几种药物对脂多糖诱导奶牛乳腺上皮细胞(BMECs)分泌炎性因子的影响
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摘要
通过脂多糖(lipopolysaccharide,LPS)诱导奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)建立体外炎症模型,探究不同药物对炎症模型细胞分泌炎性因子的抑制作用,为阐明药物抗炎机制奠定基础。本研究通过CCK-8法筛选LPS的最佳致炎剂量,确定细胞炎症模型;将氟尼辛葡甲胺、亚硫酸氢钠穿心莲内酯、苦木、莲胆作用于炎症模型细胞,以地塞米松作为阳性对照,ELISA法检测各组细胞培养液上清中炎性因子TNF-α、IL-6和IL-1β的表达变化情况。研究发现,经LPS(10mg/L)刺激BMECs 24h,炎症模型组细胞培养上清中TNF-α和IL-1β的含量显著高于空白对照组(P<0.05),IL-6的含量极显著高于空白对照组(P<0.01);氟尼辛葡甲胺(10mg/L)、亚硫酸氢钠穿心莲内酯(5mg/L)、苦木(5mg/L)、莲胆(5mg/L)在使用质量浓度下均能不同程度地抑制炎症模型组BMECs分泌TNF-α、IL-6和IL-1β,其中亚硫酸氢钠穿心莲内酯对炎症细胞具有很好的抗炎作用,为以后体内的抗炎作用研究奠定基础。
To explore inhibitory effect of different drug on secreting inflammatory cytokines via inflammation cell model and further study mechanism of anti-inflammatory drugs,an inflammation model was built in vitro by inducing bovine mammary epithelial cells(BMECs)with lipopolysaccharide.In this study,the optimum dose causing inflamed was screened by CCK-8,and the inflammatory cell model was determined.The flunixin meglumine,sodium bisulfite paniculta lactone,Picrasma quassioides,Liandan were used in experimental group,and the dexamethasone was used in the positive control,the expression of inflammatory factor TNF-α,IL-6 and IL-1βin the cell culture supernatant in each grup were detected by ELISA method.The study found that with the BMECs stimulated by LPS(including 10 mg/L)about 24 h,the content of TNF-αand IL-1βin the cell culture supernatant was higher in experimental group than blank control group(P<0.05),the content of IL-6 was significantly higher than that of blank control group(P<0.01);The flunixin meglumine(10 mg/L),sodium bisulfite paniculta lactone(5 mg/L),Picrasma quassioides(5 mg/L),Liandan(5 mg/L)in proper concentrations can inhibit the secretion of TNF-α,IL-6 and IL-1βin the BMECs in different level.In addition,sodium bisulfite paniculta lactone had a good anti-inflammatory effect,it laid a solid foundation for the study of the anti-inflammatory effects in the future.
To explore inhibitory effect of different drug on secreting inflammatory cytokines via inflammation cell model and further study mechanism of anti-inflammatory drugs,an inflammation model was built in vitro by inducing bovine mammary epithelial cells(BMECs)with lipopolysaccharide.In this study,the optimum dose causing inflamed was screened by CCK-8,and the inflammatory cell model was determined.The flunixin meglumine,sodium bisulfite paniculta lactone,Picrasma quassioides,Liandan were used in experimental group,and the dexamethasone was used in the positive control,the expression of inflammatory factor TNF-α,IL-6 and IL-1βin the cell culture supernatant in each grup were detected by ELISA method.The study found that with the BMECs stimulated by LPS(including 10 mg/L)about 24 h,the content of TNF-αand IL-1βin the cell culture supernatant was higher in experimental group than blank control group(P<0.05),the content of IL-6 was significantly higher than that of blank control group(P<0.01);The flunixin meglumine(10 mg/L),sodium bisulfite paniculta lactone(5 mg/L),Picrasma quassioides(5 mg/L),Liandan(5 mg/L)in proper concentrations can inhibit the secretion of TNF-α,IL-6 and IL-1βin the BMECs in different level.In addition,sodium bisulfite paniculta lactone had a good anti-inflammatory effect,it laid a solid foundation for the study of the anti-inflammatory effects in the future.
引文
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[4]GENINI S,BADAOUI B,SCLEP G,et al.Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources[J].BMC Genomics,2011,12(1):225.
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[9]李玉,贺胜男,丁娜娜,等.穿心莲内酯对LPS诱导的小鼠乳腺炎的抗炎作用[J].中国兽医科学,2017,47(9):1200-1206.
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[12]卢金霞,何芳,刘丛玲,等.BMECs模型的建立与鉴定[J].黑龙江畜牧兽医,2017(9):30-33.
[13]田璐,林晶晶,侯瑛倩,等.脂多糖对小鼠乳腺组织中Toll-like Receptor 4表达的影响[J].中国兽医学报,2012,32(6):902-905.
[14] GENINI S,BADAOUI B,SCLEP G.et al.Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources[J].BMC Genomics,2011,12(1):225.
[15] WEI Z,YAO M,LI Y,et al.Inhibition of lipopolysaccharide(LPS)-induced inflammatory responses by selenium in bovine mammary epithelial cells in primary culture[J].Inflammation,2015,38(1):152.
[16] MIERNIKIEWICZ P,KLOPOT A,SOLUCH R,et al.T4 Phage tail adhesin Gp12counteracts LPS-induced inflammation in vivo[J].Front Microbiol,2016(7):1112.
[17] RIOLLET C,RAINARD P,POUTREL B.Cell subpopulations and cytokine expression in cow milk in response to chronic Staphylococcus aureus infection[J].J Dairy Sci,2001,84(5):1077-1084.
[18]路孝兵,奶牛乳房炎中药“蒲和饮”透皮剂的研制及临床疗效试验[D].重庆:西南大学,2014.
[19]刘艳,罗永江,辛蕊华,等.中药对牛免疫调节作用研究进展[J].动物医学进展,2016,37(6):95-98.
[20]张霞,吴迪,王家泰,等.穿心莲破坏内毒素作用的体外实验研究[J].中国中西医结合急救杂志,2000,7(4):212-214.
[21]李欣,李中改,张小艺,等.金英黄归汤对LPS介导乳腺上皮细胞TLR4信号通路中相关因子的影响[J].畜牧兽医学报,2016,47(3):609-614.
[22]陈莉,曾文谊.糖皮质激素类药物的合理使用[J].实用药物与临床,2012,15(1):39-40.
[2]赵洁,杨选明,都培双,等.T淋巴细胞抑制急性感染的炎症反应[J].生物化学与生物物理进展,2008,35(2):129-132.
[3]SOUZA F N D,HEINEMANN M B,GIDLUND M A,et al.The innate immunity in bovine mastitis:The role of pattern-recognition receptors[J].Am J Immunol,2012,8(4):166-178.
[4]GENINI S,BADAOUI B,SCLEP G,et al.Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources[J].BMC Genomics,2011,12(1):225.
[5]PAULRUD C O.Basic concepts of the bovine teat canal[J].Vet Res Commun,2005,29(3):215-245.
[6]PORCHERIE A,CUNHA P,TROTEREAU A,et al.Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells[J].Vet Res,2012,43(1):14.
[7]倪志宇.CCK-8对炎症反应中促炎及抗炎性细胞因子表达的调控机制研究[D].河北石家庄:河北医科大学,2005.
[8]唐后庆,吴广军.糖皮质激素在牲畜治疗中需谨慎使用[J].畜牧兽医科技信息,2010(8):48.
[9]李玉,贺胜男,丁娜娜,等.穿心莲内酯对LPS诱导的小鼠乳腺炎的抗炎作用[J].中国兽医科学,2017,47(9):1200-1206.
[10]焦伟华.中药苦木抗炎活性成分研究[D].辽宁沈阳:沈阳药科大学,2010.
[11]苏欣.穿心莲及其制剂的药理作用和临床研究进展[J].黑龙江科技信息,2015(9):72-73.
[12]卢金霞,何芳,刘丛玲,等.BMECs模型的建立与鉴定[J].黑龙江畜牧兽医,2017(9):30-33.
[13]田璐,林晶晶,侯瑛倩,等.脂多糖对小鼠乳腺组织中Toll-like Receptor 4表达的影响[J].中国兽医学报,2012,32(6):902-905.
[14] GENINI S,BADAOUI B,SCLEP G.et al.Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources[J].BMC Genomics,2011,12(1):225.
[15] WEI Z,YAO M,LI Y,et al.Inhibition of lipopolysaccharide(LPS)-induced inflammatory responses by selenium in bovine mammary epithelial cells in primary culture[J].Inflammation,2015,38(1):152.
[16] MIERNIKIEWICZ P,KLOPOT A,SOLUCH R,et al.T4 Phage tail adhesin Gp12counteracts LPS-induced inflammation in vivo[J].Front Microbiol,2016(7):1112.
[17] RIOLLET C,RAINARD P,POUTREL B.Cell subpopulations and cytokine expression in cow milk in response to chronic Staphylococcus aureus infection[J].J Dairy Sci,2001,84(5):1077-1084.
[18]路孝兵,奶牛乳房炎中药“蒲和饮”透皮剂的研制及临床疗效试验[D].重庆:西南大学,2014.
[19]刘艳,罗永江,辛蕊华,等.中药对牛免疫调节作用研究进展[J].动物医学进展,2016,37(6):95-98.
[20]张霞,吴迪,王家泰,等.穿心莲破坏内毒素作用的体外实验研究[J].中国中西医结合急救杂志,2000,7(4):212-214.
[21]李欣,李中改,张小艺,等.金英黄归汤对LPS介导乳腺上皮细胞TLR4信号通路中相关因子的影响[J].畜牧兽医学报,2016,47(3):609-614.
[22]陈莉,曾文谊.糖皮质激素类药物的合理使用[J].实用药物与临床,2012,15(1):39-40.