毛蕊异黄酮对PM2.5介导炎症反应的作用及其机制
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摘要
探索毛蕊异黄酮(Calycosin)对PM2.5介导炎症反应的作用以及其机制,小鼠肺泡上皮细胞MLE12培养基中加入0～1mmol/L Calycosin,24h后MTT检测细胞活性。MLE12细胞经Calycosin预处理1h后,加入PM2.5至终浓度25μg/cm~2,共同孵育24h后,3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)法检测细胞活性,实时定量PCR检测细胞中白介素IL-6和IL-8mRNA,酶联免疫吸附法(ELISA)检测细胞上清液IL-6和IL-8含量,免疫荧光染色检测p65表达。MLE12细胞经0～1mmol/L Calycosin处理12h后,检测p-AMPK含量。MLE12细胞经腺苷单磷酸激活蛋白激酶(AMPK)抑制剂Compound C(10μmol/L)预处理1h,再加入Calycosin 100μmol/L处理1h,最后加入PM2.5(25μg/cm~2)刺激,24h后进行细胞活性和炎症反应检测。结果显示:0～1mmol/L Calycosin对MLE12细胞活性无明显影响,而Calycosin浓度大于500μmol/L具有明显的细胞毒性。Calycosin可明显减轻PM2.5暴露对细胞活性的损伤,抑制PM2.5诱导的IL-6和IL-8mRNA表达以及IL-6和IL-8蛋白的释放,并呈剂量依赖性。50或100μmol/L Calycosin可明显抑制PM2.5诱导的p65由细胞质进入细胞核。Calycosin可增加MLE12细胞内p-AMPK含量,并呈剂量依赖性。Compound C可抑制Calycosin减轻PM2.5介导细胞毒性、炎症因子增加和NF-κB激活的作用。由此推断:Calycosin在体外通过激活AMPK通路,抑制NF-κB的激活从而减轻PM2.5诱导的细胞损伤和炎症反应。
To explore the effect of Calycosin on PM2.5-mediated inflammatory response and the underlying mechanism.Calycosin was added to MLE12 cell culture medium at a final concentration of0-1 mmol/L.Cell viability was then measured by MTT after 24 hours.MLE12 cells were pretreated with Calycosin for 1 hour.Subsequently,PM2.5 was added at a final concentration of 25μg/cm~2.After incubation for 24 hours,MTT assay was performed to evaluate the cell viability and the expression of IL-6 and IL-8 mRNA were detected by real-time quantitative PCR.The levels of IL-6 and IL-8 protein in supernatant were detected by enzyme-linked immuno sorbent assay and the p65 expression were examined using immunofluorescence staining.MLE cells were treated with 0-100μmol/L of Calycosin for 12 hand the p-AMPK levels were evaluated using Western blot.MLE cells were pretreated with AMPC inhibitor Compound C(10μmol/L)for 1 h and then treated with 100μmol/L of Calycosin for 1 hour.Finally,PM2.5(25μg/cm~2)was added and the cell viablity and inflammatory response were detected after 24 h.Results showed that:Calycosin treatment at a concention of 1-100μmol/L had no significant effect on cell viability,whereas the cell viablity significantly decreased in MLE12 cells treated with Calycosin at a concentration of higher than 500μmol/L.Calycosin inhibited the expreession of IL-6 and IL-8 mRNA and the release of IL-6 and IL-8 protein induced by PM2.5 in a dose-dependent manner.50 or 100μmol/L of Calycosisn can significantly reduced the PM2.5 induced p65 translocation from the cytoplasm into the nucleus.Calycosin treatment can increase the content of p-AMPK in MLE12 cells in a dose-dependent manner.Compound C pre-treatment can abrogate the inhibitory effect of Calycosin on PM2.5-mediated cytotoxicity,inflammatory response and NF-κB activation.The result can draw the conclusion that Calycosin can inhibit the activation of NF-κB to decrease PM2.5-induced cell injury and inflammatory response in vitro by activating AMPK pathway.
To explore the effect of Calycosin on PM2.5-mediated inflammatory response and the underlying mechanism.Calycosin was added to MLE12 cell culture medium at a final concentration of0-1 mmol/L.Cell viability was then measured by MTT after 24 hours.MLE12 cells were pretreated with Calycosin for 1 hour.Subsequently,PM2.5 was added at a final concentration of 25μg/cm~2.After incubation for 24 hours,MTT assay was performed to evaluate the cell viability and the expression of IL-6 and IL-8 mRNA were detected by real-time quantitative PCR.The levels of IL-6 and IL-8 protein in supernatant were detected by enzyme-linked immuno sorbent assay and the p65 expression were examined using immunofluorescence staining.MLE cells were treated with 0-100μmol/L of Calycosin for 12 hand the p-AMPK levels were evaluated using Western blot.MLE cells were pretreated with AMPC inhibitor Compound C(10μmol/L)for 1 h and then treated with 100μmol/L of Calycosin for 1 hour.Finally,PM2.5(25μg/cm~2)was added and the cell viablity and inflammatory response were detected after 24 h.Results showed that:Calycosin treatment at a concention of 1-100μmol/L had no significant effect on cell viability,whereas the cell viablity significantly decreased in MLE12 cells treated with Calycosin at a concentration of higher than 500μmol/L.Calycosin inhibited the expreession of IL-6 and IL-8 mRNA and the release of IL-6 and IL-8 protein induced by PM2.5 in a dose-dependent manner.50 or 100μmol/L of Calycosisn can significantly reduced the PM2.5 induced p65 translocation from the cytoplasm into the nucleus.Calycosin treatment can increase the content of p-AMPK in MLE12 cells in a dose-dependent manner.Compound C pre-treatment can abrogate the inhibitory effect of Calycosin on PM2.5-mediated cytotoxicity,inflammatory response and NF-κB activation.The result can draw the conclusion that Calycosin can inhibit the activation of NF-κB to decrease PM2.5-induced cell injury and inflammatory response in vitro by activating AMPK pathway.
引文
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[12]杜冬华,利凯,周静,等.盐酸小檗碱对E.coli诱导的断乳仔兔腹泻转录因子NF-κB信号通路的影响[J].中国兽医学报,2018,38(4):741-745.
[13]王自力,薛长定,牟唐维,等.黄芪生脉饮对模拟运输应激大鼠肺脏组织炎性细胞因子mRNA表达及NF-κB信号通路的影响[J].中国兽医学报,2018,38(1):183-188.
[2]HOESEL,BASTIAN,SCHMID,et al.The complexity of NF-kappaB signaling in inflammation and cance[J].Mol Cancer,2013,12(1):86.
[3]SONG L,LI D,LI X,et al.Exposure to PM2.5induces aberrant activation of NF-kappaB in human airway epithelial cells by downregulating miR-331expression[J].Environ Toxicol Pharmacol,2017,50(3):192-199.
[4]LIU B,ZHANG J Z,LIU W H,et al.,Calycosin inhibits oxidative stress-induced cardiomyocyte apoptosis via activating estrogen receptor-alpha/beta[J].Bioorg Med Chem Lett,2016,26(1):181-185.
[5]XING Y F,XU Y H,SHI M H,et al.The impact of PM2.5on the human respiratory system[J].J Thorac Dis,2016,8(1):69-74.
[6]QUAN G H,WANG H,Cao J,et al.Calycosin suppresses RANKL-mediated osteoclastogenesis through inhibition of MAPKs and NF-kappaB[J].Int J Mol Sci,2015,16(12):29496-24507.
[7]SU X,HUANG Q,CHEN J,et al.Calycosin suppresses expression of pro-inflammatory cytokines via the activation of p62/Nrf2-linked heme oxygenase 1in rheumatoid arthritis synovial fibroblasts[J].Pharmacol Res,2016,113(Pt A):695-704.
[8]LAWRENCE T.The nuclear factor NF-kappaB pathway in inflammatio[J].Cold Spring Harb Perspect Biol,2009,1(6):a001651.
[9]WEI R,GUAN L F,ZHANG F,et al.PM2.5-induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF-kappaB-dependent pathway[J].J Appl Toxicol,2016,36(1):48-59.
[10] FERGUSON M D,MIGLIACCIO C,WARD T,et al.Comparison of how ambient PMc and PM2.5influence the inflammatory potential[J].Inhal Toxicol,2013,25(14):766-773.
[11] DAGHER Z,GARON G,BILLET S,et al.Role of nuclear factor-kappa B activation in the adverse effects induced by air pollution particulate matter(PM2.5)in human epithelial lung cells(L132)in culture[J].J Appl Toxicol,2007,27(3):284-290.
[12]杜冬华,利凯,周静,等.盐酸小檗碱对E.coli诱导的断乳仔兔腹泻转录因子NF-κB信号通路的影响[J].中国兽医学报,2018,38(4):741-745.
[13]王自力,薛长定,牟唐维,等.黄芪生脉饮对模拟运输应激大鼠肺脏组织炎性细胞因子mRNA表达及NF-κB信号通路的影响[J].中国兽医学报,2018,38(1):183-188.