胰岛素样生长因子-Ⅰ对乙酰水杨酸诱导小肠上皮细胞损伤的修复作用
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摘要
本实验研究胰岛素样生长因子-Ⅰ(insulin-like growth factors,IGF-Ⅰ)对乙酰水杨酸(acetylsalicylic acid,ASA)所致的小肠上皮细胞(intestinal epithelial cells,IEC-6)损伤的影响。通过构建ASA诱导IEC-6损伤模型,采用CCK-8法检测IGF-Ⅰ对IEC-6损伤的修复作用;同时测定细胞培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)活力,以及细胞中丙二醛(malondialdelyde,MDA)浓度、碱性磷酸酶(alkaline phosphatase,ALP)活力、细胞总蛋白质量浓度和Na+,K+-ATP酶活力。结果表明:与对照组相比,不同质量浓度的IGF-Ⅰ均可以显著促进细胞的增殖(P<0.05);与损伤模型组相比,IGF-Ⅰ能提高细胞存活率,降低细胞培养液中LDH活力,降低细胞ALP活力和MDA浓度,提高细胞总蛋白质量浓度,提高Na~+,K~+-ATP酶活力。IGF-Ⅰ对ASA所致损伤的IEC-6可以促进增殖,抑制脂膜氧化,维持细胞形态的完整性,具有损伤修复作用。
The present study was designed to investigate the repairing effect of insulin-like growth factors-Ⅰ(IGF-Ⅰ) on acetylsalicylic acid(ASA)-induced damage of intestinal epithelial cells(IEC-6). The cell damage model was induced by ASA. The CCK-8 assay was used to evaluate the proliferation of IEC-6. After being treated with ASA and IGF-Ⅰ, the cells were collected for determining the activities of lactate dehydrogenase(LDH) and alkaline phosphatase(ALP), and the culture supernatant for analysis of Na+,K+-ATPase and alkaline phosphatase(ALP) and the contents of total protein and malondialdelyde(MDA). We found that compared with the control group, IGF-Ⅰ at all concentrations tested promoted cell proliferation(P < 0.05). IGF-Ⅰ increased the cell survival rate, decreased the LDH activity in the culture supernatant and reduced the ALP activity and MDA content in the cells, as well as elevated the total protein content and Na~+,K~+-ATPase activity. According to our results, IGF-Ⅰ can promote the proliferation of IEC-6 damaged by ASA, inhibit membrane lipid peroxidation, maintain the morphological integrity and consequently repair cell damage.
The present study was designed to investigate the repairing effect of insulin-like growth factors-Ⅰ(IGF-Ⅰ) on acetylsalicylic acid(ASA)-induced damage of intestinal epithelial cells(IEC-6). The cell damage model was induced by ASA. The CCK-8 assay was used to evaluate the proliferation of IEC-6. After being treated with ASA and IGF-Ⅰ, the cells were collected for determining the activities of lactate dehydrogenase(LDH) and alkaline phosphatase(ALP), and the culture supernatant for analysis of Na+,K+-ATPase and alkaline phosphatase(ALP) and the contents of total protein and malondialdelyde(MDA). We found that compared with the control group, IGF-Ⅰ at all concentrations tested promoted cell proliferation(P < 0.05). IGF-Ⅰ increased the cell survival rate, decreased the LDH activity in the culture supernatant and reduced the ALP activity and MDA content in the cells, as well as elevated the total protein content and Na~+,K~+-ATPase activity. According to our results, IGF-Ⅰ can promote the proliferation of IEC-6 damaged by ASA, inhibit membrane lipid peroxidation, maintain the morphological integrity and consequently repair cell damage.
引文
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[16]WARZECHA Z,DEMBINSKI A,CERANOWICZ P,et al.IGF-1stimulates production of interleukin-10 and inhibits development of caerulein-induced pancreatitis[J].Journal of Physiology and Pharmacology,2003,54(4):575-590.
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[18]李顺,黄品婕,葛缅,等.类胰蛋白酶通过激活PAR-2促进肠粘膜上皮IEC-6细胞损伤[J].中国病理生理杂志,2015,31(3):530-533.DOI:10.3969/j.issn1000-4718.2015.03.025.
[19]WU Z Q,HAN X D,WANG Y,et al.Interleukin-1 receptor antagonist reduced apoptosis and attenuated intestinal mucositis in a 5-fluorouracil chemotherapy model in mice[J].Cancer Chemotherapy and Pharmacology,2011,68(1):87-96.DOI:10.1007/s00280-010-1451-5.
[20]WU H,WANG L,ZHANG DM,et al.PRDM5 promotes the apoptosis of epithelial cells induced by IFN-gamma during Crohn’s disease[J].Pathology Research and Practice,2017,213(6):666-673.DOI:10.1016/j.prp.2016.12.004.
[21]TAMAKI K,OTAKA A,SHIBUYA T,et al.Traditional herbal medicine,rikkunshito,induces HSP60 and enhances cytoprotection of small intestinal mucosal cells as a nontoxic chaperone inducer[J].Evidence-Based Complementary and Alternative Medicine,2011,2012(3):278958.DOI:10.1155/2012/278958.
[22]王秋颖.碱性磷酸酶特性及其应用的研究进展[J].中国畜牧兽医,2011,38(1):157-160.
[23]NODA S,YAMADA A,TANABE R,et al.Menaquinone-4(vitamin K2)up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells[J].Nutrition Research,2016,36(11):1269-1276.DOI:10.1016/j.nutres.2016.10.001.
[24]ESCOBAR C,BYER K J,KHASKHELI H,et al.Apatite induced renal epithelial injury:insight into the pathogenesis of kidneystones[J].Journal of Urology,2008,180(1):379-387.DOI:10.1016/j.juro.2008.02.041.
[25]WANG X,MARTINEZ M A,DAI M H,et al.Permethrininduced oxidative stress and toxicity and metabolism.a review[J].Environmental Research,2016(149):86-104.DOI:10.1016/j.envres.2016.05.003.
[26]LIN S M,LUO L,YE Y T.Effects of dietary protein to energy ratio and N-Phy-P levels on nitrogen and phosphorus excretion and activities of GOT and GPT in Eriocheir sinensis[J].Journal of Fishery Sciences of China,2001,8:62-66.
[27]张婷,罗宇良.甲氰菊酯对鲤鳃Na+,K+-ATP酶活性及组织损伤的影响[J].南方水产科学,2013,9(6):41-46.
[28]樊凯芳,唐迎雪,曹淑霞.三化汤对脑缺血-再灌注老龄大鼠胃肠组织Na+-K+-ATP酶活性及Ca2+-ATP酶活性的影响[J].时珍国医国药,2009,20(6):1367-1368.DOI:10.3969/j.issn.1008-0805.2009.06.034.
[29]SATOH H,AMAGASE K,TAKEUCHI K.Exacerbation of nonsteroidal anti-inflammatory drug-induced small intestinal lesions by antisecretory drugs in rats:the role of intestinal motility[J].Journal of Pharmacology and Experimental Therapeutics,2012,343(2):270-277.DOI:10.1124/jpet.112.197475.
[30]TAKEUCHI K,HASE S,MIZOGUCHI H,et al.Protection by aspirin of indomethaein-induced small intestinal damage in rats:mediation by salicylic acid[J].Journal of Physiology Paris,2001,95:51-58.DOI:10.1016/S0928-4257(01)00009-2.
[31]QUARONI A,WANDS J,TRELSTAD R L,et al.Epithelioid cell cultures from rats small intestine:characterization by morphologic and immunologic criteria[J].Journal of Cell Biology,1979,80(2):248-265.DOI:10.1083/jcb.80.2.248.
[32]WOOD S R,ZHAO Q,SMITH L H,et al.Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression[J].Tissue and Cell,2003,35(1):47-58.DOI:10.1016/S0040-8166(02)00103-9.
[33]GENDRON F P,MONGRAIN S,LAPRISE P,et al.The CDX2transcription factor regulates furin expression during intestinal epithelial cell differentiation[J].American Journal of PhysiologyGastrointestinal and Liver Physiology,2006,290(2):G310-G318.DOI:10.1152/ajpgi.00217.2005.
[34]XU L,HE S S,LI D Y,et al.Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells(IEC-6)[J].Molecular Biology,2016,50(2):270-277.DOI:10.7868/S0026898416020269.
[35]MEI Chen,HE Shasha,YIN Peng,et al.Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury[J].Journal of Zhejiang University Science B,2016,17(6):413-424.DOI:10.1631/jzus.B1500261.
[2]COLLEONI J L,RIBEIRO F N,MOS P C A,et al.Venous thromboembolism prophylaxis after total knee arthroplasty(TKA):aspirin vs.rivaroxaban[J].Revista Brasileira de Ortopedia,2017,53(1):22-27.DOI:10.1016/j.rboe.2017.11.007.
[3]KIRSTEN B D.Aspirin use for the primary prevention of cardiovascular disease and colorectal cancer:US preventive services task force recommendation statement[J].Annals of Internal Medicine,2017,164(12):836-845.DOI:10.7326/M16-0577.
[4]罗佳,王惠吉,郝瑞瑞.非甾体抗炎药物所致肠道损伤[J].中国药物警戒,2011,8(5):302-305.
[5]GOLDSTEIN J L,SCHEIMAN J M,FORT J G,et al.Aspirin use in secondary cardiovascular protection and the development of aspirin-associated erosions and ulcers[J].Journal of Cardiovascular Pharmacology,2016,68(2):121-126.DOI:10.1097/FJC.0000000000000387.
[6]DOUTHWAITE A H,LINTOTT G A M.Gastroscopic observation of the effect of aspirinand certain other substances on the stomach[J].Lancet,1938,232:1222-1224.DOI:10.1016/S0140-6736(00)78970-7.
[7]BJARNASON I,ZANELLI G,PROUSEP,et al.Effect of nonsteroidal anti-inflammatory drugs on the human small intestine[J].Drugs,1986,32(1):35-41.DOI:10.2165/00003495-198600321-00007.
[8]ENDO H,SAKAI E,KATO T,et al.Small bowel injury in low-dose aspirin users[J].Journal of Gastroenterology,2015,50(4):378-386.DOI:10.1007/s00535-014-1028-x.
[9]雷洪军,单晶,孙晓滨,等.阿司匹林相关性胃肠黏膜病变研究现状[J].实用医院临床杂志,2016,13(6):156-159.
[10]FUQUAY J W,FOX P F,MCSWEENEY P L H.Encyclopedia of dairy sciences[M].2nd ed.London:Elsevier,2011:10.
[11]DAI Z Q,WU F,LI Y H,et al.IGF-IEc expression,regulation and biological function in different tissues[J].Growth Hormone and IGFResearch,2010,20(4):275-281.DOI:10.1016/j.ghir.2010.03.005.
[12]沈峰,王恬.IGF-I在胃肠道中的稳定性及其对肠道生长发育的影响[J].家畜生态,2004(4):131-133.
[13]张榕,施茵,关鑫.GH-SOCS2-IGF-1轴对溃疡性结肠炎肠黏膜屏障的影响[J].世界华人消化杂志,2010(23):2442-2447.
[14]MYLONAS P G,MATSOUKA P T,PAPANDONIOU E V,et al.Growth hormone and insulin-like growth factor I protect intestinal cells from radiation induced apoptosis[J].Molecular&Cellular Endocrinology,2000,160(1):115-122.
[15]WILKINS H R,OHNEDA K,KEKU T O,et al.Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice[J].American Journal of Physiology Gastrointestinal&Liver Physiology,2002,283(2):G457-G464.
[16]WARZECHA Z,DEMBINSKI A,CERANOWICZ P,et al.IGF-1stimulates production of interleukin-10 and inhibits development of caerulein-induced pancreatitis[J].Journal of Physiology and Pharmacology,2003,54(4):575-590.
[17]KUEMMERLE J F.Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity[J].American Journal of Physiology Gastrointestinal and Liver Physiology,2004,228(1):G101-111.DOI:10.1152/ajpgi.00032.2004.
[18]李顺,黄品婕,葛缅,等.类胰蛋白酶通过激活PAR-2促进肠粘膜上皮IEC-6细胞损伤[J].中国病理生理杂志,2015,31(3):530-533.DOI:10.3969/j.issn1000-4718.2015.03.025.
[19]WU Z Q,HAN X D,WANG Y,et al.Interleukin-1 receptor antagonist reduced apoptosis and attenuated intestinal mucositis in a 5-fluorouracil chemotherapy model in mice[J].Cancer Chemotherapy and Pharmacology,2011,68(1):87-96.DOI:10.1007/s00280-010-1451-5.
[20]WU H,WANG L,ZHANG DM,et al.PRDM5 promotes the apoptosis of epithelial cells induced by IFN-gamma during Crohn’s disease[J].Pathology Research and Practice,2017,213(6):666-673.DOI:10.1016/j.prp.2016.12.004.
[21]TAMAKI K,OTAKA A,SHIBUYA T,et al.Traditional herbal medicine,rikkunshito,induces HSP60 and enhances cytoprotection of small intestinal mucosal cells as a nontoxic chaperone inducer[J].Evidence-Based Complementary and Alternative Medicine,2011,2012(3):278958.DOI:10.1155/2012/278958.
[22]王秋颖.碱性磷酸酶特性及其应用的研究进展[J].中国畜牧兽医,2011,38(1):157-160.
[23]NODA S,YAMADA A,TANABE R,et al.Menaquinone-4(vitamin K2)up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells[J].Nutrition Research,2016,36(11):1269-1276.DOI:10.1016/j.nutres.2016.10.001.
[24]ESCOBAR C,BYER K J,KHASKHELI H,et al.Apatite induced renal epithelial injury:insight into the pathogenesis of kidneystones[J].Journal of Urology,2008,180(1):379-387.DOI:10.1016/j.juro.2008.02.041.
[25]WANG X,MARTINEZ M A,DAI M H,et al.Permethrininduced oxidative stress and toxicity and metabolism.a review[J].Environmental Research,2016(149):86-104.DOI:10.1016/j.envres.2016.05.003.
[26]LIN S M,LUO L,YE Y T.Effects of dietary protein to energy ratio and N-Phy-P levels on nitrogen and phosphorus excretion and activities of GOT and GPT in Eriocheir sinensis[J].Journal of Fishery Sciences of China,2001,8:62-66.
[27]张婷,罗宇良.甲氰菊酯对鲤鳃Na+,K+-ATP酶活性及组织损伤的影响[J].南方水产科学,2013,9(6):41-46.
[28]樊凯芳,唐迎雪,曹淑霞.三化汤对脑缺血-再灌注老龄大鼠胃肠组织Na+-K+-ATP酶活性及Ca2+-ATP酶活性的影响[J].时珍国医国药,2009,20(6):1367-1368.DOI:10.3969/j.issn.1008-0805.2009.06.034.
[29]SATOH H,AMAGASE K,TAKEUCHI K.Exacerbation of nonsteroidal anti-inflammatory drug-induced small intestinal lesions by antisecretory drugs in rats:the role of intestinal motility[J].Journal of Pharmacology and Experimental Therapeutics,2012,343(2):270-277.DOI:10.1124/jpet.112.197475.
[30]TAKEUCHI K,HASE S,MIZOGUCHI H,et al.Protection by aspirin of indomethaein-induced small intestinal damage in rats:mediation by salicylic acid[J].Journal of Physiology Paris,2001,95:51-58.DOI:10.1016/S0928-4257(01)00009-2.
[31]QUARONI A,WANDS J,TRELSTAD R L,et al.Epithelioid cell cultures from rats small intestine:characterization by morphologic and immunologic criteria[J].Journal of Cell Biology,1979,80(2):248-265.DOI:10.1083/jcb.80.2.248.
[32]WOOD S R,ZHAO Q,SMITH L H,et al.Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression[J].Tissue and Cell,2003,35(1):47-58.DOI:10.1016/S0040-8166(02)00103-9.
[33]GENDRON F P,MONGRAIN S,LAPRISE P,et al.The CDX2transcription factor regulates furin expression during intestinal epithelial cell differentiation[J].American Journal of PhysiologyGastrointestinal and Liver Physiology,2006,290(2):G310-G318.DOI:10.1152/ajpgi.00217.2005.
[34]XU L,HE S S,LI D Y,et al.Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells(IEC-6)[J].Molecular Biology,2016,50(2):270-277.DOI:10.7868/S0026898416020269.
[35]MEI Chen,HE Shasha,YIN Peng,et al.Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury[J].Journal of Zhejiang University Science B,2016,17(6):413-424.DOI:10.1631/jzus.B1500261.