Boswellic Acid And Arsenic Trioxide With Wu Duicheng Fibroblasts, Thp-1 Cells Mmps And Activity Regulation

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.