参附注射液通过调节核内HMGB1和NF-κB结合发挥抗炎活性的机制

Anti-inflammatory Mechanism of Shenfu Injection by Regulating the Combination of Nuclear HMGB1 and NF-κB Pathway

探讨参附注射液(Shenfu injection,SFI)对脂多糖(lipopolysaccharides,LPS)诱导的RAW264.7细胞的抑炎作用及其潜在机制。采用LPS诱导野生型RAW264.7细胞制作炎症细胞模型,经参附注射液不同剂量进行干预,RT-qPCR、WB和ELISA检测细胞内促炎因子,包括诱导型一氧化氮合酶(induced nitric oxide synthase,iNOS)、白介素-1β(interleukin-1β,IL-1β)和肿瘤坏死因子α(tumor necrosis factorα,TNF-α)的转录、翻译和分泌水平,评价参附注射液的抗炎作用;构建高迁移率组蛋白B1(high mobility group protein B1,HMGB1)过表达RAW264.7细胞(HMGB1^(+)-RAW264.7)和HMGB1点突变RAW264.7细胞(HMGB1^(m)-RAW264.7),通过免疫荧光检测野生型、HMGB1^(+)和HMGB1^(m)-RAW264.7细胞中HMGB1的核移位情况,采用哺乳动物双杂交法和免疫共沉淀法检测HMGB1、P65和P50的结合率,以探讨核内HMGB1在炎症反应中的作用及SFI抗炎的潜在分子机制。结果表明:SFI可抑制LPS诱导的RAW264.7细胞中iNOS、IL-1β和TNF-α的转录、表达和分泌(P<0.01)。SFI能抑制HMGB1向核外迁移,使其高表达于核内。核内高表达HMGB1可使RAW264.7细胞对LPS的耐受度增加。在1μg/mL浓度LPS诱导下,HMGB1^(+)-RAW264.7细胞中TNFα和IL-1β的转录和表达水平均升高,且SFI能逆转这一现象。HMGB1^(m)-RAW264.7细胞中TNFα和IL-1β与对照组无差异。SFI可增加核内HMGB1与P65、HMGB1与P50的结合率(P<0.01),减少P50与P65的结合率。试验结果表明:SFI可抑制LPS诱导的RAW264.7细胞炎症反应;其作用途径可能是通过抑制HMGB1出核,竞争性结合P65和P50合,减少P65-P50二聚体的形成,进而抑制核因子κB(nuclear factor kappa-B,NF-κB)通路的激活。

To explore the anti-inflammatory effect and potential mechanism of Shenfu injection(SFI)on lipopolysaccharides(LPS)induced RAW264.7 cells.Inflammatory cell model in wild type RAW264.7 cells were induced by using LPS.Pro-inflammatory factors(including iNOS,IL-1βand TNF-α)were tested by using RT-qPCR,WB and ELISA to evaluate the anti-inflammatory effect of SFI.HMGB1^(+)(over-expressing HMGB1)-RAW264.7 cells and HMGB1^(m)(mutation of nuclear localization sites to prevent the extra-nuclear migration)-RAW264.7 cells were constructed to explore the potential mechanism.After stimulating by LPS,the level of HMGB1,TNF-α,IL-1βand iNOS were tested by RT-qPCR and WB,and the translocation of HMGB1 were tested by immunofluorescence in wild type,HMGB1^(+)and HMGB1^(m)-RAW264.7 cells.Then mammalian two-hybrid approach and co-immunoprecipitation method were carried out to detect the binding rates among HMGB1,P65 and P50.The results indicate that the expression of iNOS,IL-1β,and TNF-αin LPS induced RAW264.7 cells were inhibited by SFI(P<0.01).The extracellular migration of HMGB1 was inhibited also by SFI,then resulting in the high expression of HMGB1 in the nucleus.the tolerance of RAW264.7 cells to LPS was increased by the high expression of HMGB1 in the nucleus.The TNF-αand IL-1βof HMGB1^(+)-RAW264.7 cells induced by LPS(1μg/mL)were both increased,which was reversed by SFI.The TNF-αand IL-1βof HMGB1^(m)-RAW264.7 cells had no difference with control group.SFI increased the binding rate of HMGB1 to P65 and HMGB1 to P50 in the nucleus(P<0.01),and reduced the binding rate of P50 to P65.The results show that the above results suggested that the inflammatory response of LPS induced RAW264.7 cell was inhibited by SFI.Its pathway of action may be through inhibiting the translocation of HMGB1,competitively binding to P65 and P50,as well as reducing the formation of P65-P50 dimers.