参苓白术散抗炎症性肠病作用与调节肠上皮细胞自噬的关系探讨
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摘要
目的:探讨参苓白术散通过调节肠上皮细胞自噬对5%葡聚糖硫酸钠(DSS)所致小鼠炎症性肠病(IBD)的改善作用。方法:84只BALB/c小鼠随机分为7组,每组12只。除正常组之外,5%DSS自由饮用7 d诱导急性炎症性肠病,治疗组分别给予参苓白术散高、中、低剂量(12,6,3 g·kg~(-1)·d~(-1)),美沙拉嗪(2 g·kg~(-1)·d~(-1)),自噬诱导剂雷帕霉素(4 mg·kg~(-1)·d~(-1))灌胃。苏木素-伊红(HE)染色观察小鼠结肠组织的病理形态学变化;透射电镜检测肠上皮细胞自噬体形成情况;蛋白免疫印迹法(Western blot)检测自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ,磷脂酰肌醇-3激酶(PI3K),哺乳动物雷帕霉素靶蛋白(m TOR),泛素结合蛋白~(-1)(p62),Beclin1磷酸化,ULK1,4EBP蛋白表达。结果:与正常组比较,模型组小鼠结肠黏膜上皮细胞广泛缺失,腺体大多数不完整,呈结肠炎改变,LC3-Ⅱ含量明显降低(P <0. 05),自噬不明显,而自噬水平增加,结肠组织中自噬通路蛋白PI3K,mTOR,p-p62磷酸化程度及ULK1蛋白表达明显升高(P <0. 05),Beclin1磷酸化程度及4EBP蛋白表达明显降低(P <0. 05);与模型组比较,参苓白术散中、高剂量组,美沙拉嗪组及雷帕霉素组结肠组织明显改善,LC3-Ⅱ含量均明显升高(P <0. 05),参苓白术散高、中、低剂量组,美沙拉嗪组及其对应的抑制剂组可明显抑制PI3K,mTOR,p-p62磷酸化,降低ULK1蛋白表达,促进Beclin1磷酸化及升高4EBP蛋白表达(P <0. 05)。结论:参苓白术散抗DSS诱导的IBD的作用与调节肠上皮细胞自噬通路蛋白PI3K,mTOR,p62的磷酸化有关。
Objective: To investigate the effect of Shenling Baizhu San on inflammatory bowel disease(IBD) induced by 5% dextran sodium sulfate(DSS) in mice by regulating autophagy of intestinal epithelial cells.Method: The 84 BALB/c mice were randomly divided into 7 groups with 12 mice in each group. In addition to the normal group,5% DSS was freely drunk for 7 days to induce acute inflammatory bowel disease. In the treatment group,high, medium and low doses of shenlingbaishu powder(12, 6, 3 g ·kg~(-1)·d~(-1)), mesalazine(2 g·kg~(-1)·d~(-1)) and autophagy inducer rapamycin(4 mg·kg~(-1)·d~(-1)) were given by gavage. Hematoxylin-eosin(HE) staining was used to observe the morphological changes of colon tissues in mice. The autophagosome formation of intestinal epithelial cells was detected by transmission electron microscopy. Western blot was used to detect the ratio of autophagy related protein LC3-Ⅱ/LC3-Ⅰ,phosphatidylinositol-3 kinase(PI3 K),mammalian target of rapamycin(m TOR),ubiquitin-binding protein~(-1)(p62),Beclin1 phosphorylation,ULK1,4 EBP protein expression. Result: Compared with normal group,model group mice colonic mucosa epithelial cells are widely missed,most incomplete glands,change in colitis,LC3-Ⅱ content decreased significantly(P < 0. 05),autophagy is not obvious,and an increased level of autophagy,autophagy pathways in the colon tissue protein PI3 K,m TOR,p-p62 phosphorylation degree and the ULK1 protein expression increased significantly(P < 0. 05)),Beclin1 phosphorylation and 4 ebp protein expression significantly decreased(P < 0. 05)). Compared with model group,Shenling Baizhu San high dose group,salad lamictal and rapamycin group improved colon tissue obviously,LC3-Ⅱlevels were higher significantly(P < 0. 05),and Shenling Baizhu San high,medium and low dose group,salad oxazine group and its corresponding inhibitor can obviously inhibit PI3 K,m TOR,p-p62 phosphorylation,reduce ULK1 protein expression,promote Beclin1 phosphorylation and 4 EBP protein expression(P < 0. 05). Conclusion:The effect of Shenling Baizhu San on DSS-induced IBD is related to the regulation of the phosphorylation of PI3 K,m TOR and p62 proteins in the autophagy pathway of intestinal epithelial cells.
Objective: To investigate the effect of Shenling Baizhu San on inflammatory bowel disease(IBD) induced by 5% dextran sodium sulfate(DSS) in mice by regulating autophagy of intestinal epithelial cells.Method: The 84 BALB/c mice were randomly divided into 7 groups with 12 mice in each group. In addition to the normal group,5% DSS was freely drunk for 7 days to induce acute inflammatory bowel disease. In the treatment group,high, medium and low doses of shenlingbaishu powder(12, 6, 3 g ·kg~(-1)·d~(-1)), mesalazine(2 g·kg~(-1)·d~(-1)) and autophagy inducer rapamycin(4 mg·kg~(-1)·d~(-1)) were given by gavage. Hematoxylin-eosin(HE) staining was used to observe the morphological changes of colon tissues in mice. The autophagosome formation of intestinal epithelial cells was detected by transmission electron microscopy. Western blot was used to detect the ratio of autophagy related protein LC3-Ⅱ/LC3-Ⅰ,phosphatidylinositol-3 kinase(PI3 K),mammalian target of rapamycin(m TOR),ubiquitin-binding protein~(-1)(p62),Beclin1 phosphorylation,ULK1,4 EBP protein expression. Result: Compared with normal group,model group mice colonic mucosa epithelial cells are widely missed,most incomplete glands,change in colitis,LC3-Ⅱ content decreased significantly(P < 0. 05),autophagy is not obvious,and an increased level of autophagy,autophagy pathways in the colon tissue protein PI3 K,m TOR,p-p62 phosphorylation degree and the ULK1 protein expression increased significantly(P < 0. 05)),Beclin1 phosphorylation and 4 ebp protein expression significantly decreased(P < 0. 05)). Compared with model group,Shenling Baizhu San high dose group,salad lamictal and rapamycin group improved colon tissue obviously,LC3-Ⅱlevels were higher significantly(P < 0. 05),and Shenling Baizhu San high,medium and low dose group,salad oxazine group and its corresponding inhibitor can obviously inhibit PI3 K,m TOR,p-p62 phosphorylation,reduce ULK1 protein expression,promote Beclin1 phosphorylation and 4 EBP protein expression(P < 0. 05). Conclusion:The effect of Shenling Baizhu San on DSS-induced IBD is related to the regulation of the phosphorylation of PI3 K,m TOR and p62 proteins in the autophagy pathway of intestinal epithelial cells.
引文
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[2]Lee E J,Tournier C.The requirement of uncoordinated51-like kinase 1(ULK1)and ULK2 in the regulation of autophagy[J].Autophagy,2011,7(7):689-695.
[3]Tanida I,Ueno T,Kominami E.LC3 and autophagy[J].Methods Mol Biol,2008,445:77-88.
[4]Noureldein M H,Eid A A.Gut microbiota and m TORsignaling:insight on a new pathophysiological interaction[J].Microb Pathog,2018,118:98-104.
[5]LIU Y H,DING Y,GAO C C,et al.Functional macrophages and gastrointestinal disorders[J].World JGastroenterol,2018,24(11):1181-1195.
[6]Martini E,Krug S M,Siegmund B,et al.Mend your fences:the epithelial barrier and its relationship with mucosal immunity in inflammatory bowel disease[J].Cell Mol Gas Hep,2017,4(1):33-46.
[7]HU C A,HOU Y,YI D,QIU Y,et al.Autophagy and tight junction proteins in the intestine and intestinal diseases[J].Anim Nutr,2015,1(3):123-127.
[8]Massey D C,Parkes M.Genome-wide association scanning highlights two autophagy genes,ATG16L1 and IRGM,as being significantlu associated with Crohn's disease[J].Autophagy,2007,3(6):649-651.
[9]刘玉晖,胡婕,易文凤,等.参苓白术散治疗炎症性肠病与肠上皮细胞紧密连接的关系探讨[J].中国实验方剂学杂志,2015,21(3):130-133.
[10]刘玉晖,刘志勇,廖旺娣,等.参苓白术散抗脂多糖致肠隐窝上皮细胞损伤的作用及机制[J].中药新药与临床药理,2016,27(1):1-5.
[11]Kanauchi O,Nakamura T,Agata K,et al.Effects of germinated barley food stuff on dextran sulfate sodiuminduced colitis in rats[J].J Gastroenterol,1998,33:179-188.
[12]Massey D C,Parkes M.Genome-wide association scanning highlights two autophagy genes,ATG16L1 and IRGM,as being significantlu associated with Crohn's disease[J].Autophagy,2007,3(6):649-651.
[13]Adolph T E,Tomczak M F,Niederreiter L,et al.Paneth cells as a site of origin for intestinal inflammation[J].Nature,2013,503(7475):272-276.
[14]Kundu M.ULK1,mammalian target of rapamacin,and mitochondria:linking nutrient availability and autophagy[J].Antioxid Redox Signal,2011,14(10):1953-1958.
[15]徐昌君,王鹏飞,刘杨,等.黄芪甲苷对特发性肺纤维化自噬活性作用及PI3K/Akt/m TOR信号调控的影响[J].中国实验方剂学杂志,2017,23(18):75-82.
[16]YUE Z,JIN S,YANG C,et al.Beclin1,an autophagy gene essential for early embryonic development,is a haploinsufficient tumor suppressor[J].Proc Natl Acad Sci USA,2003,100(25):15077-15082.
[17]Haussinger D.The role of cellular hydration in the regulation of cell function[J].Biochem J,1996,31(3):697.
[18]刘玉晖,严雪梅,佟阳,等.参苓白术散通过MAPK信号通路对炎症性肠炎小鼠的作用[J].时珍国医国药,2014,25(4):793-795.