白芍总苷对HaCaT细胞表达白细胞介素18的影响及相关信号通路的研究

Effect of total glucosides of paeony on the expression of interleukin-18 in human HaCaT keratinocytes and its related signaling pathways

目的 观察白芍总苷（TGP）对角质形成细胞（KC）表达白细胞介素（IL）-18的影响,并初步探讨ERK1/2、JNK1/2信号通路在其中的作用.方法 将部分HaCaT细胞分为3个组,即对照组加入0.031％二甲基亚砜的细胞培养液,TGP组分别加入6种不同浓度的TGP（0.5、2.5、12.5、62.5、125.0、312.5 mg/L）,抑制剂组分别加入10μmol/L ERKl/2抑制剂PD98059和JNK1/2抑制剂SP600125预处理2h后,再加入125 mg/LTGP.细胞继续培养48 h.实时反转录（RT）-PCR方法和ELISA方法检测HaCaT细胞IL-18的表达.部分HaCaT细胞分为两组,一组用125 mg/L TGP分别处理15,30,60 min,另一组分别用10μmol/L ERKl/2抑制剂PD98059和JNK1/2抑制剂SP600125预处理后再加入125 mg/L TGP分别处理15,30,60 min.免疫印迹技术观察两组HaCaT细胞ERK1/2、JNK1/2磷酸化水平.结果 TGP在低浓度（0.5、2.5 mg/L）时对HaCaT细胞IL-18mRNA和蛋白的表达有促进作用,62.5～125.0 mg/L时可抑制IL-18 mRNA和蛋白的表达,125 mg/L TGP抑制作用最强.TGP（125 mg/L）作用15 min后磷酸化ERKl/2蛋白表达达到高峰,表达水平为0.448±0.018,与对照组（0.204±0.005）比较,差异有统计学意义（P＜0.01）;30 min后表达水平降低至0.213±0.005,60 min后为0.217±0.005,与对照组相比差异无统计学意义（均P＞0.05）.PD98059预处理组磷酸化ERK1/2表达水平为0.237±0.010,与单独125 mg/L TGP给药组相比,差异有统计学意义（P＜0.01）.125 mg/L TGP对JNK蛋白的磷酸化作用不明显,各组相比差异无统计学意义（P＞0.05）.结论 TGP可抑制IL-18 mRNA和蛋白的表达,ERK1/2信号途径可能介导这一抑制作用.

Objective To evaluate the effect of total glucosides of paeony （TGP） on the expression of interleukin-18 （IL-18） in human HaCaT keratinocytes,and to explore the roles of extracelluar signal-regulated protein kinase1/2 （ERK1/2） and c-Jun N-terminal kinase 1/2 （JNK1/2） signaling pathways in the effect.Methods Some cultured human HaCaT keratinocytes were classified into three groups：control group treated with dimethyl sulfoxide （0.031％）,TGP groups treated with 6 different concentrations （0.5,2.5,12.5,62.5,125.0 and 312.5 mg/L） of TGP respectively,inhibitor groups treated with TGP of 125 mg/L after 2-hour pretreatment with PD98059 （an ERK1/2 inhibitor） and SP600125 （a JNK1/2 inhibitor） of 10 μmol/L respectively.After additional culture for 48 hours,reverse transcription （RT）-PCR was performed to measure the mRNA expression level of IL-18,and enzymelinked immunosorbent assay （ELISA） to determine the level of IL-18 protein in the culture supematant of HaCaT cells.Some HaCaT keratinocytes were classified into two groups to be treated with TGP of 125 mg/L for 15,30 and 60 minutes with or without the pretreatment with PD98059 and SP600125 of 10 μmol/L; then,Western blot was carried out to determine the phosphorylation levels of ERK1/2 and JNK1/2 in HaCaT cells.Results The levels of IL-18 mRNA and protein in culture supernatant were significantly increased by TGP of 0.5 and 2.5 mg/L,but decreased by TGP of 62.5 and 125.0 mg/L,and TGP of 125.0 mg/L showed the strongest inhibitory effect.After treatment with TGP of 125.0 mg/L,the level of phosphorylated ERK1/2 in HaCaT cells peaked at 15 minutes （0.448 ± 0.018）,decreased to 0.213 ± 0.005 at 30 minutes and 0.217 ± 0.005 at 60 minutes,with significant differences between TGP-treated and untreated cells at 15 minutes （0.448 ± 0.018 vs.0.204 ± 0.005,P＜ 0.05） but not at 30 or 60 minutes （both P ＞ 0.05）.The phosphorylation level of ERK1/2 was 0.237 ± 0.010 in HaCaT cells pretreated with PD98059 prior to the treatment with TGP,significantly different from that in HaCaT cells treated with TGP only （P ＜0.01）.TGP of 125.0 mg/L had no obvious effect on JNK phosphorylation,and there was no significant difference in the level of phosphorylated JNK1/2 between HaCaT cells untreated and those treated with TGP of 125.0 mg/L for different durations （all P ＞ 0.05）.Conclusions TGP can inhibit the expression of IL-18 mRNA and protein in HaCaT cells,likely through the ERK1/2 signaling pathway.