CAJ | 학술논문

目的探讨金属蛋白酶(MPs)家族的MMPs和ADAMs两亚类成分在前体TNF-α (mTNF-α)向成熟型TNF-α (sTNF-α)转化过程所起的作用,MMPs在炎症过程中活性的变化和中药RDQ抗炎保护功效的分子机制,为人工干预炎症过程提供依据和手段.方法以IPS刺激HL-60细胞为天然模型,用细胞学和分子生物学技术在蛋白质和mRNA水平上研究MMPs、ADAM17、TNF-α在炎症过程中的表达和活性变化.结果 ①LPS刺激后,HL50细胞上清中MMPs的活性随刺激时间延长逐渐减弱,胞浆裂解物中的MMPs则随刺激时间延长活性增加但LPS刺激对MMPs mRNA的表达影响不大;②LPS刺激能使HL-60细胞中ADAM17 mRNA和TNF-α mRNA表达在6 h同时达高峰;③RDQ能明显降低LPS诱导的ADAM17 mRNA的增加,并能抑制sTNF-α的分泌.结论 ①ADAM17对sTNF-α的产生起主导作用;②纯中药注射液热毒清(RDQ)能调节TACE的表达,最终抑制TNF-α的分泌.
목적 탐토금속단백매(MPs)가족적MMPs화ADAMs량아류성분재전체TNF-α (mTNF-α)향성숙형TNF-α (sTNF-α)전화과정소기적작용,MMPs재염증과정중활성적변화화중약RDQ항염보호공효적분자궤제,위인공간예염증과정제공의거화수단.방법 이IPS자격HL-60세포위천연모형,용세포학화분자생물학기술재단백질화mRNA수평상연구MMPs、ADAM17、TNF-α재염증과정중적표체화활성변화.결과 ①LPS자격후,HL50세포상청중MMPs적활성수자격시간연장축점감약,포장렬해물중적MMPs칙수자격시간연장활성증가단LPS자격대MMPs mRNA적표체영향불대;②LPS자격능사HL-60세포중ADAM17 mRNA화TNF-α mRNA표체재6 h동시체고봉;③RDQ능명현강저LPS유도적ADAM17 mRNA적증가,병능억제sTNF-α적분비.결론 ①ADAM17대sTNF-α적산생기주도작용;②순중약주사액열독청(RDQ)능조절TACE적표체,최종억제TNF-α적분비.
Objective This study was aimed to investigate the altered activity of two subclasses of metalloproteinases (MPs) family,matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases( ADAMs),during the transformation of precursor TNF-α (mTNF-α) to mature TNF-α (sTNF-α),and to explore the innate inhibitory effect of the Chinese herbal Re Du Qing (RDQ) on inflammation.Methods HL-60 cells were treated in vitro by lipopolvsaccharide (LPS) and RDQ.MPs activity was measured on SDS-PAGE substrate gel embedded with gelatin (zymography, Gelatin-PAGE).The mRNA expression of MMPs (MMP2,MMP7 and MMP9),ADAM17 (also called TNF-α converting enzyme,TACE),and sTNF-α, at various time points after LPS stimulation, was examined by Dot blot hybridization and RT-PCR,respectively.The amount of released sTNF-α was detected by MTT colorimetric assay.RT-PCR was used to assess the inhibitory effect of RDQ on TACE at mRNA level.Result Zymographic analysis revealed that the activity of MMPs in HL-60 cells gradually decreased with the time of LPS stimulation, whereas the alteration of MMPs at mRNA expression level was not significant, as detected by RT-PCR.Dot-blot results indicated that the expression of TACE and TNF-α at mRNA level increased simultaneously.The Chinese herb RDQ displayed obvious inhibitry effect on the cytotoxicity of sTNF-α induced by LPS stimulation at the transcriptional level of TACE gene.Conclusion TACE subclass of MPs family plays a crucial role in controlling the secretion of TNF-α induced by LPS.The Chinese herb RDQ can regulate TACE mRNA expression and inhibit sTNF-α secretion.TACE may serve as a novel target for inflammatory therapy.