乳香酸与三氧化二砷配伍对成纤维细胞和THP-1细胞基质金属蛋白酶产生及活性的调节作用

Acetyl-11-keto-beta-boswellic acid and arsenic trioxide regulate the productions and activities of matrix metalloproteinases in human skin fibroblasts and human leukemia cell line THP-1

目的:探讨乳香、砒石主要单体成分11-羰基-β-乙酰乳香酸(acetyl-11-keto-beta-boswellic acid,AKBA)与三氧化二砷(arsenic trioxide,ATO)配伍对人皮肤成纤维细胞(human skin fibroblast,HSFb)、人单核细胞系THP-1细胞基质金属蛋白酶(matrix metalloproteinase,MMP)-1、MMP-2和MMP-9的调节作用,揭示其在促进创面愈合中的作用。方法:模拟创面炎症微环境建立3个细胞模型:由肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)刺激的HSFb模型,佛波酯(phorbol-12-myristate-13-acetate,PMA)活化的THP-1细胞模型和HSFb、THP-1共培养细胞模型。AKBA、ATO与这3个细胞模型共同孵育24 h后,分别用酶联免疫吸附测定(enzyme-linkedi mmunosorbent assay,ELISA)法和明胶酶谱法检测细胞培养上清中MMP-1、MMP-2和MMP-9的含量及MMP-2和MMP-9的活性;逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)检测细胞中MMP-1、MMP-2和MMP-9 mRNA的表达情况;ELISA法检测各细胞模型炎性因子TNF-α和白细胞介素-1β(interleukin-1beta,IL-1β)的分泌量;蛋白印迹法检测细胞中细胞外信号调节蛋白激酶1/2(extracellular signal-regulated kinases 1 and 2,ERK1/2)及p38丝裂原激活的蛋白激酶(p38mitogen-activated proteinkinase,p38 MAPK)磷酸化水平。结果:AKBA与ATO配伍对TNF-α刺激后的HSFb、PMA活化的THP-1细胞以及细胞共培养体系产生的MMP-1、MMP-2和MMP-9分别在活性、含量和mRNA水平有一定抑制作用(P<0.05,P<0.01);同时,AKBA与ATO配伍可降低THP-1细胞和共培养体系中TNF-α和IL-1β的分泌(P<0.01),并降低HSFb和THP-1细胞中ERK1/2及p38 MAPK磷酸化水平(P<0.05,P<0.01)。结论:AKBA与ATO配伍应用可能是通过抑制炎症状态下细胞炎症因子释放及抑制p38 MAPK信号转导通路,使成纤维细胞和炎性细胞产生MMP减少,从而发挥活血化腐、促进创面愈合的作用。

Objective： In order to reveal the treatment mechanism of Chinese medicine with the effect of activating blood and resolving putridity,we selected acetyl-11-keto-beta-boswellic acid（AKBA） and arsenic trioxide（ATO）,the main monomeric components of frankincense and arsenolite which are two most commonly used Chinese medicine with effect of activating blood and resolving putridity.We combined AKBA and ATO as a compound,and explored its regulatory role in productions and activities of matrix metalloproteinase（MMP）-1,MMP-2 and MMP-9 in human skin fibroblasts（HSFbs） and human acute monocytic leukemia cell line THP-1 in inflammatory state.Methods： In order to simulate the inflammatory micro-environment of chronic wounds,we established 3 cell models： HSFb model activated by tumor necrosis factor-alpha（TNF-？？,THP-1 cell model activated by phorbol-12-myristate-13-acetate（PMA） and HSFb-THP-1 cell coculture system.AKBA and ATO were cocultured with these cell models.Enzyme-linked immunosorbent assay（ELISA）,gelatin zymography assay and reverse transcription-polymerase chain reaction（RT-PCR） were used to test the secretions,activities and mRNA expressions of MMP-1,MMP-2 and MMP-9.In the study of the regulatory mechanism of AKBA and ATO on MMPs,AKBA and ATO were cocultured with the cell models.ELISA was used to test the secretions of TNF-？？and interleukin-1beta（IL-？？ and Western blot was used to test the phosphorylation levels of extracellular signal-regulated kinases 1 and 2（ERK1/2） and p38 mitogen-activated proteinkinase（p38MAPK）.Results： Compound of AKBA and ATO inhibited MMP-1,MMP-2 and MMP-9 mRNA expressions,secretions and activities respectively in HSFbs and THP-1 cells in inflammatory state（P0.05,P0.01）.Also compound of AKBA and ATO inhibited secretions of TNF-？？and IL-1？ in THP-1 cells and cell coculture system（P0.01）.It also decreased the phosphorylation of ERK1/2 and p38 MAPK in HSFbs and THP-1 cells（P0.05,P0.01）.Conclusion： The combined use of AKBA and ATO which in line with the rule of activating blood and resolving putridity inhibits fibroblasts and inflammatory cells in producing MMPs in inflammatory state through inhibiting the release of inflammatory factors and MAPK cascade pathway.