[Effects of zhuhong ointment on MMPs activities and production by HSF].

HuaFu Shengji is the primary traditional Chinese medicine (TCM) therapy for treating chronic skin ulcer. The high activities of the protein enzyme in the wound fluids is one of the main cause of healing delay. In order to investigate the effect of TCM Zhuhong ointment for promoting wound healing. This research focused on its influence on matrix metalloproteinase (MMP) activities in wound fluids with TCM Yang syndromes, directly on the activated MMP-1,2 activities in vitro and on MMP-1,-2,-9 production by HSF. 8 wound fluid samples were collected, which were diagnosed Yang Syndromes in TCM. Wound fluid activities of MMP-2 and MMP-9 were measured by gelatin zymogram assay. MMP-1 and MMP-2 activities in vitro were measured by substrate cleavage. CCK-8 was used to observe the toxicity of Zhuhong ointment on HSF. MMP-1,-2,-9 production by HSF were detected by confocal microscope. Zhuhong ointment from 1 to 25 g x L(-1) obviously inhibited MMP-2 activity in wound fluid. When Zhuhong ointment was over 5 g x L(-1), it showed significantly inhibitory effect on wound fluid MMP-9 activity. In vitro study, when the mercury concentration was 320 mg x L(-1), Zhuhong ointment solution directly inhibited both MMP-1 activity and MMP-2. But mercury concentration from 0.51-2.56 mg x L(-1), it could activate MMP-1 activity, and from 0.51-64 mg x L(-1), activate MMP-2 activity instead. The mercury concentration when Zhuhong ointment saturated in DMEM was 39.6 mg x L(-1). When the mercury concentration was over 1.23 mg x L(-1), Zhuhong ointment showed toxicity to HSF. At 1.23, 0.62, 0.31 mg x L(-1) of mercury concentration, it increased MMP-1 expression by HSF, and at 1.23, 0.62 mg x L(-1), decreased MMP-2 expression. However, at 1.23, 0.62, 0.31 mg x L(-1), it decreased MMP-9 expression. At higher concentration, Zhuhong ointment can inhibit MMP-2, MMP-9 activities in wound fluid with dose-dependent way and show a direct inhibitory effect on activated MMP-1 and MMP-2 in vitro. But at a lower concentration, it showed two-way adjustment, with increased MMP-1, MMP-2 activities and its expression by HSF and decreased MMP-9 activity.

Arsenic trioxide regulates the production and activities of matrix metalloproteinases-1, -2, and -9 in fibroblasts and THP-1.

BACKGROUND: The elevated matrix metalloproteinase (MMP) activity is an important cause of chronic wound healing failure. Arsenolite, whose main component is arsenic trioxide (As2O3), is a common traditional Chinese medicine wildly used in treating chronic wounds; it can remove necrotic tissue and promote tissue regeneration. This research was designed to evaluate the effects of As2O3 on production and activities of MMP-1, MMP-2 and MMP-9, and on regulation of its signal transduction pathway in human skin fibroblasts (HSFb) and human monocyte line (THP-1 cells) that were in an inflammatory state. METHODS: We established three cell models; HSFb activated by TNF-α, THP-1 cells activated by phorbol 12-myristate 13-acetate (PMA) and an HSFb-THP-1 co-culture system. Three cell models was cultured with As2O3 for 24 hours. The levels of MMP-1, MMP-2, MMP-9, TNF-α and IL-1ß in the cell culture supernatants were assayed by ELISA. The mRNA expressions of MMP-1, MMP-2 and MMP-9 were determined by RT-PCR. The activities of MMP-2 and MMP-9 were tested by Gelatin zymography assays. The phosphorylation levels of ERK1/2 and p38MAPK were assayed by Western blotting. RESULTS: As2O3 inhibited the expression of MMP-1, MMP-2 and MMP-9 mRNA, the secretion and activity of MMP-1, MMP-2 and MMP-9 in HSFb and THP-1 cells in the inflammatory state (P < 0.05 and P < 0.01 respectively). It also inhibited the secretion of TNF-α and IL-1ß in THP-1 cells and in the co-culture system (P < 0.05 and P < 0.01, respectively). It also decreased the phosphorylation of ERK1/2 and p38 MAPK in HSFb and THP-1 cells (P < 0.05 and P < 0.01, respectively). CONCLUSIONS: As2O3, as a main component of arsenolite, can inhibit the production of MMPs by HSFb and THP-1 cells in an inflammatory state through inhibiting the release of inflammatory factors and the activation of the MAPK cascade pathway. This may be a possible mechanism for arsenolite healing chronic wounds.

Salvianolic acid B in vitro inhibited matrix metalloproteinases-1, -2, and -9 activities.

OBJECTIVE: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all extracellular matrix components. The proteolytic property of the MMPs is important during wound healing to remove debris and facilitate cell migration. Targeting towards the decreased MMPs activities is a new treatment strategy for healing chronic wounds. Salvia miltiorrhiza is a popular Chinese herb that could promote chronic ulcers healing for topical use. Salvianolic acid B (Sal B) is the most abundant bioactive component in Salvia miltiorrhiza. The research was designed to explore the inhibitory effects of Sal B on MMP-1, MMP-2 and MMP-9 activities. METHODS: Pure human interstitial collagenase (MMP-1) or gelatinase A (MMP-2) was activated by p-aminophenylmercuric acetate (APMA), and was incubated with Sal B for 1 h. The activities were observed by quenched fluorescent substrate. Gelatinase B (MMP-9) is rich in polymorphonuclear neutrophils (PMN), so the rat PMN was used as a source of MMP-9 for MMPs activity assays. In vitro MMP-9 from rats' PMN lysate was incubated with Sal B for 1 h, and its activity was tested by gelatin zymography. RESULTS: Sal B dose-dependently inhibited the human MMP-1 and MMP-2 activities in the range of 0.002 4 to 0.3 g/L, with 50% inhibiting concentration (IC(50)) of (0.090<0.015) g/L and (0.080<0.005) g/L respectively. In the range of 0.003 to 0.3 g/L, Sal B could inhibit the MMP-9 activity (P<0.01). CONCLUSION: The broad-spectrum inhibitory effects of Sal B on MMPs may reveal one of the mechanisms for the effects of Salvia miltiorrhiza on chronic wounds.