清肠栓对LPS诱导Caco-2细胞损伤的保护作用
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摘要
目的探讨清肠栓(QCS)含药血清对脂多糖(LPS)诱导Caco-2细胞损伤模型的保护作用。方法收集对数生长期细胞,分为5个组,即正常对照组、LPS组,以及QCS低剂量组(2.5%含药血清)、中剂量组(5%含药血清)、高剂量组(10%含药血清)。采用MTT法检测细胞活力;ELISA法测定培养细胞上清液IL-8、IL-17和PGE_2含量,以及MDA、GSH和SOD水平;划痕实验检测细胞体外迁移能力;Western blot技术检测RhoA、Rac1蛋白的表达。结果 QCS能拮抗LPS诱导的Caco-2细胞活性下降;与LPS组比较,QCS可显著减少LPS诱导的IL-8、IL-17和PGE_2分泌(P<0.05),并减少LPS诱导的MDA释放,增加GSH和SOD的分泌(P<0.01);划痕实验48 h时QCS组的划痕宽度明显变小,细胞迁移能力增强(P<0.001),划痕的修复加快,且呈浓度依赖性改变;Western blot结果显示,QCS能有效逆转LPS所致RhoA、Rac1蛋白表达的抑制(P<0.05)。结论 QCS含药血清对LPS诱导的Caco-2细胞损伤有保护作用,可通过抑制炎症因子、抗氧化应激和促进结肠上皮细胞迁移来提高Caco-2的生存率。
Objective To investigate the protective effects of Qingchang Suppository(QCS) medicated serum on lipopolysaccharide(LPS)-induced Caco-2 cell injury model. Methods Caco-2 cells in logarithmic growth phase were collected and divided into five groups: normal control group, LPS group, low-dose QCS group(2.5% QCS medicated serum), medium-dose QCS group(5% QCS medicated serum) and high-dose QCS group(10% QCS medicated serum). MTT assay was used to detect cell viability; ELISA was used to determine the content of IL-8, IL-17 and PGE_2, and the levels of MDA, GSH and SOD in the supernatant; scratch assay was employed to test cell migration ability in vitro; Western blot was used to detect the expression of RhoA and Rac1 proteins. Results QCS antagonized the decrease in Caco-2 cell viability induced by LPS. Compared with the LPS group, QCS significantly reduced the secretion of IL-8, IL-17 and PGE_2 induced by LPS(P<0.05), lowered the release of MDA induced by LPS and increased the secretion of GSH and SOD(P<0.01); the scratch width in the QCS group decreased significantly and the cell migration ability increased at 48 h(P<0.001), which speeded up the repair of scratches in a concentration-dependent manner; Western blot results showed that QCS could effectively reverse LPS-induced inhibition of RhoA and Rac1 protein expression(P<0.05). Conclusion Qingchang Suppository medicated serum protects Caco-2 cells from LPS-induced injury and increases cell survival rate by inhibiting inflammatory factors and antioxidant stress and promoting migration of colonic epithelial cells.
Objective To investigate the protective effects of Qingchang Suppository(QCS) medicated serum on lipopolysaccharide(LPS)-induced Caco-2 cell injury model. Methods Caco-2 cells in logarithmic growth phase were collected and divided into five groups: normal control group, LPS group, low-dose QCS group(2.5% QCS medicated serum), medium-dose QCS group(5% QCS medicated serum) and high-dose QCS group(10% QCS medicated serum). MTT assay was used to detect cell viability; ELISA was used to determine the content of IL-8, IL-17 and PGE_2, and the levels of MDA, GSH and SOD in the supernatant; scratch assay was employed to test cell migration ability in vitro; Western blot was used to detect the expression of RhoA and Rac1 proteins. Results QCS antagonized the decrease in Caco-2 cell viability induced by LPS. Compared with the LPS group, QCS significantly reduced the secretion of IL-8, IL-17 and PGE_2 induced by LPS(P<0.05), lowered the release of MDA induced by LPS and increased the secretion of GSH and SOD(P<0.01); the scratch width in the QCS group decreased significantly and the cell migration ability increased at 48 h(P<0.001), which speeded up the repair of scratches in a concentration-dependent manner; Western blot results showed that QCS could effectively reverse LPS-induced inhibition of RhoA and Rac1 protein expression(P<0.05). Conclusion Qingchang Suppository medicated serum protects Caco-2 cells from LPS-induced injury and increases cell survival rate by inhibiting inflammatory factors and antioxidant stress and promoting migration of colonic epithelial cells.
引文
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[21] WU H,LUO T,LI Y M,et al.Granny Smith apple procyanidin extract upregulates tight junction protein expression and modulates oxidative stress and inflammation in lipopolysaccharide- induced Caco- 2 cells[J].Food Funct,2018,9(6):3321- 3329.
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[23] RIDLEY A J.Rho GTPase signalling in cell migration[J].Curr Opin Cell Biol,2015(36):103- 112.
[24] SADOK A,MARSHALL C J.Rho GTPases:masters of cell migration[J].Small GTPases,2014(5):e29710.
[25] SANTOS M F,MCCORMACK S A,GUO Z,et al.Rho proteins play a critical role in cell migration during the early phase of mucosal restitution[J].J Clin Invest,1997,100(1):216- 225.
[26] VIDMAR J,CHINGWARU C,CHINGWARU W.Mammalian cell models to advance our understanding of wound healing:a review[J].J Surg Res,2017(210):269- 280.
[27] NIGRO E,SCHETTINO P,POLITO R,et al.Adiponectin and colon cancer:evidence for inhibitory effects on viability and migration of human colorectal cell lines[J].Mol Cell Biochem,2018,448(1- 2):125- 135.
[2] 中华医学会消化病学分会炎症性肠病学组.炎症性肠病诊断与治疗的共识意见(2018年北京)[J].中国实用内科杂志,2018,38(9):796- 813.
[3] ZENG Z,ZHU Z,YANG Y,et al.Incidence and clinical characteristics of inflammatory bowel disease in a developed region of Guangdong Province,China:a prospective population- based study[J].J Gastroenterol Hepatol,2013,28(7):1148- 1153.
[4] YANG H,LI Y,WU W,et al.The incidence of inflammatory bowel disease in Northern China:a prospective population- based study[J].PLoS One,2014,9(7):e101296.
[5] WANG Y,OU Y Q.Ulcerative colitis in China:retrospective analysis of 3 100 hospitalized patients[J].J Gastroenterol Hepatol,2007,22(9):1450- 1455.
[6] DAI Y C,TANG Z P,MA G T,et al.A review of Qingchang Shuan for treatment of ulcerative colitis[J].J Tradit Chin Med,2010,30(3):237- 240.
[7] 谢建群,张涛,郑昱,等.清肠栓对大鼠溃疡性结肠炎caspase- 3影响的研究[J].中成药,2008,30(3):340- 343.
[8] 卢璐,谢建群,郭春荣.中药清肠栓对溃疡性结肠炎大鼠结肠黏膜紧密连接蛋白ZO- 1、occludin的影响[J].世界华人消化杂志,2011,19(22):2322- 2327.
[9] SUN B,YUAN J,WANG S,et al.Qingchang Suppository ameliorates colonic vascular permeability in dextran- sulfate- sodium- induced colitis[J].Front Pharmacol,2018(9):1235.
[10] WAN P,CHEN H,GUO Y,et al.Advances in treatment of ulcerative colitis with herbs:from bench to bedside[J].World J Gastroenterol,2014,20(39):14099- 14104.
[11] 李超男,龚雨萍.清肠栓治疗溃疡性结肠炎的临床和实验研究进展[J].临床医药文献杂志,2018,5(9):181- 185.
[12] 龚雨萍,柳文,马贵同,等.清肠栓治疗溃疡性结肠炎的随机对照研究[J].上海中医药大学学报,2007,6(21):33- 36.
[13] ZHOU Y X,XIN H L,RAHMAN K,et al.Portulaca oleracea L:a review of phytochemistry and pharmacological effects[J/OL].Biomed Res Int,2015[2015-01-26].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321094.
[14] 王天宁,刘玉婷,肖凤琴,等.马齿苋化学成分及药理活性的现代研究整理[J].中国实验方剂学杂志,2018,24(6):224- 234.
[15] LAI J L,LIU Y H,LIU C,et al.Indirubin inhibits LPS- induced inflammation via TLR4 abrogation mediated by the NF- κB and MAPK signaling pathways[J].Inflammation,2017,40(1):1- 12.
[16] 庞丹清,陈勇,刘玟君,等.三七药理作用研究进展[J].大众科技,2018,20(9):49- 51.
[17] XU Y,TAN H Y,LI S,et al.Panax notoginseng for inflammation- related chronic diseases:a review on the modulations of multiple pathways[J].Am J Chin Med,2018,46(5):971- 996.
[18] GUO S,AL- SADI R,SAID H M,et al.Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR- 4 and CD14[J].Am J Pathol,2013,182(2):375- 387.
[19] LAMBERT G P.Stress- induced gastrointestinal barrier dysfunction and its inflammatory effects[J].J Anim Sci,2009,87(14 Suppl):E101- E108.
[20] PISTOL G C,MARIN D E,DRAGOMIR C,et al.Synbiotic combination of prebiotic grape pomace extract and probiotic Lactobacillus sp.reduced important intestinal inflammatory markers and in- depth signalling mediators in lipopolysaccharide- treated Caco- 2 cells[J].Br J Nutr,2019,121(3):291- 305.
[21] WU H,LUO T,LI Y M,et al.Granny Smith apple procyanidin extract upregulates tight junction protein expression and modulates oxidative stress and inflammation in lipopolysaccharide- induced Caco- 2 cells[J].Food Funct,2018,9(6):3321- 3329.
[22] BOUREUX A,VIGNAL E,FAURE S,et al.Evolution of the Rho family of ras- like GTPases in eukaryotes[J].Mol Biol Evol,2007,24(1):203- 216.
[23] RIDLEY A J.Rho GTPase signalling in cell migration[J].Curr Opin Cell Biol,2015(36):103- 112.
[24] SADOK A,MARSHALL C J.Rho GTPases:masters of cell migration[J].Small GTPases,2014(5):e29710.
[25] SANTOS M F,MCCORMACK S A,GUO Z,et al.Rho proteins play a critical role in cell migration during the early phase of mucosal restitution[J].J Clin Invest,1997,100(1):216- 225.
[26] VIDMAR J,CHINGWARU C,CHINGWARU W.Mammalian cell models to advance our understanding of wound healing:a review[J].J Surg Res,2017(210):269- 280.
[27] NIGRO E,SCHETTINO P,POLITO R,et al.Adiponectin and colon cancer:evidence for inhibitory effects on viability and migration of human colorectal cell lines[J].Mol Cell Biochem,2018,448(1- 2):125- 135.
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