连翘脂素对LPS诱导RAW264.7细胞炎症反应的影响
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摘要
为研究连翘脂素的抗炎效应及其抗炎机制,以地塞米松作为阳性对照,建立脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7炎症模型,检测炎症因子的释放及相关蛋白和mRNA的表达,以期提高对连翘脂素抗炎作用的全面认识并为连翘脂素临床开发提供有力的科学依据。实验采用Griess法检测细胞上清液中NO含量,ELISA法检测TNF-α和IL-6的含量,Western blot法检测i NOS、COX-2蛋白的表达,RT-qPCR法检测i NOS、COX-2 mRNA的表达。与LPS组比较,连翘脂素组和地塞米松组可以明显降低LPS诱导的RAW 264.7细胞释放NO、TNF-α和IL-6的量,并呈现浓度依赖关系。Westren blot和RT-qPCR结果显示连翘脂素能抑制LPS诱导的i NOS、COX-2的蛋白表达以及mRNA的表达,并呈浓度依赖关系。实验研究表明连翘脂素能够明显抑制LPS诱导的RAW264.7细胞炎症因子的释放,i NOS、COX-2蛋白及mRNA的表达从而抑制炎症反应。
To study the anti-inflammatory effect and anti-inflammatory mechanism of Phillygenin,dexamethasone as a positive control,established a lipopolysaccharide(LPS)-induced RAW264.7 inflammation model,to detect the release of inflammatory factors and the expression of related proteins and mRNA,in order to improve the comprehensive understanding of the antiinflammatory effect of Phillygenin and provide a strong scientific basis for the clinical development of Phillygenin.The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent.The levels of TNF-α and IL-6 in culture media were measured with ELISA kits.Western blot assay was performed to illustrate the inhibitory effects of Phillygenin on i NOS and COX-2.RT-qPCR was detected for mRNA expression of i NOS and COX-2.Treatment with Phillygenin or Dexamethasone dose-dependently inhibited the production expression of NO,TNF-α and IL-6 in RAW264.7 macrophages.Western blot and RT-qPCR assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of i NOS and COX-2.In conclusion,Phillygenin exhibited obvious anti-inflammatory effect and its mechanism may be related to its regulation on inflammatory cytokines,and inhibition of the expression of i NOS and COX-2.
To study the anti-inflammatory effect and anti-inflammatory mechanism of Phillygenin,dexamethasone as a positive control,established a lipopolysaccharide(LPS)-induced RAW264.7 inflammation model,to detect the release of inflammatory factors and the expression of related proteins and mRNA,in order to improve the comprehensive understanding of the antiinflammatory effect of Phillygenin and provide a strong scientific basis for the clinical development of Phillygenin.The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent.The levels of TNF-α and IL-6 in culture media were measured with ELISA kits.Western blot assay was performed to illustrate the inhibitory effects of Phillygenin on i NOS and COX-2.RT-qPCR was detected for mRNA expression of i NOS and COX-2.Treatment with Phillygenin or Dexamethasone dose-dependently inhibited the production expression of NO,TNF-α and IL-6 in RAW264.7 macrophages.Western blot and RT-qPCR assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of i NOS and COX-2.In conclusion,Phillygenin exhibited obvious anti-inflammatory effect and its mechanism may be related to its regulation on inflammatory cytokines,and inhibition of the expression of i NOS and COX-2.
引文
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2 Jonathan C.The immunopathogenesis of sepsis[J].Nature,2002,420:885-891.
3 Zheng LL,Zhang GJ,Wang JJ,et al.Biological anti-inflammatory effects in active components of Flos Chrysanthemi Indic[J].Tianjin J Tradit Chin Med(天津中医药),2011,8:251-253.
4 Zhang ZY,Zhang GJ,Wang JJ,et al.Extract technique of water-soluble polysaccharide of Lysimachia foenum-graecum Hance optimized by RSM and its anti-inflammatory actions[J].Tianjin J Tradit Chin Med(天津中医药),2013,30:235-238.
5 Chinese Pharmacopoeia Commission.Pharmacopoeia of the People’s Republic of China:VolⅠ(中华人民共和国药典:第一部)[M].Beijing:China Medical Science Press,2015:170-171.
6 Pan XL.Effect of forsythin on LPS-induced inflammatory response and its molecular mechanism[D].Nanjing:Nanjing Normal University(南京师范大学),2014.
7 Zhou L.Experimental study on anti-endotoxin effect and mechanism of forsythiaside A[D].Nanchang:Nanchang University(南昌大学),2013.
8 Lu S,Chen SN,Guan JY,et al.Effect of forsythiaside on the function of RAW264.7 cells under endotoxin[J].Chin Agric Bull(中国农学通报),2012,28(20):58-62.
9 Nishibe S,Sakushima A,Kitagawa S,et al.Phenolic compounds from forsythia Leaves(III):on the comparison of constituents between hybrid and parents[J].Japan J Pharmacog,1988,42:324-328.
10 Maiada MA,Rahman,Paul M,et al.Lignans of forsythia intermedia[J].Phytochemistry,1990,29:1971-1980.
11 Su BN,Zhu QX,Gao K,et al.Ligand and phenylpropanoid glycosides from lancea tibetica and their antitumor activity[J].Planta Medica,1999,65:558-561.
12 Zhao CY,Qiu R,Zheng RL.In vitro antitumor activities of furan lignans[J].J Lanzhou Univ Med:Nat Sci(兰州大学学报:自科版),2000,36(4):66-68.
13 Liu XZ,Sheng N,Huo HL.Metabolites of phillyrin by rat intestinal flora[J].Chin J Pharm(中国医药工业杂志),2012,43:455-458.
14 Liu XZ,Sheng N,Huo HL,et al.Metabolites of forsythin in rat intestinal bacteria[J].Chin J Pharm(中国医药工业杂志),2012,43:455-458.
15 Quan YY,Yuan A,Gong XH,et al.Basic screening study on anti-inflammatory pharmacodynamics of forsythia[J].Nat Prod Res Dev(天然产物研究与开发),2017,29:435-438.
16 Hung CY,Tsai YC,Li KY.Phenolic antioxidants isolated from the flowers of osmanthus fragrans[J].Molecules,2012,17:10724-10737.
17 Kang WY,Wang JM.In vitro antioxidant properties and in vivo lowering blood lipid of forsythia suspense leave[J].MCRE,2010,19:617-628.
18 Yu YH,Shi Z,LI YJ,et al.Inhibition of i NOS and COX-2 expression by ginsenoside Rd in LPS-stimulated RAW264.7cells[J].Chin J Immun(中国免疫学杂志),2014,30:209-212.
19 Zhag XH,Dong L,Yang YX,et al.Effect of ethanol component of Zijinlong on secretion of inflammatory factors in RAW264.7 cells induced by lipopolysaccharide[J].Chin Jof Exper Trad Med Formu(中国实验方剂学杂志),2014,20:149-152.
20 Lei XH,Bao M,Zhen HN.Effect of TNF-αon expression of connective tissue growth factor in rat lung fibroblasts[J].Chin J Biochem Pharm(中国生化药物杂志),2010,31:340-342.
21 Shi ZR.Expression of IL-2,IL-8,TNF-αand MMP-9 in stable phase of chronic obstructive pulmonary disease[J].Chin J Biochem Pharm(中国生化药物杂志),2010,31:422-423.
22 Wang D,Dubois RN.The role of COX-2 in intestinal inflammation and colorectal cancer[J].Oncogene,2010,29:781-788.
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