Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation

The mechanism of indomethacin-induced gastric ulcer healing by ellagic acid (EA) in experimental mice model is described in our study. Ulcer index (UI) and myeloperoxidase (MPO) activity of the stomach tissues showed maximum ulceration on the third day after indomethacin (18 mg/kg, single dose) administration. Preliminary observation of UI and MPO activity suggests that EA possesses ulcer-healing activity. Other anti-ulcer parameters such as the levels of prostaglandin E(2), cyclooxygenase (COX) 1 and 2 enzymes, anti-inflammatory cytokines [interleukin (IL)-4 and -5], pro-angiogenic factors, e.g. vascular endothelial growth factor, hepatocyte growth factor (HGF), and endothelial growth factor (EGF) were down-regulated by indomethacin. EA (7 mg/kg/day) treatment for 3 days shifted the indomethacin-induced pro-inflammatory biochemical parameters to the healing side. These activities were correlated with the ability of EA to alter the COX-2-dependent healing pathways. The ulcer-healing activity of EA was, however, compromised by pre-administration of the specific COX-2 inhibitor, celecoxib, and NS-398. Taken together, these results suggested that the EA treatment accelerates ulcer healing by inducing IL-4, EGF/HGF levels and enhances COX-2 expression.     

Curcumin influences hepatic expression patterns of matrix metalloproteinases in liver toxicity

Hepatic fibrosis is a result of an imbalance between enhanced matrix synthesis and diminished breakdown of connective tissue proteins, the net result of which is increased deposition of Extra Cellular Matrix. In this concept Matrix Metalloproteinases play an important role because their activity is largely responsible for extra cellular matrix breakdown. In the present study we have tested the influence of curcumin, the active principle of turmeric, on matrix metalloproteinase expression during alcohol and thermally oxidised sunflower oil induced liver toxicity. Male albino Wistar rats were used for the study. The matrix metalloproteinase expressions were found to be increased significantly in alcohol as well as thermally oxidised sunflower oil groups and on treatment with curcumin there was a significant decrease. In alcohol + thermally oxidised sunflower oil group, we found a significant decrease in matrix metalloproteinase activities. Administration of curcumin significantly improved their activities. From the results obtained, we could conclude that curcumin influences the hepatic matrix metalloproteinases and effectively protects liver against alcohol and delta PUFA induced toxicity.     

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing

The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1–7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4–6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1–6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds. © 1996 Wiley-Liss, Inc.     

Expression of matrix metalloproteinases MMP-2 and MMP-9 in gastric cancer and their relation to claudin-4 expression

Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 can degrade type IV collagen of extracellular matrix and basal membranes. Claudin-4 is a member of a large family of transmembrane proteins, claudins, essential in the formation and maintenance of tight junctions. Claudin-4 has been shown to activate MMP-2, indicating that claudin-mediated increased cancer cell invasion might be mediated through the activation of MMP proteins. To explore the roles of MMP-2, MMP-9 and claudin-4 in gastric cancer, we selected 88 cases and then analyzed the expression of these proteins using immunohistochemistry. We found that all of MMP-2, MMP-9 and claudin-4 expressions were significantly higher in intestinal-type than in diffuse-type gastric cancer. On further analysis, testing the relationship between MMP-2 and MMP-9 expression with claudin-4 expression, claudin-4 expression was significantly associated with MMP-9 expression, but not with MMP-2 expression. The results showed that MMP-2, MMP-9 and claudin-4 expression may be phenotypic features, distinguishing intestinal-type and diffuse-type gastric cancer. Possibly, claudin-4 played a role in determining MMP-9 activity which favored intestinal-type gastric cancer to distal metastasis.     

Alterations of matrix metalloproteinases,gastric mucin and prostaglandin E2 levels by pectic polysaccharide of swallow root (Decalepis hamiltonii) during ulcer healing

Gastric ulcer is a multi-step disease caused due to imbalance between mucosal defense and aggressive factors. Available anti-ulcer drugs although effective at various steps of ulcer pathogenecity, pose adverse effects. Pectic polysaccharide (SRPP) from swallow root (Decalepis hamiltonii) – previously shown to possess ulcer preventive effect against swim stress and ethanol induced gastric ulcers. In the current study, alteration of matrix metalloproteinases, gastric mucin and prostaglandin E₂ levels during polysaccharide mediated ulcer healing was determined in acetic acid induced gastric ulcer model in Wistar albino rats. Results indicated the potential ulcer healing effect of SRPP as evidenced by ∼90% reduction in ulcer index; improvement in the antioxidant defense such as increase of glutathione levels together with significant reduction in lipid and protein oxidation and protection to damaged gastric mucin. Further, histological studies substantiated the result of the recovery of mucin that was eroded during ulceration, rejuvenation of mucosal epithelium and enhancement of high molecular mass mucin as opposed to the degraded ∼55 kDa mucin that appeared only during ulcer condition. Matrix metalloproteinases (MMPs) that are involved in tissue injury was found to be modulated by SRPP treatment in addition to increased cytoprotectivity due to enhanced synthesis of PGE₂ that necessitates the active proliferation of gastric mucin cells. Further, reduction in ∼3 folds of galectin-3, an inflammatory marker suggests gastro protection against acid induced inflammation and gastric wall damages. Overall, studies show the effectiveness of SRPP in inhibiting MMPs and galectin-3 levels which were up-regulated during ulcer conditions. In addition SRPP ensured cytoprotectivity and rejuvenation of mucosal barrier via PGE₂ trigger leading to ulcer healing.     

Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartum

The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6-12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steroidogenic enzymes, mediate fetal membrane detachment after parturition.     

Expression of matrix metalloproteinases 2 and 9 in the mouse uterus during implantation and oil-induced decidualization

During implantation, matrix metalloproteinases are believed to play roles in the tissue remodelling that accompanies decidualization in the endometrium and in embryo invasion. The objective of this study was to characterize further the expression of matrix metalloproteinases 2 and 9 in the mouse uterus during early pregnancy and oil-induced decidualization. mRNA encoding matrix metalloproteinase 2 was detected in pregnant uteri and uteri undergoing oil-induced decidualization by northern blot analyses. The steady-state concentrations of mRNA encoding matrix metalloproteinase 2 did not change significantly in implantation compared with inter-implantation areas on days 5-8 of pregnancy but were significantly lower in stimulated compared with non-stimulated uterine horns during artificially induced decidualization. mRNA encoding matrix metalloproteinase 9 was also detected in uteri undergoing oil-induced decidualization but not in pregnant uteri. Its concentration was significantly greater in uterine horns undergoing oil-induced decidualization compared with control horns. Immunoreactive matrix metalloproteinases 2 and 9 were detected in the uterus during early pregnancy and oil-induced decidualization by immunohistochemistry, localized to the endometrial stroma, but the staining progressively became weaker and was absent in areas that had undergone decidualization. By day 8 of pregnancy and 72 h after the induction of decidualization, matrix metalloproteinase 2 and 9 proteins remained mainly in the region of non-decidualized stromal cells adjacent to the myometrium. In implantation segments, they were also localized to the region of the trophoblast giant cells. The second objective of the present study was to determine whether endometrial stromal cells isolated from uteri sensitized for decidualization express matrix metalloproteinases 2 and 9. Northern blot analyses and gelatin zymography showed that these cultured cells expressed matrix metalloproteinase 2 and 9, and that transforming growth factor beta1 significantly increased matrix metalloproteinase 9 expression. The results of the present study further characterize matrix metalloproteinases 2 and 9 expression in the uterus during implantation and artificially induced decidualization.     

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes.We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca²⁺ chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca²⁺ mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFκB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.     

Gastroprotective action of glucocorticoids during the formation and the healing of indomethacin-induced gastric erosions in rats.

The aim of the present study consisted of the investigation of glucocorticoid role in the formation and the healing of indomethacin-induced (25 mg/kg, s.c.) gastric erosions in rats. The effect of deficiency of glucocorticoid production followed by corticosterone replacement on the formation and the healing of the gastric erosions was evaluated. Glucocorticoid production was decreased by adrenalectomy or by delayed inhibitory action after a single pharmacological dose of cortisol (300 mg/kg i.p.) injected 1 week before the onset of ulcerogenic stimulus. Indomethacin induced corticosterone rise and caused gastric erosions. The loss of indomethacin-induced plasma corticosterone rise potentiated the formation of indomethacin-induced erosions in both models. The area of gastric erosions in rats with glucocorticoid deficiency was considerably larger than that in control animals 4 h after indomethacin administration as well as during 48 h after the drug administration (period of erosion healing). Injecting corticosterone in rats with glucocorticoid deficiency significantly decreased the formation of indomethacin-induced gastric erosions and promoted their healing. Thus, the present data support the gastroprotective action of glucocorticoids in the formation and in the healing of indomethacin-induced mucosal injury.     

Biphasic activity of resveratrol on indomethacin-induced gastric ulcers

Resveratrol showed biphasic activity in indomethacin-induced gastric ulcerated mice. A protective effect at a lower dose (2 mg kg⁻¹) and a contraindicative effect at a higher dose of Resveratrol (10 mg kg⁻¹) were observed. This phenomenon was possibly controlled by a COX-1 and eNOS balance, which ultimately maintained angiogenesis in Resveratrol-treated pre-ulcerated mice. The lower dose of Resveratrol (2 mg kg⁻¹) augmented eNOS expression without altering COX-1 expression, but, at a higher dose (10 mg kg⁻¹), Resveratrol predominantly suppressed COX-1 expression, which significantly reduced both PGE₂ synthesis and angiogenesis. Thus it ultimately resulted in delay healing of indomethacin-induced gastric ulcers. Hence, it could be concluded that COX-1 and eNOS acted as key regulatory factors switching the biphasic effects of Resveratrol in indomethacin-induced ulcerated mice.     

Novel role of famotidine in downregulation of matrix metalloproteinase-9 during protection of ethanol-induced acute gastric ulcer

Gastric ulcer is a multifaceted process including acid secretion, reactive oxygen species generation, prostaglandin inhibition, and extracellular matrix (ECM) degradation. Matrix metalloproteinases (MMPs) have the ability to cleave and remodel the ECM. We investigated the activity and expression of MMP-9 and -2 in ethanol-induced acute gastric ulceration in rats. We found that severity of gastric ulcer was strongly correlated with increasing doses of ethanol and increased secretion of proMMP-9. ProMMP-9 was upregulated approximately 25-fold at maximum ulcer index. Increased secretion of proMMP-9 was associated with increased expression of tumor necrosis factor-alpha and interleukin-6. We examined the effect of H(2)-receptor antagonists and antioxidants on proMMP-9 secretion and synthesis during prevention of ethanol-induced gastric ulcer. Our data reveal that famotidine dose dependently blocked increased secretion and synthesis of proMMP-9 during gastroprotection and arrested infiltration of inflammatory cells as well as oxidative stress in rat gastric tissues. Similar to H(2)-receptor antagonists, N-acetylcysteine and dimethyl sulfoxide, well-known antioxidants, inhibited proMMP-9 upregulation to the control level. In conclusion, ethanol-induced gastric ulceration is associated with increased expression of proMMP-9 that can be attenuated by H(2)-receptor antagonists and antioxidants. These findings furnish a novel MMP-9-mediated pathway and its inhibition via proinflammatory cytokines by famotidine in ethanol-induced gastric ulceration.     

Quercetin downregulates matrix metalloproteinases 2 and 9 proteins expression in prostate cancer cells (PC-3)

Background: Cancer metastasis, involving multiple processes and various cytophysiological changes, is a primary cause of cancer death and may complicate the clinical management, even lead to death. Quercetin is a flavonoid and widely used as an antioxidant and recent studies have revealed its pleiotropic anticancer and antiproliferative capabilities. Gelatinases A and B (matrixmetalloproteinases 2 and 9) are enzymes known to involve in tumor invasion and metastases. In this study, we observed the precise involvement of quercetin role on these proteinases expression and activity. Design and methods: PC-3 cells were treated with quercetin at various concentrations (50 and 100 μM), for 24 h period and then subjected to western blot analysis to investigate the impact of quercetin on matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9) expressions. Conditioned medium and cell lysate of quercetin-treated PC-3 cells were subjected to western blot analysis for proteins expression of MMP-2 and MMP-9. Gelatin zymography was also performed in quercetin treated PC-3 cells. Results: The results showed that quercetin treatment decreased the expressions of MMP-2 and MMP-9 in dose-dependent manner. The level of pro-MMP-9 was found to be high in the 100 μM quercetin-treated cell lysate of PC-3 cells, suggesting inhibitory role of quercetin on pro-MMP-9 activation. Gelatin zymography study also showed the decreased activities of MMP-2 and MMP-9 in quercetin treated cells. Conclusion: Hence, we speculated that inhibition of metastasis-specific MMPs in cancer cells may be one of the targets for anticancer function of quercetin, and thus provides the molecular basis for the development of quercetin as a novel chemopreventive agent for metastatic prostate cancer.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

Curcumin differentially regulates TGF-beta1, its receptors and nitric oxide synthase during impaired wound healing

Wound healing is a highly ordered process, requiring complex and coordinated interactions involving peptide growth factors of which transforming growth factor-beta (TGF-beta) is one of the most important. Nitric oxide is also an important factor in healing and its production is regulated by inducible nitric oxide synthase (iNOS). We have earlier shown that curcumin (diferuloylmethane), a natural product obtained from the plant Curcuma longa, enhances cutaneous wound healing in normal and diabetic rats. In this study, we have investigated the effect of curcumin treatment by topical application in dexamethasone-impaired cutaneous healing in a full thickness punch wound model in rats. We assessed healing in terms of histology, morphometry, and collagenization on the fourth and seventh days post-wounding and analyzed the regulation of TGF-beta1, its receptors type I (tIrc) and type II (tIIrc) and iNOS. Curcumin significantly accelerated healing of wounds with or without dexamethasone treatment as revealed by a reduction in the wound width and gap length compared to controls. Curcumin treatment resulted in the enhanced expression of TGF-beta1 and TGF-beta tIIrc in both normal and impaired healing wounds as revealed by immunohistochemistry. Macrophages in the wound bed showed an enhanced expression of TGF-beta1 mRNA in curcumin treated wounds as evidenced by in situ hybridization. However, enhanced expression of TGF-beta tIrc by curcumin treatment observed only in dexamethasone-impaired wounds at the 7th day post-wounding. iNOS levels were increased following curcumin treatment in unimpaired wounds, but not so in the dexamethasone-impaired wounds. The study indicates an enhancement in dexamethasone impaired wound repair by topical curcumin and its differential regulatory effect on TGF-beta1, it's receptors and iNOS in this cutaneous wound-healing model.     

Downregulation of endothelin-1 by interleukin-4 during gastric ulcer healing.

We investigated the course of events associated with gastric ulcer healing by analyzing mucosal expression of interleukin-4 (IL-4), endothelin-1 (ET-1), tumor necrosis factor-alpha (TNF-alpha), and the activity of constitutive (cNOS) and inducible nitric oxide synthase (NOS-2). Ulcer onset was characterized by a massive epithelial apoptosis associated with a 5.7-fold increase in TNF-alpha, a 17.5-fold increase in NOS-2, and a 3.9-fold increase in ET-1, while mucosal expression of cNOS showed a 7.6-fold drop and IL-4 fell by 37.2%. Healing was accompanied by a rapid raise in IL-4; decrease in apoptosis, TNF-alpha, ET-1, and NOS-2; and a slow recovery in cNOS. The expression of IL-4 returned to control levels by the 7th day of healing and that of ET-1 and TNF-alpha by the 14th day, while apoptotic DNA fragmentation and the activity of NOS-2 remained significantly elevated beyond the 14-day period. The results suggest that a decrease in the mucosal level of IL-4 at ulcer onset may well be a key factor causing dysregulation of ET-1 production, induction of TNF-alpha, and triggering the apoptotic events that affect the efficiency of mucosal repair.     

The role of matrix metalloproteinases 2 and 9 during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry.     

Dynamics of matrix metalloproteinases activity of primary murine embryonic fibroblasts during cultivation

Embryonic cells regulate the expression of matrix metalloproteinases (MMP) providing remodulation of extracellular matrix, which in turn provides the changes in cell adhesion and migration during the cell development and differentiation. In present work we studied the changes of gelatinases (MMP-2 and MMP-9) and collagenases (MMP-1 and MMP-8) activities in the process of cultivating the primary murine embryonic fibroblasts (MEF). Cultivation was continued for 6 passages, after that the culture died in time. According to gelatin and collagen zymography results, drastic changes of all MMPs activities occurred during the third passage of cell cultivation. The MMP-1 and MMP-9 activity appears in the middle of cultivation and then disappeared at the end. The most important event MEF cultivation is appearance and subsequent reservation of collagenase MMP-8 and active form of gelatinase MMP-2.