,HFD组小鼠体重及血清中TG、TC水平明显增高;给予20%L-Arg刺激后,与NCD相比,NSAP组血清AMY、TNF-α、LPS和IL-6含量显著升高(P<0.05),结肠Claudin、Occludin、ZO-1蛋白表达水平显著下降(P<0.05),TLR4、NF-κB蛋白表达显著增加(P<0.05),LC3Ⅱ蛋白表达明显减少(P<0.05);与NSAP组相比,HSAP组小鼠体重及血清中TG、TC、AMY、TNF-α、LPS和IL-6含量明显升高(P<0.05),结肠Claudin、Occludin、ZO-1表达显著下降(P<0.05),同时结肠TLR4、NF-κB蛋白表达显著增加,结肠LC3II蛋白表达明显减少(P<0.05)。结论高脂饲喂小鼠肠黏膜屏障应激能力减弱,可能与TLR4/NF-κB信号通路介导的自噬相关。Objective To investigate the potential mechanism of intestinal mucosal barrier injury caused by intestinal epithelial cell autophagy in high-fat-diet fed mice.Methods Forty specific pathogenfree mice were randomly divided into normal control group(NCD group),high fat diet group(HFD group),normal severe acute pancreatitis group(NSAP group),and high fat diet plus severe acute pancreatitis group(HSAP group),with 10 mice in each group.The normal control group was fed with normal diet,and the highfat diet group was fed with high-fat diet for 8 weeks.Mice in the NSAP and HSAP groups were intraperitoneally injected with 20%L-arginine(L-Arg)(3.5 g/kg,1 hour interval,2 times in total)as a stress stimulus,while rats in the NCD and HFD groups were intraperitoneally injected with the same amount of normal saline.ELISA method was used to detect the serum TC,TG,AMY,LPS,IL-6,the content of TNF-α.HE staining was used to observe the pancreas,colon and lung tissue pathological changes.Immunohistochemistry was used to observe the distribution of tight junction proteins Claudin,ZO-1,Occludin,autophagy-related protein LC3Ⅱ,and pathway-related proteins TLR4 and NF-kB in colon epithelium.Western Blot was used to detect the expression levels of Claudin,ZO-1,Occludin,TLR4,NF-kB and LC3Ⅱin colon.Results Compared with NCD group,HFD mice body weight and serum TG,TC level increased obviously.After 20%L-Arg stimulation,compared with NCD group,the levels of serum AMY,TNF-α,LPS and IL-6 in NSAP group were significantly increased(P<0.05),and the expression levels of colon Claudin,Occludin and ZO-1 protein were significantly decreased(P<0.05).TLR4,NF-κB protein expression significantly increased(P<0.05),LC3II protein expression decreased significantly(P<0.05).Compared with the NSAP group,the body weight and serum levels of TG,TC,AMY,TNF-α,LPS and IL-6 in the HSAP group were significantly increased(P<0.05),and the expression of Claudin,Occludin and ZO-1 in the colon was significantly decreased(P<0.05).At the same time,the expression of TLR
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