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.
引文
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