野漆树苷对LPS诱导的RAW264.7细胞的抗炎作用及机制探究
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摘要
目的:探究野漆树苷(Rhoifolin,RHO)对细菌脂多糖(lipopolysaccharide,LPS)诱导的小鼠单核巨噬细胞RAW264.7细胞炎症模型释放炎性因子的影响,并探讨其作用机制.方法:用脂多糖(0.5μg·mL~(-1))刺激体外生长良好的RAW264.7细胞24h建立体外细胞炎症模型,以MTT法测定不同浓度RHO对RAW264.7细胞的毒性作用,用Griess试剂法检测一氧化氮(Nitric Oxide NO)的含量,RT-PCR法检测细胞中肿瘤坏死因子α(Tumor Necrosis Factor,TNF-α),白介素1β(Interleukin-1β,IL~(-1)β),白介素6(Interleukin,IL-6)的含量,采用Western Blot法检测MAPK信号通路中相关蛋白的含量.结果:与空白对照组相比,在LPS诱导下,RAW264.7细胞分泌致炎因子NO,TNF-α,IL~(-1)β,IL-6(P<0.01);与模型组相比,25~100μmol·L~(-1)的RHO可明显下调LPS诱导的RAW264.7细胞释放炎症因子NO,TNF-α,IL~(-1)β和IL-6(P<0.05,P<0.01),并呈现良好的剂量依赖关系;野漆树苷还不同程度的抑制了Erk和JNK激酶的磷酸化.结论:RHO可以抑制LPS所致的RAW264.7细胞炎症反应,减少炎症因子NO分泌,抑制TNF-α,IL~(-1)β和IL-6mRNA的合成,抑制JNK/SAPK及Erk信号通路可能是其抗炎作用机制之一.
This study performed to investigate the effect of Rhoifolin(RHO)on the secretion of inflammatory factors in monocyte-macrophage RAW264.7 cells induced by lipopolysaccharide(LPS),and to explore its mechanism.RAW264.7 cells were cultured with lipopolysaccharide(0.5μg·ml-1)for 24 hto establish the model of in vitro cell inflammation.MTT assay was used to determine the toxic effects of different concentrations of RHO on RAW264.7 cells.The levels of nitric oxide(NO)was determined by Griess reagent;the level of TNF-α,interleukin-1β(IL~(-1)β)and interleukin-6(IL-6)in the cells were measured by RT-PCR;Western Blot was used to detect the content of related proteins in MAPK signal pathway.Data showed that compared with the control group,LPS could induce the secretion of inflammatory factors NO,TNF-α,IL~(-1)βand IL-6(P<0.01)in RAW264.7 cells.Compared with the model group,25~100μmol·L1 RHO significantly down-regulated LPS-induced release of inflammatory cytokines NO,TNF-α,IL~(-1)βand IL-6(P<0.05,P<0.01)and showed well dose-dependent relationship.In conclution,Rhoifolin also inhibited the phosphorylation of Erk and JNK kinase to some extent,RHO can inhibit the inflammatory response of RAW264.7 cells induced by LPS,and its anti-inflammatory mechanism may be related to the decrease of NO secretion,inhibition the level of TNF-α,IL~(-1)βand IL-6 mRNA,and inhibit JNK/SAPK and Erk signaling pathways.
This study performed to investigate the effect of Rhoifolin(RHO)on the secretion of inflammatory factors in monocyte-macrophage RAW264.7 cells induced by lipopolysaccharide(LPS),and to explore its mechanism.RAW264.7 cells were cultured with lipopolysaccharide(0.5μg·ml-1)for 24 hto establish the model of in vitro cell inflammation.MTT assay was used to determine the toxic effects of different concentrations of RHO on RAW264.7 cells.The levels of nitric oxide(NO)was determined by Griess reagent;the level of TNF-α,interleukin-1β(IL~(-1)β)and interleukin-6(IL-6)in the cells were measured by RT-PCR;Western Blot was used to detect the content of related proteins in MAPK signal pathway.Data showed that compared with the control group,LPS could induce the secretion of inflammatory factors NO,TNF-α,IL~(-1)βand IL-6(P<0.01)in RAW264.7 cells.Compared with the model group,25~100μmol·L1 RHO significantly down-regulated LPS-induced release of inflammatory cytokines NO,TNF-α,IL~(-1)βand IL-6(P<0.05,P<0.01)and showed well dose-dependent relationship.In conclution,Rhoifolin also inhibited the phosphorylation of Erk and JNK kinase to some extent,RHO can inhibit the inflammatory response of RAW264.7 cells induced by LPS,and its anti-inflammatory mechanism may be related to the decrease of NO secretion,inhibition the level of TNF-α,IL~(-1)βand IL-6 mRNA,and inhibit JNK/SAPK and Erk signaling pathways.
引文
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[15]Eun-Ha Joh,Wan Gu,Dong-Hyun Kim.Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4in NF-κB and MAPK pathways[J].Biochemical Pharmacology,2012,84(3):331-340.
[2]江泽波,胡金萍,温晓文,等.猪苓多糖对巨噬细胞RAW264.7的双向免疫调节作用[J].免疫学杂志,2014,30(12):1033-1038.
[3]周博文.化州橘红治疗慢支肺气肿成分[J].中草药,1992(7):350.
[4]张秀明,陈志霞,林励.毛橘红与光橘红的化痰及抗炎作用比较研究[J].中药材,2004,27(02):122-123.
[5]肖崇厚,杨松松.中药化学[M].上海:上海科学技术出版社.1997:265.
[6]林励,陈志霞,袁旭江,等.两种化橘红的质量鉴别[J].广州中医药大学学报,2004,21(4):308-312.
[7]Eldahshan O A,Azab S.Anti-inflammatory effect of Apigenin-7-neohesperidoside(Rhoifolin)in carrageenin-induced rat edema model[J].Journal of Applied Pharmatical Science,2012,2(8):74-79.
[8]张秀明,陈志霞,林励.毛橘红与光橘红的化痰及抗炎作用比较研究[J].中药材,2004,27(02):122-123.
[9]Occhiuto F,Circosta C,De Pasquale A,et al.Comparative hemody-namic effects of the flavonoids rhoifolin and vitexin in the dog[J].Phytotherapy Research,1990,4(3):118-20.
[10]Kiem P,Nguyen TM,Minh C,et al.Two new c-glucosyl benzoic acids and flavonoids from Mallotus nanus and their antioxidant activity[J].Archives of Pharmacal Research,2010,33(2):203-208.
[11]Rao YK,Lee MJ,Chen K,et al.Insulin-Mimetic Action of Rhoifolin and Cosmosiin Isolated from Citrus grandis(L.)Osbeck Leaves:Enhanced Adiponectin Secretion and Insulin Receptor Phosphorylation in 3T3-L1Cells[J].Evidence-Based Complementary and Alternative Medicine,2011:1-9.
[12]Kyriakis J M,Avruch J.Mammalian Mitogen-Activated Protein Kinase Signal Transduction Pathways Activated by Stress and Inflammation[J].Physiol.Rev.2001,81(2):807-869.
[13]Luedde T,Trautwein C.Intracellular survival pathways in the liver[J].Liver International Official Journal of the International Association for the Study of the Liver,2006,26(10):1163.
[14]Guiqing Kong,Xiao Huang,Lipeng Wang,et al.Astilbin alleviates LPS-induced ARDS by suppressing MAPK signaling pathway and protecting pulmonary endothelial glycocalyx[J].International Immunopharmacology,2016,36:51-58.
[15]Eun-Ha Joh,Wan Gu,Dong-Hyun Kim.Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4in NF-κB and MAPK pathways[J].Biochemical Pharmacology,2012,84(3):331-340.