乳香酸与三氧化二砷配伍对成纤维细胞、THP-1细胞MMPs产生及活性的调节作用

【作者】 梁雅慧；

【导师】 黄启福； 李萍；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 基质金属蛋白酶家族（MMPs）是一类活性依赖于金属锌离子的蛋白酶,它基本上可以降解绝大多数细胞外基质成分。MMPs水解蛋白质的特性有利于创伤愈合过程中坏死组织的清除,以及细胞的迁移。目前大量报道证实,与急性创面比较,慢性皮肤溃疡创面MMP-1、MMP-2、MMP-9以及其它一些MMPs表达明显增加,而TIMP-1、TIMP-2的表达下降。创面的高MMPs活性是慢性皮肤溃疡愈合迟缓的原因之一,MMPs的过多积累及其活性的过度增高会使生长因子降解,也会破坏能够促进细胞迁移和维持组织完整性的基质蛋白,最终会抑制细胞的迁移和血管的形成。所以,降低创面的高MMPs活性是治疗慢性皮肤溃疡的新途径。目的活血生肌、化腐生肌是中医治疗慢性皮肤溃疡的主要治则,外用活血化腐中药在中医治疗慢性皮肤溃疡中发挥着举足轻重的作用。本课题选用常用的外用活血化腐类中药代表药乳香与砒石中的主要单体成分:乳香酸（AKBA）与三氧化二砷（As₂O₃）配伍组成复方,以其为研究对象,探索其对慢性皮肤溃疡创面中活性升高的主要几种基质金属蛋白酶:MMP-1,MMP-2和MMP-9活性的是否具有调节作用,揭示中医活血生肌、化腐生肌治疗法则的疗效机制。方法为了模拟慢性创面炎症微环境,我们建立了三个细胞模型:由TNF-α活化的人皮肤成纤维细胞（HSFb）模型;PMA诱导THP-1成为巨噬细胞的细胞模型和成纤维细胞-THP-1共培养细胞模型。利用ELISA法、明胶酶谱法和RT-PCR法观察AKBA与As₂O₃配伍或单独作用对这三个细胞模型中MMPs和TIMPs的分泌量、酶活性和mRNA表达的影响。在对MMPs调节机制的研究中,利用ELISA法、激光共聚焦显微镜观察及westernblot法观察了AKBA与As₂O₃配伍或单独作用对各细胞模型中炎性因子TNF-α和IL-β的分泌量、胞浆钙离子浓度和ERK1/2及p38MAPK磷酸化水平的影响。利用明胶酶谱法及底物裂解法在蛋白水平观察AKBA与As₂O₃对MMP-1、MMP-2和MMP-9活性的直接影响。结果通过mRNA水平、蛋白水平和酶活性水平的检测发现具有活血化腐作用的AKBA与As₂O₃配伍的复方组对活化后的HSFb细胞中MMP-1和MMP-2的产生有一定抑制作用（P＜0.05）,对TIMP-1的产生有一定的升高作用（P＜0.05）,但对TIMP-2无明显作用（P＞0.05）;对经PMA诱导后的THP-1细胞MMP-9的产生有抑制作用（P＜0.01）。AKBA单独作用对活化后的HSFb中MMP-2,TIMP-1的产生基本都具有促进作用（P＜0.05,P＜0.01）,对TIMP-2的产生无影响（P＞0.05）;而对于活化的THP-1细胞,AKBA对MMP-9的产生表现出抑制作用（P＜0.05）。As₂O₃单独作用对活化后的HSFb中MMP-1、MMP-2的产生表现出抑制作用（P＜0.05,P＜0.01）,对TIMP-1和TIMP-2无影响（P＞0.05）;对于活化的THP-1细胞,As₂O₃对MMP-9的产生表现出抑制作用（P＜0.01）。在共培养细胞模型中,AKBA与As₂O₃配伍可使细胞共培养模型中MMP-1、MMP-2和MMP-9的分泌量及MMP-2和MMP-9的活性明显下降（P＜0.05,P＜0.01）,对TIMP-1和TIMP-2的分泌无影响（P＞0.05）。AKBA或As₂O₃单独作用均能能抑制MMP-1、MMP-2和MMP-9的分泌,及MMP-2和MMP-9的活性（P＜0.05,P＜0.01）,对TIMP-1和TIMP-2的分泌无影响（P＞0.05）。在AKBA与As₂O₃对MMPs调节作用的机制的研究中显示,AKBA与As₂O₃配伍组可以降低HSFb细胞中钙离子的浓度（P＜0.01）,降低细胞中ERK1/2及p38 MAPK磷酸化水平（P＜0.05）。AKBA单独作用可使HSFb细胞中钙离子浓度和细胞中ERK1/2及p38 MAPK磷酸化水平进一步升高（P＜0.01,P＜0.05）。As₂O₃的作用与AKBA的作用相反,它可以降低HSFb细胞中钙离子的浓度（P＜0.01）,降低细胞中ERK1/2及p38 MAPK磷酸化水平（P＜0.05）。AKBA与As₂O₃配伍作用或者单独作用都可使活化后的THP-1细胞及HSFb-THP-1细胞共培养模型分泌TNF-α和IL-1β减少（P＜0.05,P＜0.01）,使活化后的THP-1细胞胞浆中钙离子浓度降低（P＜0.01）,ERK1/2及p38 MAPK磷酸化水平也降低（P＜0.05,P＜0.01）。AKBA和As₂O₃在蛋白水平上对MMP-1、MMP-2和MMP-9活性的直接调节的研究中发现:AKBA可以直接抑制MMP-1、MMP-2和MMP-9的活性,呈剂量依赖性（P＜0.01）;而As₂O₃对MMP-1和MMP-2的活性无直接影响（P＞0.05）,而高浓度的As₂O₃可略升高MMP-9的活性（P＜0.01）。结论活血化腐法则的AKBA与As₂O₃配伍应用可以降低炎症状态下成纤维细胞、巨噬细胞以及两种细胞共培养模型中MMPs的产生,其中具有活血作用的中药乳香中的活性成分AKBA还可以直接抑制MMP-1、MMP-2和MMP-9的活性。这两方面的综合作用可能抑制慢性创面的高酶活性状态,从而发挥活血化腐中药治疗慢性皮肤溃疡的作用。更多还原

【Abstract】 Matrix metalloproteinases（MMPs） are a family of zinc-dependent endopeptidases, which can degrade essentially all ECM components.The proteolytic property of the MMPs is important during wound healing to remove debris and facilitate cell migration.It has been consistently reported the levels of MMP-1,MMP-2,MMP-9 and other MMPs elevated dramatically in chronic wounds compared with acute wounds.The excessive accumulation and activation of MMPs suppress cell proliferation and angiogenesis due to destruction of growth factors and matrix proteins that provide necessary substrates for cell migration and integrity of the tissue.The high MMPs activity leads to the wounds close failure.Targeting towards the decreasing MMPs activity is a new treatment approach for healing chronic wounds.Huoxue Shengji and Huafu Shengji are main rules for Chinese medicine to heal chronic skin ulcer,the Chinese medicine with the effect of Huoxue and Huafa are playing an important role in treatment of chronic skin ulcer.In order to reveal the treatment mechanism of the Chinese medicine with the effect of Huoxue and Huafa,we selected frankincense acid （AKBA） and arsenic trioxide（As₂O₃）,the main monomer components of frankincense and arsenolite which are two commonly topical used Chinese medicine with effect of Huoxue Huafu.We combined AKBA and As₂O₃ as a compound,and explored its regulatory role on the activities of MMP-1,MMP-2 and MMP-9,which elevated dramatically in chronic skin ulcer.MethodsIn order to simulate the inflammatory micro-environment of chronic wounds,we established three cell models:activated human dermal fibroblasts（HSFb） by TNF-α,THP-1 cells by PMA and HSFb-THP-1 cell co-culture system.AKBA and As₂O₃ were alone or combined cultured with these cell models.ELISA,Gelatin zymography assays and RT-PCR were used to test the secretion,activities and mRNA expression of MMPs and TIMPs.In the study of the regulatory mechanism of AKBA and As₂O₃ on MMPs,AKBA and As₂O₃ were alone or combined cultured with the cell models.ELISA was used to test to the secretion of TNF-αand IL-β,Laser scanning confocal microscope was used to test the intracellular calcium ion concentration and western blot was used to test the phosphorylation level of ERK 1/2 and p38MAPK.Gelatin zymography and substrate cleavage were used to observe the direct effect of AKBA and As₂O₃ on MMP-1,MMP-2 and MMP-9 activities on the protein level. ResultsThrough the tests on the level of mRNA,protein and activities,in the activated HSFb, the compound of AKBA and As₂O₃ which with the effect of Huoxue Huafu inhibited the produce of MMP-1 and MMP-2（P＜0.05）,promoted the produce of TIMP-1（P＜0.05）,but had no effect on TIMP-2（P＞0.05）;In THP-1 which induced by PMA,the compound of AKBA and As₂O₃ inhibited the produce of MMP-9（P＜0.01）.AKBA alone promoted the produce of MMP-2 and TIMP-1 in activated HSFb（P＜0.05,P＜0.01）,had no effect on TIMP-2（P＞0.05）;It also inhibited the produce of MMP-9 in activated THP-1（P＜0.05）. As₂O₃ alone inhibited the product of MMP-1 and MMP-2 in activated HSFb（P＜0.05, P＜0.01）,had no effect on TIMP-1 and TIMP-2（P＞0.05）;It also inhibited the produce of MMP-9 in activated THP-1（P＜0.01）.In the HSFb-THP-1 cell co-culture system,the compound of AKBA and As₂O₃ inhibited the secretion of MMP-1,MMP-2 and MMP-9,and inhibited the activities of MMP-2 and MMP-9（P＜0.05,P＜0.01）,but had no effect on secretion of TIMP-1 and TIMP-2（P＞0.05）. AKBA or As₂O₃ alone inhibited the secretion of MMP-1,MMP-2 and MMP-9,and inhibited the activities of MMP-2 and MMP-9（P＜0.05,P＜0.01）,but had no effect on secretion of TIMP-1 and TIMP-2（P＞0.05）.In the study of the regulatory mechanism of AKBA and As₂O₃ on MMPs,the compound of AKBA and As₂O₃ decreased the intracellular calcium ion concentration and the phosphorylation level of ERK1/2 and p38MAPK（P＜0.01,P＜0.05）.AKBA alone increased the intracellular calcium ion concentration and the phosphorylation level of ERK1/2 and p38MAPK（P＜0.01,P＜0.05）.As₂O₃ alone had contrary effects with AKBA,it decreased the intracellular calcium ion concentration and the phosphorylation level of ERK1/2 and p38MAPK（P＜0.01,P＜0.05）.AKBA and As₂O₃ combined or alone decreased the secretion of TNF-αand IL-1βin activated THP-1 and HSFb-THP-1 cell co-culture system（P＜0.05, P＜0.01）,also decreased the intracellular calcium ion concentration and the phosphorylation level of ERK1/2 and p38MAPK in activated THP-1（P＜0.01,P＜0.05）.On the protein level,AKBA dose-dependently inhibited the activities of MMP-1, MMP-2 and MMP-9 directly（P＜0.01）;As₂O had no effect on the activities of MMP-1 and MMP-2（P＞0.05）,but As₂O₃ increased the activity of MMP-9 in high concentration（P＜0.01）.ConclusionThe combined used of AKBA and As₂O₃ which in line with the rule of Huoxue Huafu inhibited the produce of MMPs by HSFb,THP-1 and the cell co-culture system in inflammatory state.AKBA the bioactive compound of frankincense dose-dependently inhibited the activities of MMP-1,MMP-2 and MMP-9 directly.The combined effects of these two areas may inhibit the high level MMPs activities in chronic skin ulcer,and educe the active role of Chinese medicine with Huoxue Huafu effect in healing chronic skin ulcer.更多还原