甘草酸通过c-Jun氨基末端蛋白激酶通路缓解脂多糖诱导的IPEC-J2细胞炎症反应

Glycyrrhizin Alleviates Lipopolysaccharide⁃Induced Inflammatory Response in IPEC⁃J2 Cells via c⁃Jun N⁃Terminal Protein Kinase Pathway

本试验旨在研究甘草酸(GL)缓解脂多糖(LPS)诱导的IPEC-J2细胞炎症的分子机制。选择IPEC-J2细胞作为研究对象,采用CCK-8法检测GL在不同浓度(0、10、50、100、200、500μmol/L)下处理不同时间(12、24、48 h)后的细胞活力,从而确定合适的GL处理条件。试验共设4个组,分别为对照组[IPEC-J2细胞生长24 h后,经二甲基亚砜(DMSO)处理12 h再经杜氏磷酸缓冲液(DPBS)共同处理12 h]、LPS组(IPEC-J2细胞生长24 h后,经DMSO处理12 h再经10μg/mL的LPS共同处理12 h)、GL组(IPEC-J2细胞生长24 h后,经50μmol/L的GL处理12 h再经DPBS共同处理12 h)和GL+LPS组(IPEC-J2细胞生长24 h后,经50μmol/L的GL处理12 h再经10μg/mL的LPS共同处理12 h)。采用酶联免疫吸附试验(ELISA)法检测LPS和GL处理后细胞上清液中白细胞介素-1β(IL-β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)以及高迁移率族蛋白1(HMGB1)的水平,实时荧光定量PCR(RT-qPCR)检测细胞中IL-1β、IL-6和TNF-α的mRNA表达水平,蛋白质免疫印迹(Western blotting)法和免疫荧光染色法检测细胞中Toll样受体4(TLR4)信号通路相关蛋白的表达水平。结果显示:1)与未添加GL相比,浓度为50μmol/L的GL处理24 h对IPEC-J2细胞活力无显著影响(P>0.05),故后续选择浓度为50μmol/L的GL处理24 h作为GL的适宜处理条件。2)与对照组相比,LPS组中IL-6的水平和mRNA表达水平显著升高(P<0.05);与LPS组相比,GL+LPS组中IL-6的水平和mRNA表达水平显著降低(P<0.05)。3)与对照组相比,LPS刺激显著增加了c-Jun氨基末端蛋白激酶1/3(p-JNK1/3)和c-Jun的蛋白表达水平(P<0.05);与LPS组相比,GL预处理显著降低了p-JNK1/3和c-Jun的蛋白表达水平(P<0.05),但对p38丝裂原活化蛋白激酶(p38MAPK)、核因子-κB p65(NF-κB p65)的蛋白表达水平无显著影响(P>0.05)。4)LPS组细胞核中磷酸化c-Jun(p-c-Jun)荧光强度显著高于对照组(P<0.05),且GL预处理可使细胞核中p-cJun荧光强度显著下降(P<0.05)。综上所述,GL可以通过下调IL-6基因表达以及c-Jun氨基末端蛋白激酶(JNK)信号通路相关蛋白表达,缓解LPS引起的IPEC-J2细胞的炎症反应。

This study was to elucidate the molecular mechanisms of glycyrrhizin(GL)alleviating lipopolysac⁃charide(LPS)⁃induced inflammatory response in IPEC⁃J2 cells.The IPEC⁃J2 cells were selected as the study object.The viability of IPEC⁃J2 cells after incubation with GL of different concentrations(0,10,50,100,200 and 500μmol/L)for different time(12,24 and 48 h)was measured by CCK⁃8 assay to determine the appropriate treatment condition of GL.There were four groups in this study,namely control group[IPEC⁃J2 cells were grown for 24 h and then treated with dimethyl sulfoxide(DMSO)for 12 h followed by Dulbecco’s phosphate buffered saline(DPBS)co⁃treatment for 12 h],LPS group(IPEC⁃J2 cells were grown for 24 h and then treated with DMSO for 12 h followed by LPS co⁃treatment with 10μg/mL for 12 h),GL group(IPEC⁃J2 cells were grown for 24 h and then treated with 50μmol/L GL for 12 h followed by DPBS co⁃treat⁃ment for 12 h)and GL+LPS group((IPEC⁃J2 cells were grown for 24 h and then treated with 50μmol/L GL for 12 h followed by 10μg/mL LPS co⁃treatment for 12 h).Enzyme linked immunosorbent assay(ELISA)method was used to detect the levels of interleukin⁃1β(IL⁃1β),interleukin⁃6(IL⁃6),tumor necrosis factor⁃α(TNF⁃α)and high mobility group protein 1(HMGB1)in the supernatant of IPEC⁃J2 cells treated with LPS and GL.The mRNA expression levels of IL⁃1β,IL⁃6 and TNF⁃αin cells were detected by real⁃time fluorescent quantitative PCR(RT⁃qPCR).Expression levels of proteins related with Toll⁃like receptor 4(TLR4)signal pathway were detected by Western blotting method and immunofluorescence staining method.The results showed as follows:1)compared with no GL added,which was treated with 50μmol/L GL for 24 h had no significant effect on the viability of IPEC⁃J2 cells(P>0.05),so the appropriate concentration and time for GL were 50μmol/L and 24 h,respectively.2)Compared with the control group,the level and mRNA expression level of IL⁃6 in the LPS group were significantly increased(P<0.05);compared with the LPS group,the level and mRNA expression level of IL⁃6 in the GL+LPS group were significantly decreased(P<0.05).3)Com⁃pared with the control group,LPS stimulation significantly increased the protein expression levels of phospho⁃rylated c⁃Jun N⁃terminal protein kinase 1/3(p⁃JNK1/3)and c⁃Jun in cells(P<0.05);compared with the LPS group,pretreatment with GL could down⁃regulate the protein expression levels of p⁃JNK1/3 and c⁃Jun in cells(P<0.05),but had no significant effects on the protein expression levels of p38 mitogen⁃activated protein kinase(p38 MAPK)and nuclear factor kappa⁃B p65(NF⁃κB p65)in cells(P>0.05).4)The fluorescence intensity of phosphorylated c⁃Jun(p⁃c⁃Jun)in cell nucleus in the LPS group was significantly decreased com⁃pared with the control group(P<0.05),while pretreatment with GL could significantly down⁃regulate the flu⁃orescence intensity of p⁃c⁃Jun in cell nucleus(P<0.05).In conclusion,GL alleviates LPS⁃induced inflamma⁃tory response in IPEC⁃J2 cells by down⁃regulating the gene expression of IL⁃6 and related protein expression of c⁃Jun N⁃terminal protein kinase(JNK)signaling pathway.