Arsenic and its compounds, such as realgar and arsenolite are famous traditional Chinese medicines for treatment of a variety of illnesses. It has a remarkable effect on healing chronic wounds.
Chronic wounds in the elderly are a major health problem resulting in distress and disability and are an increasing burden for health care providers. Chronic wounds encompass a spectrum of diseases and have three principal forms: pressure sores, venous ulcers and diabetic ulcers. Matrix metalloproteases (MMPs) are a family of neutral proteases that play a vital role throughout the entire wound healing process. They regulate inflammation, degrade the extracellular matrix (ECM) to facilitate the migration of cells and remodel the new ECM. However, excessive MMP activity contributes to the development of chronic wounds.1 Inferred from analyses of wound fluids, MMPs account for 90% of total proteinase activity in chronic wounds of various etiologies.2,3
Considering the beneficial effects of arsenolite on promoting chronic ulcer healing, this research was intended to explore the possible effects of arsenic trioxide (As2O3) on MMPs activities and provide new therapeutic application for clinical treatment.
Drugs and reagents
Arsenious injection (the concentration of As2O3 was 1 mg/ml) was provided by Harbin Yida Medicine Company (China), Lot number: 20070601. PMA was purchased from Sigma (USA), and TNF-α from Peprotech, Inc. (USA). Anti-vimentin mouse monoclonal antibody and anti-β-actin rabbit monoclonal antibody were from Santa Cruz Biotechnology Inc. (USA), and the Cell Counting kit-8 (CCK-8) from Dojindo Laboratories (Japan). ELISA kits for human MMP-1, MMP-2 and MMP-9 were from R&D Systems (USA), RapidBio Lab and Jingmei Biotech (China), respectively. Trizol reagent was from Invitrogen (USA), and PCR kits from TaKaRa (China). Anti-phospho-ERK1/2 rabbit monoclonal antibody, anti-phospho-p38 MAPK rabbit monoclonal antibody and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Biotechnology Inc. (USA).
Human dermal fibroblasts were isolated and purified from foreskin biopsies.3 Biopsies, from patients undergoing circumcision in the Department of Surgery, Beijing Traditional Chinese Medicine Hospital, were soaked in the PBS containing 0.1% bromogeramine for 30 minutes. After removing the epidermis and fatty tissue, the dermis was minced to 1–2 mm3 pieces with a razor blade. Seed the dermis pieces in the 25 cm2 culture bottle, turn over the culture bottle, then add 1 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin, then cultured at 37°C with 5% CO2. After 4 hours, the culture bottle was reversed to make the DMEM immerse the pieces. The next day, another 5 ml DMEM was added. The dermis pieces were cultured for 3–14 days until spindle-shaped fibroblasts became the dominance cells. The fibroblasts were identified with anti-vimentin antibody by immunocytochemistry (Figure 1). Fibroblasts between 6 and 10 passages were used for experiments.
The human monocyte line (THP-1 cells, purchased from Cell Center of Peking Union Medical College, China) were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2.
As2O3 cytotoxicity on human skin fibroblasts (HSFb) and THP-1 cells
The As2O3 cytotoxicity on HSFb and THP-1 cells were assayed by CCK-8 following the manufacture's instruction. 3×103 cells/well HSFb were seeded into 96-well plates for adherent to plate bottom overnight. The medium was then changed to serum-free medium and cells were cultured for 24 hours at 37°C. 2×104 cells/well THP-1 cells were cultured with serum-free medium in 96-well plates for 24 hours at 37°C. Then the HSFb and THP-1 cells were treated with a series of concentrations of As2O3 for another 24 hours. At 4 hours before the experiment ended, 10 μl CCK-8 was added to each well and incubated at 37°C. The results were measured with a micro-plate reader at λ=450 nm.
Assays for MMP-1, MMP-2, MMP-9, TNF-α and IL-1β secretion in cell culture supernatants
The 5×104 cells/well of HSFb were cultured with 10% FBS in 6-well plates. After three days, cells were starved in serum-free medium for 24 hours, then treated with 20 ng/ml TNF-α alone or 20 ng/ml TNF-α together with 1.25 μmol/L As2O3 in fresh serum-free medium.
The 5×105 cells/well of THP-1 cells starved in serum-free medium for 24 hours were seeded into 6-well plates. Then they were treated with 1×10−7 mol/L PMA alone or 1×10−7 mol/L PMA together with 1.25 μmol/L As2O3 in fresh serum-free medium.
Cell co-culture systems: THP-1 cells pretreated with 1×10−7 mol/L PMA for 24 hours were harvested by re-suspension and centrifuged. Then 5×105 cells/well of THP-1 cells were co-cultured with a confluent monolayer of HSFb in 6-well plates, and 1.25 μmol/L As2O3 was added. Untreated THP-1 cells were co-cultured with HSFb as the control group.
All the cell culture supernatants were collected 24 hours later. All samples were stored at -80°C.
ELISA kits were used to quantify MMP-1, MMP-2, MMP-9, TNF-α and IL-1β in the cell culture supernatants following the manufacture's instruction.
Assays for MMP-1, MMP-2 and MMP-9 mRNA expression in HSFb and THP-1 cells
MMP-1, MMP-2 and MMP-9 mRNA levels were observed by semi-quantitative RT-PCR. 5×104 cells/well of HSFb were cultured at 37°C with 10% FBS in 6-well plates. After three days the cells were starved in serum-free medium for 24 hours, then treated with 20 ng/ml TNF-α alone or 20 ng/ml TNF-α together with 1.25 μmol/l As2O3 in fresh serum-free medium for 24 hours. 5×105 cells/well of THP-1 cells in 6-well plates were starved in serum-free medium for 24 hours, then treated with 1×10−7 mol/L PMA alone or 1×10−7 mol/L PMA together with 1.25 μmol/L As2O3 for 24 hours. Total RNA was extracted from HSFb and THP-1 cells using Trizol reagent and was reverse transcribed into cDNA. The resulting cDNA was used as a template for PCR.
The primers used for MMP-1, MMP-2 and MMP-9 mRNA amplification were (F) 5'-AGA TGC TGA AAC CCT GAA-3' and (R) 5'-TGA TGT CTG CTT GAC CCT-3' (242 bp), (F) 5'-AGA TCT TCT TCT TCA AGG ACC GGT T-3' and (R) 5'-GGC TGG TCA GTG GCT TGG GGT A-3' (225 bp), and (F) 5'-CTT TGA CAG CGA CAA GAA GTG-3' and (R) 5'-AGG GCG AGG ACC ATA GAG G-3' (208 bp), respectively. PCR conditions were as follows: 95°C for 2 minutes, followed by 30 cycles at 95°C for 30 seconds, 56°C for 45 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. Simultaneously, transcripts encoding β-actin were assayed in all samples and served as internal controls using the primers (F) 5'-CGG GAA ATC GTG CGT GAC-3' and (R) 5'-TGG AAG GTG GAC AGC GAG G-3' (443 bp). The PCR products were resolved in 1.5% agarose gel for electrophoresis and were quantified under ultraviolet light.
Assays for MMP-2 and MMP-9 activities in cell culture supernatants
Gelatin zymography was used to test MMP-2 and MMP-9 activity. The cell culture supernatants were subjected to SDS-PAGE under non-reducing condition with 10 μl of supernatants loaded in each lane. After electrophoresis, the gel was washed twice with 2.5% Triton X-100 for 20 minutes at room temperature, and then incubated with incubation buffer (0.05% Tris-HCl pH 8.8, 5 mmol/L CaCl2, 0.02% NaN3) for 18–20 hours at 37°C. The gels were stained (0.25% Coomassie blue, 10% acetic acid and 45% methanol) for 1 hour and then destained (7.5% acetic acid and 25% methanol) for 1 hour. Proteolytic activity was seen as clear bands of degraded protein on a uniformly blue background.
Gels were scanned and analyzed using the Image masterVDS (Ampharmacia, Franch). The image of the gel was inverted to reveal dark bands on a white background. The molecular weight, area, and optical density of each band were determined. The relative amounts of the proteinases were determined by multiplying the area of each band by the optical density.
Aassys for the phosphorylation level of ERK and p38MAPK in HSFb and THP-1 cells
SDS-PAGE and Western blotting were used to detect the phosphorylation level in HSFb and THP-1 cells. Experimental conditions were the same as MMP-1, MMP-2 and MMP-9 mRNA expression assay. The total protein of HSFb and THP-1 cells was extracted with RIPA lysis buffer. Equal amounts of protein from each group were added to 10% polyacrylamide gel. After electrophoresis, proteins were blotted onto PVDF sheets at 100 V for 2 hours. Blots were blocked with blocking buffer (1×TBS, 0.1% Tween-20, 5% milk) at room temperature for 1 hour. Thereafter the blot was probed with primary antibodies of anti-p-ERK (1:1000 dilutions), anti-p-p38MAPK (1:1000 dilution) or anti-β-actin (1:500 dilution) at 4°C overnight. Blots were incubated with an HRP-conjugated goat anti-rabbit IgG (1:2000 dilutions) second antibody at room temperature for 2 hours, the immunoblots were visualized by an enhanced chemiluminescence (ECL) kit according to the manufacturer's instructions.
All data were expressed as mean±standard deviation (SD). Each treated sample was compared with the control using one-way analysis of vaviance (ANOVA) with SPSS 13.0 (SPSS Inc., USA). A P value of <0.05 was considered statistically significant.
Cytotoxicity of As2O3 on HSFb and THP-1 cells
For HSFb the concentrations of As2O3 higher than 5 μmol/L showed cytotoxicity. But for the THP-1 cells the concentrations higher than 2.5 μmol/L were cytotoxic (data not shown). A dose of 1.25 μmol/L of As2O3 was chose in the following experiments, which showed no cytotoxicity.
Effect of As2O3 on MMP-1, MMP-2, MMP-9, TNF-α and IL-1β secretion
MMP-1 and MMP-2 were detected in HSFb culture supernatants by ELISA, while MMP-9 was detected in THP-1 cell culture supernatants. MMP-1, MMP-2 and MMP-9 were found in co-culture system supernatants. TNF-α increased HSFb secretion of both MMP-1 and MMP-2, PMA increased MMP-9 secretion by THP-1 cells, and MMP-1, MMP-2 and MMP-9 secretion in the co-culture system. As2O3 inhibited the activated HSFb secretion of MMP-1 and MMP-2, as well as the activated secretion of MMP-9 by THP-1 cells. In the cell co-culture system As2O3 also showed inhibitory effects on MMP-1, MMP-2 and MMP-9 secretion (Table 1). Compared with control group, THP-1 cells and the cell co-culture system activated by PMA secreted more TNF-α and IL-1β, but As2O3 inhibited their secretion.
Effect of As2O3 on MMP-1, MMP-2 and MMP-9 mRNA expression in HSFb and THP-1
After activation by TNF-α, HSFb expressed higher level of MMP-1 and MMP-2 mRNA, and after activation by PMA, THP-1 cells expressed higher level MMP-9 mRNA. As2O3 inhibited the expression of MMP-1, MMP-2 mRNA in HSFb and the expression of MMP-9 mRNA in THP-1 cells (Figure 2).
Effect of As2O3 on MMP-2 and MMP-9 activities in cell culture supernatants
After treatment with TNF-α or PMA, both MMP-2 and MMP-9 activities were increased in the culture supernatants of HSFb or THP-1 cells compared with the control group. Meanwhile As2O3 inhibited both of these activities. Both MMP-2 and MMP-9 activities increased in the cell co-culture system when THP-1 cells were pretreated by PMA. After treatment with As2O3, enzyme activity decreased (Figure 3).
Effect of As2O3 on the phosphorylation of ERK1/2 and p38 MAPK in HSFb and THP-1 cells
The phosphorylation of ERK1/2 and p38 MAPK was increased in activated HSFb and THP-1 cells. As2O3 inhibited the phosphorylation of ERK1/2 and p38 MAPK in both HSFb and THP-1 cells (Figure 4).
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, more than 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they are divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs.4 MMPs are produced by several different types of cells in skin including keratinocytes, fibroblasts, endothelial cells, neutrophils, and macrophages.5 MMP activity is specifically inhibited by the tissue inhibitors of metalloproteinases (TIMPs).
Cutaneous wound repair can be divided into a series of overlapping phases; formation of the fibrin clot, inflammatory responses, granulation tissue formation incorporating re-epithelialisation and angiogenesis and finally, matrix formation and remodeling.6,7 MMPs are an important part of the inflammatory stage of wound healing, but they become destructive to the wound matrix when a prolonged inflammatory stage predominates in a “stalled” chronic wound. The proteolytic property of the MMPs is important during wound healing to remove debris and facilitate cell migration.5 However, excessive accumulation and activation of MMPs can suppress cell proliferation and angiogenesis due to degradation of growth factors and matrix proteins that provide necessary substrates for cell migration and integrity, then the wound becomes deadlocked, unable to progress to the next healing stage.8,9
It has been consistently reported that the levels of MMP-1, MMP-2 and MMP-9 are increased, while the level of TIMP-1 is decreased in chronic wounds compared with acute wounds.10–12 Lobmann et al reported that the concentration of MMP-1, MMP-2, MMP-8 and MMP-9 were increased in biopsies of diabetic foot ulcers; in particular MMP-1 was 65-fold higher than that in traumatic wounds, and the expression of TIMP-2 was reduced in diabetic wounds compared with lesions of non-diabetic patients.8 Because of the excessive expression and activation of MMPs and the deficient level of TIMPs, MMPs are not balanced by an equal amount of TIMPs, resulting in wound healing failure.10,13–15 New treatment strategies for healing chronic wounds could target restoring the balance between MMPs and TIMPs.
The major difference between chronic wounds and normal healing wounds is the prolonged inflammatory phase. A large number of inflammatory cells infiltrate the wound bed and release inflammatory factors, which leads to a high level of MMP activity in chronic wounds. Stimulated by TNF-α and IL-1β, MMPs are secreted by inflammatory cells and connective tissue cells; including fibroblasts, cartilage cells, neutrophils, monocytes/ macrophages and other cells. In this process, the mitogen activated protein kinases (MAPKs) cascade is an important signal transduction pathway. MAPK gene family members (such as ERK1/2, p38MAPK and JNK) induce the production of MMPs through activating the transcription factor activation protein-1 (AP-1).16–18
As2O3 is indicated as a broad-spectrum anticancer medicine for a variety of cancers. It has been successfully applied in the treatment of acute promyelocyte leukemia (APL), as it can effectively induce apoptosis in APL cells.19 Recently, As2O3 was found to induce apoptosis and inhibit migration and invasion of cancer cells through inhibiting the expression of MMP-2 and MMP-9 in solid tumors.20–23 As2O3 also exerts broad anti-inflammatory effects in certain diseases, such as asthma24 and acute pancreatitis.25 These effects of As2O3 may play a positive role to control high level of MMP activity in chronic wounds. Assuming As2O3 inhibits MMPs secretion in chronic wounds, we established two cell models to mimic the inflammatory microenvironment of chronic wounds: fibroblasts were stimulated by TNF-α26 and THP-1 cells induced by PMA.27 After treatment with PMA, THP-1 cells are converted into mature cells with the function of macrophages.
In the HSFb model stimulation by TNF-α, the MMP-1 and MMP-2 mRNA, protein and enzyme activity were increased, while the phosphorylation levels of ERK1/2 and p38MAPK were also increased. However, As2O3 significantly inhibited the expression of TNF-α induced MMP-1 and MMP-2 mRNA and protein production, as well as the ERK1/2 and p38MAPK phosphorylation. In preliminary experiments, we found As2O3 was able to promote MMP-1 and MMP-2 activity in cell culture supernatant of HSFb not activated by inflammatory cytokines.28 These results show the pluripotent activity of As2O3; promoting MMP secretion in unactivated cells, while suppressing high MMPs secretion in a state of inflammation. Although MMP-9 was expressed at trace levels in normal THP-1 cells, after activation by PMA, its secretion, mRNA expression and activity were dramatically elevated. The secretion of TNF-α and IL-1β, and the phosphorylation levels of ERK1/2 and p38MAPK were also increased. As2O3 showed significant inhibition effect on the production of MMP-9, the secretion of TNF-α and IL-1β, and the phosphorylation of ERK1/2 and p38MAPK.
To determine the interaction between HSFb and THP-1 cells, we established a co-culture system.29 Compared with the control group, the secretions of MMP-1, MMP-2 and MMP-9 in the co-culture were 5–40 folds higher than in untreated HSFb or THP-1 cells. Stimulated by PMA, the secretions of MMP-1, MMP-2 and MMP-9 from cells in co-culture were 5–12 folds higher than from the activated HSFb or THP-1 cells, and the secretions of TNF-α and IL-1β were 3–8 folds higher than from the activated THP-1 cells. This may result from autocrine or paracrine stimulation of HSFb and THP-1 cells in the inflammatory state, which expands the inflammatory response and the secretion of MMP-1, MMP-2 and MMP-9. However, the enhanced secretion of MMP-1, MMP-2, MMP-9, TNF-α and IL-1β can all be inhibited by As2O3.
In conclusion, PMA activation of THP-1 cells leads to the release of inflammatory factors. As2O3 could inhibite the release of inflammatory factors and the production of MMPs. Along with the decrease of inflammatory factors, the phosphorylation of the MAPK pathway proteins ERK1/2 and p38MAPK will also be reduced. As2O3 can also inhibit the production of MMPs through directly inhibiting the phosphorylation of ERK1/2 and p38MAPK in HSFb. These may be the mechanisms of arsenolite healing chronic wounds.
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In the original article entitled Characteristics of bone tunnel changes after anterior cruciate ligament reconstruction using Ligament Advanced Reinforcment System artificial ligament published in November 20 issue, 2012 (Chin Med J 2012; 125 (22): 3961–3965), there is an error in the author information: the information of corresponding author is changed to be “Dr. LIU Hao-yuan, Department of Orthopedics, Chenggong Hospital of Xiamen University (174th Hospital of People's Liberation Army), Xiamen, Fujian 361003, China (Tel: 86–592–6335707. Email: [email protected]); Dr. KANG Yi-fan, Department of Orthopedics, Shanghai Changhai Hospital, the Second Military Medical University, Shanghai 200433, China (Tel: 86–21–81873392. Fax: 86–21–81873398. Email: [email protected])”.