白藜芦醇对中波紫外线致人角质形成细胞损伤的防护作用

目的:研究白藜芦醇(resveratrol,Res)对中波紫外线(UVB)照射体外培养的人永生化角质形成细胞(HaCaT)的损伤的保护作用。方法:体外培养HaCaT细胞,MTT法检测不同浓度Res对HaCaT细胞增殖活性的影响。UVB照射及Res处理24 h后,采用RT-PCR方法测定细胞中MMP-1,MMP-9和TIMP-1mRNA的相对含量。结果:UVB 30 mJ.cm-2照射后,HaCaT细胞产生的MMP-9 mRNA含量显著增加(P<0.05),TIMP-1 mRNA明显减少(P<0.05);UVB 90 mJ.cm-2照射HaCaT细胞后其MMP-1 mRNA增加(P<0.05)。与UVB照射组相比较,0.5 mmol.L-1Res能显著抑制UVB诱导的HaCaT细胞产生的MMP-1,MMP-9 mRNA含量增加以及UVB对TIMP-1 mRNA的抑制作用(P<0.05)。结论:Res可以显著抑制UVB照射诱导的HaCaT细胞产生的MMP-1,MMP-9及TIMP-1 mRNA含量的变化,对皮肤光老化可有一定的防护作用。     

Trichloroethanol up-regulates matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in HaCaT cells

Occupational trichloroethylene (TCE) exposure could induce generalized skin hypersensitivity reactions complicated with severe liver dysfunctions. Active extracellular matrix degradation and remodeling are involved in the skin hypersensitivity reaction induced by chemical exposure. In the present study, we have compared the effects of in vitro exposure to trichloroethanol (TCOH) and trichloroacetic acid (TCA) of a keratinocyte cell line (HaCaT). The modulation of matrix metalloproteinases (MMPs) was selected as marker of sensitization. HaCaT cells were treated with different concentrations of TCOH or TCA up to 6 days. The gelatinolyic activities of MMP-2 and MMP-9 were detected by gelatin-zymography. MMP-2, tissue inhibitor of metalloproteinase (TIMP)-2, MMP-9 and TIMP-1 mRNAs were analyzed by real-time PCR and MMP-9 and TIMP-1 proteins were tested by Western blotting. A dose–effect relationship between TCOH treatment and MMP-9 activity, mRNA and protein expression levels was found in HaCaT cells. TCOH also induced up-regulation of TIMP-1 mRNA and protein. We found no such effects in HaCaT cells treated with TCA. Moreover, previously published literatures on patch tests suggested that TCOH could induce moderately positive reactions at low concentrations in hypersensitivity patients caused by occupational TCE exposure. In summary, these observations indicated that TCOH might play an important role in TCE-induced skin hypersensitivity.     

Salmon and king crab trypsin stimulate interleukin-8 and matrix metalloproteinases via protease-activated receptor-2 in the skin keratinocytic HaCaT cell line

Occupational skin symptoms are prevalent among the workers of the seafood processing industry. In this study we investigate the role of salmon (Salmo salar) and king crab trypsin (Paralithodes camtschaticus) as inducers of inflammation in skin via secretion of inflammatory mediators. Human skin keratinocytes (HaCaT cells) were exposed to purified salmon and king crab trypsin. We observed that salmon trypsin enhanced the secretion of IL-8 and MMP-2 and crab trypsin enhanced the secretion of IL-8, MMP-2 and MMP-9 in a dose dependent manner. As protease activated receptors (PAR)-2 in skin are known to play an important role in physiology and pathology, we explored the involvement of these receptors in mediating the release of interleukin (IL)-8 and matrix metalloproteinase (MMP)-2 and -9 subsequent to exposure of skin keratinocytes to salmon and crab trypsin. In addition we observed that salmon and crab trypsin exhibit individual differences in stimulating the release of these inflammatory mediators. Finally, using specific small interfering RNA (siRNA) against PAR-2, we confirmed that the increase in secretion of IL-8, MMP-2 and MMP-9 in skin keratinocytes following exposure to salmon and crab trypsin was mediated via activation of PAR-2. These results suggest that exposure to proteases from the seafood may lead to inflammatory reactions in skin.     

中波紫外线对角质形成细胞MMP-9及TIMP-1的影响

【摘要】目的：研究中波紫外线(UVB)照射对HaCaT细胞中基质金属蛋白酶-9（MMP-9）和基质金属蛋白酶抑制剂-1（TIMP-1）mRNA表达的影响。方法：培养HaCaT细胞,绘制细胞生长曲线,经不同剂量（0、30、60、90 mJ/cm2）UVB照射;用MTT方法测定UVB照射后细胞的增殖活性,反转录-聚合酶链反应（RT-PCR）方法测定UVB照射后HaCaT细胞中MMP-9 mRNA 和 TIMP-1 mRNA的表达。结果：随着照光剂量的增大,细胞的增殖活性逐渐降低,而细胞的增殖抑制率逐渐增加,不同照射剂量各组间OD值差异有统计学意义（P<0.05）;随着照光剂量的增大,MMP-9 mRNA的表达逐渐增加,各组之间比较差异均有统计学意义（P<0.05）;TIMP-1mRNA的表达逐渐降低,与0 mJ/cm2比较差异具有统计学意义（P<0.05）。结论：UVB照射可诱导HaCaT细胞损伤和细胞凋亡,MMP-9和TIMP-1可能与光老化的发生有一定关系。     

P物质受体在人表皮角质形成细胞和真皮成纤维细胞中的表达调控

目的考察P物质受体(Neurokinin1R,NK1R)在正常人表皮角质形成细胞和真皮成纤维细胞中的表达调控特征。方法培养人表皮角质形成细胞株HaCaT细胞和真皮成纤维细胞,应用免疫组织化学法检测NK1R在两种细胞中的表达;采用RTPCR方法检测NK1R在两种细胞mRNA水平的表达特征,并采用流式细胞术定量检测在不同刺激因素及药物作用下两种细胞中NK1R的表达调控情况。结果NK1R在HaCaT细胞及真皮成纤维细胞中均有表达,阳性部位位于细胞膜及细胞质。NK1RmRNA在HaCaT细胞上的表达水平要高于在真皮成纤维细胞上的表达。P物质和IFNγ可以上调NK1R在两种细胞上的表达,而LPS抑制了NK1R的表达;抗组胺药盐酸西替利嗪和P物质受体特异性拮抗剂SpantideI可以降低两种细胞上NK1R的表达。结论皮肤角质形成细胞和真皮成纤维细胞在细胞、蛋白及mRNA水平均有NK1R的表达,而且这种表达可被过敏炎症因子调控,提示角质形成细胞和真皮成纤维细胞参与了皮肤免疫调控,神经肽P物质可能在皮肤过敏性炎症中起重要作用。     

Matrix metalloproteinase expression and activity in human airway smooth muscle cells

Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells. MMPs and TIMPs were examined using quantitative real-time RT-PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity. The most abundant MMPs were pro-MMP-2, pro- MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4. In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling.     

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.     

Expression of matrix metalloproteinases and their inhibitors by rat NK cells: inhibition of their expression by genistein

In this study, we describe rat NK cell-derived MMPs including membrane-type MMPs (MT-MMPs) and tissue inhibitors of MMP (TIMPs). RT-PCR analysis from cDNA of rat A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-7, MMP-10, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. The RNK-16 cells expressed mRNA for MMP-7, MMP-10, MMP-11, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2, in addition to MMP-3 and MMP-13. Western blot analysis confirmed proteins for MT1-MMP and MT2-MMP in RNK-16 cells. TIMP-1 in rat A-NK cells was present at molecular mass of 34-kDa protein which may represent a highly glycosylated form. Genistein, a natural isoflavone found in soybeans, inhibited proliferation of RNK-16 cells in dosage dependent manner. In addition, it down-regulated the expression of MMP-13, MT1-MMP, TIMP-1 and TIMP-2. Moreover, genistein greatly impaired the ability of RNK-16 cells to invade through a model basement membrane. This effect might be mediated by the observed down-regulation of MMP-13 and MT1-MMP.     

Matrix metalloproteinases of human NK cells

We have previously reported that MMP-2 and MMP-9 are present in rat A-NK cells, and have recently documented that additional MMPs are present in rodent A-NK cells. To our knowledge only proMMP-9 has previously been reported for human NK and A-NK cells. Herein, we report for the first time the presence of MMP-2 and MT1-MMP in human NK cells. The importance of these enzymes for the migration of A-NK cells into tumor metastases is of great potential relevance. MMPs may be rate limiting in A-NK cells, following their adoptive transfer, to traverse basement membrane and accumulate within established cancer metastases, a likely pre-requisite to their cytolytic function. Human NK cells express and produce MMP-2, MMP-9, MT1-MMP and the inhibitor TIMP-1. Moreover, human A-NK cells degrade the extracellular matrix equivalent (Matrigel) in a seemingly IL-2 dependent manner. It is therefore likely that A-NK cell MMPs play crucial roles in contributing to A-NK cell localisation and positioning the cells in vivo to allow for triggering their cytolytic potential.     

Consistent alterations of circulating matrix metalloproteinases levels in untreated hypertensives and in spontaneously hypertensive rats: a relevant pharmacological target

Inconsistent matrix metalloproteinases (MMPs) levels have been reported in hypertension, with higher, similar and lower MMPs levels reported in hypertensives compared with normotensives. Differences between studies may reflect lack of control of drug effects, accompanying diseases and pre-analytical issues. We compared MMP-2, MMP-8 and MMP-9 levels in 38 untreated hypertensive patients (with no other diseases) with those found in 33 normotensive controls. We also studied endogenous MMPs inhibitors (TIMP-1, TIMP-2 and alpha-2-macroglobulin-A2M). Additionally, we assessed MMPs and A2M levels in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. We hypothesized that similar MMPs/endogenous inhibitors' profiles would be found in this animal model of hypertension and in clinical hypertension. MMPs, TIMPs and A2M were measured in plasma samples with commercially available ELISA and gelatin zymography. We found unaltered MMP-2, MMP-8, TIMP-1, TIMP-2 and A2M levels in hypertension. However, hypertensives had higher MMP-9 levels and MMP-9/A2M ratios than normotensives. Moreover, while we found similar MMP-2 and A2M levels in SHR and WKY rats, we found higher MMP-9 levels and MMP-9/A2M ratios in SHR versus WKY rats. These findings show consistent abnormal net plasma MMP-9 (but not MMP-2) activity in clinical and experimental hypertension. These parallel alterations in clinical hypertension and in SHR suggest an important role for MMPs in hypertension. While MMPs may be a relevant pharmacological target, antihypertensive drugs that down-regulate MMPs may offer advantages in the management of this disease.     

Altered matrix metalloproteinases and tissue inhibitors of metalloproteinases in embryos from diabetic rats during early organogenesis

Maternal diabetes increases the risks for embryo malformations. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are two relevant MMPs for embryo development. Here, we addressed whether changes in these MMPs and in tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-2 are altered in embryos and decidua from type 1 diabetic rats during early organogenesis. Our results demonstrate MMP-2 and MMP-9 overactivities and overexpression, together with increases in lipid peroxidation and nitric oxide production in embryos and decidua from diabetic animals. There is a concomitant increase in the inhibitory activity of TIMP-1 and TIMP-2 in embryos and decidua, and an increase in protein expression of embryonic TIMP-1 and TIMP-2. In situ zymography demonstrated MMPs overactivities despite increased TIMPs in embryos and decidua in maternal diabetes during early organogenesis. This study reveals that maternal diabetes leads to profound alterations in MMPs/TIMPs balance during embryo organogenesis, the gestational period during which most malformations are induced.     

Activation of latent transforming growth factor beta 1 and inhibition of matrix metalloprotease activity by a thrombospondin-like tripeptide linked to elaidic acid

Impaired wound healing and skin aging are characterized by neutral protease-mediated destruction of matrix macromolecules associated with disturbance in tissue repair. We synthesized a fatty acyl-peptide derivative at aims to simultaneously activate latent TGF-beta through its peptide domain, KFK, and inhibit MMPs through its lipophilic moiety, elaidic acid. Elaidyl-KFK as well as KFK were shown to activate LAP-TGF-beta both in vitro, using a solid phase assay with immobilized LAP-TGF-beta, and ex vivo using human dermal fibroblasts cultures. In both assays, as much as up to 10% of LAP-TGF-beta added could be recovered as active form. KQK, KQFK as well as their lipopeptide counterparts were inactive. Elaidyl-KFK-mediated LAP-TGF-beta activation led to up-regulation of collagen and TIMP-1 production and down regulation of PMA-induced MMP-1 expression in fibroblasts cultures. Those effects could be suppressed by supplementing cell culture medium with blocking TGF-beta antibody. Elaidyl-KFK inhibited MMP-2, MMP-9, MMP-3, MMP-1, in vitro with IC(50) equal to 1.2, 1.0, 0.24 and 8.9 microM, respectively. Its ex vivo inhibitory capacity, as assessed using skin tissue sections, towards the elastin-degrading capacity of MMP-9 was even more pronounced. At a 1 microM concentration, the lipopeptide decreased by up to 80% enzyme activity. Thus, "Lipospondin," i.e. elaidyl-KFK might be considered as a promising model compound to prevent age-associated dermal alterations.     

Serum MMP-8, -9 and TIMP-1 in sepsis: High serum levels of MMP-8 and TIMP-1 are associated with fatal outcome in a multicentre, prospective cohort study. Hypothetical impact of tetracyclines

Recent evidence suggests that matrix metalloproteinases (MMPs) and their endogenous inhibitors are involved in the pathogenesis of sepsis. We studied serum levels of MMP-8, MMP-9 and TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) in a multicentre, prospective cohort study of patients with sepsis treated in Intensive Care Units (ICUs). We analyzed serum samples taken on ICU admission from 248 critically ill sepsis patients. MMP-8, -9 and TIMP-1 serum levels were analyzed by enzyme-linked immunosorbent assays. Serum MMP-8, MMP-9 and TIMP-1 levels were significantly higher in patients with severe sepsis than in healthy controls. Serum MMP-8 levels among non-survivors (n = 33) were significantly (p = 0.006) higher than among survivors (n = 215). Serum TIMP-1 but not MMP-9 levels were significantly higher among non-survivors than survivors (p < 0.0001, p = 0.079, respectively). Systemic MMP-8 is upregulated in sepsis suggesting that MMP-8 may contribute to the host response during sepsis. High serum MMP-8 and TIMP-1 levels at ICU admission were seen among patients with fatal outcome. With this background, clinical studies examining the ability of MMP-inhibitors (such as the non-antimicrobial properties of tetracyclines) to diminish the MMP-mediated inflammatory response are needed to develop novel therapies in order to improve the outcome of sepsis.     

Saponins from the roots of Platycodon grandiflorum suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and NF-κB/AP-1-dependent signaling in HaCaT cells

Saponins from the roots of Platycodon grandiflorum (CKS) have been shown to exhibit many pharmacological activities, including anti-cancer and anti-inflammatory activities and antioxidant effects. However, anti-skin photoaging effects of CKS have not yet been reported. In this study, we investigated the protective effects of CKS against UVA damage on immortalized human keratinocytes (HaCaT). We then explored the inhibitory effects of CKS on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. CKS increased the cell viability and inhibited reactive oxygen species (ROS) production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with CKS inhibited UVA-induced production of MMP-1 and MMP-9. In addition, CKS decreased UVA-induced expression of the inflammatory cytokines IL-1β and IL-6. Western blot analysis further revealed that CKS markedly suppressed the enhancement of collagen degradation in UVA-exposed HaCaT cells. CKS also suppressed UVA-induced activation of NF-κB or c-Jun and c-Fos, and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1.     

Inhibition of ultraviolet-A-modulated signaling pathways by asiatic acid and ursolic acid in HaCaT human keratinocytes

Premature aging of the skin (photoaging) is a well-documented consequence of exposure to ultraviolet-A (UVA). Enhanced generation of reactive oxygen species and induction of matrix metalloproteinases (MMPs) appear to be the most important components of UVA-modulated signal transduction pathways, ultimately leading to photoaging. In this study, we investigated the effects of asiatic acid and ursolic acid, triterpene compounds, on the UVA-modulated signaling pathways using HaCaT human keratinocytes as a model cellular system. In the cells, we confirmed that UVA irradiation induced oxidative stress and increased the expression of MMP-2. Asiatic acid and ursolic acid significantly suppressed the UVA-induced reactive oxygen species production and lipid peroxidation. Pretreatment with asiatic acid or ursolic acid significantly reduced the UVA-induced activation and expression of MMP-2. In addition, UVA-induced enhanced expression of p53, a hallmark of UV-induced DNA damage and cell death, was also significantly inhibited by pretreatment with asiatic acid or ursolic acid. Taken together, these results suggest that asiatic acid and ursolic acid may be an effective inhibitor of UVA-modulated signal transduction pathways in human skin cells. These results further suggest that these agents may be useful in the prevention of UVA-induced photoaging.