中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
10期
723-727
,共5页
张洪英%王小艳%陈星宇%逄明杰%史同新
張洪英%王小豔%陳星宇%逄明傑%史同新
장홍영%왕소염%진성우%방명걸%사동신
白芍%角蛋白细胞%白细胞介素18%丝裂原激活蛋白激酶类
白芍%角蛋白細胞%白細胞介素18%絲裂原激活蛋白激酶類
백작%각단백세포%백세포개소18%사렬원격활단백격매류
RADIX PAEONIAE ALBA%Keratinocytes%Interleukin-18%Mitogen-activated protein kinases
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.
目的 觀察白芍總苷(TGP)對角質形成細胞(KC)錶達白細胞介素(IL)-18的影響,併初步探討ERK1/2、JNK1/2信號通路在其中的作用.方法 將部分HaCaT細胞分為3箇組,即對照組加入0.031％二甲基亞砜的細胞培養液,TGP組分彆加入6種不同濃度的TGP(0.5、2.5、12.5、62.5、125.0、312.5 mg/L),抑製劑組分彆加入10μmol/L ERKl/2抑製劑PD98059和JNK1/2抑製劑SP600125預處理2h後,再加入125 mg/LTGP.細胞繼續培養48 h.實時反轉錄(RT)-PCR方法和ELISA方法檢測HaCaT細胞IL-18的錶達.部分HaCaT細胞分為兩組,一組用125 mg/L TGP分彆處理15,30,60 min,另一組分彆用10μmol/L ERKl/2抑製劑PD98059和JNK1/2抑製劑SP600125預處理後再加入125 mg/L TGP分彆處理15,30,60 min.免疫印跡技術觀察兩組HaCaT細胞ERK1/2、JNK1/2燐痠化水平.結果 TGP在低濃度(0.5、2.5 mg/L)時對HaCaT細胞IL-18mRNA和蛋白的錶達有促進作用,62.5～125.0 mg/L時可抑製IL-18 mRNA和蛋白的錶達,125 mg/L TGP抑製作用最彊.TGP(125 mg/L)作用15 min後燐痠化ERKl/2蛋白錶達達到高峰,錶達水平為0.448±0.018,與對照組(0.204±0.005)比較,差異有統計學意義(P＜0.01);30 min後錶達水平降低至0.213±0.005,60 min後為0.217±0.005,與對照組相比差異無統計學意義(均P＞0.05).PD98059預處理組燐痠化ERK1/2錶達水平為0.237±0.010,與單獨125 mg/L TGP給藥組相比,差異有統計學意義(P＜0.01).125 mg/L TGP對JNK蛋白的燐痠化作用不明顯,各組相比差異無統計學意義(P＞0.05).結論 TGP可抑製IL-18 mRNA和蛋白的錶達,ERK1/2信號途徑可能介導這一抑製作用.
목적 관찰백작총감(TGP)대각질형성세포(KC)표체백세포개소(IL)-18적영향,병초보탐토ERK1/2、JNK1/2신호통로재기중적작용.방법 장부분HaCaT세포분위3개조,즉대조조가입0.031％이갑기아풍적세포배양액,TGP조분별가입6충불동농도적TGP(0.5、2.5、12.5、62.5、125.0、312.5 mg/L),억제제조분별가입10μmol/L ERKl/2억제제PD98059화JNK1/2억제제SP600125예처리2h후,재가입125 mg/LTGP.세포계속배양48 h.실시반전록(RT)-PCR방법화ELISA방법검측HaCaT세포IL-18적표체.부분HaCaT세포분위량조,일조용125 mg/L TGP분별처리15,30,60 min,령일조분별용10μmol/L ERKl/2억제제PD98059화JNK1/2억제제SP600125예처리후재가입125 mg/L TGP분별처리15,30,60 min.면역인적기술관찰량조HaCaT세포ERK1/2、JNK1/2린산화수평.결과 TGP재저농도(0.5、2.5 mg/L)시대HaCaT세포IL-18mRNA화단백적표체유촉진작용,62.5～125.0 mg/L시가억제IL-18 mRNA화단백적표체,125 mg/L TGP억제작용최강.TGP(125 mg/L)작용15 min후린산화ERKl/2단백표체체도고봉,표체수평위0.448±0.018,여대조조(0.204±0.005)비교,차이유통계학의의(P＜0.01);30 min후표체수평강저지0.213±0.005,60 min후위0.217±0.005,여대조조상비차이무통계학의의(균P＞0.05).PD98059예처리조린산화ERK1/2표체수평위0.237±0.010,여단독125 mg/L TGP급약조상비,차이유통계학의의(P＜0.01).125 mg/L TGP대JNK단백적린산화작용불명현,각조상비차이무통계학의의(P＞0.05).결론 TGP가억제IL-18 mRNA화단백적표체,ERK1/2신호도경가능개도저일억제작용.
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.
