Effects of Saikosaponin-D on syndecan-2,matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 in rats with hepatocellular carcinoma

OBJECTIVE:To investigate effects of Saikosaponin D(SSd) on syndecan-2,matrix metalloproteinases(MMPs) and tissue inhibitor of metalloproteinases-2(TIMP-2) in livers of rat with hepatocellular carcinoma(HCC).METHODS:Male SD rats were divided into control(n=10),model(n=20) and SSd(n=20) groups,and model and SSd groups given intragastric 0.2%(w/v) N-diethylnitrosamine to induce HCC.SSd group received 0.03%(w/v) SSd in saline.Liver samples were analysed immunohistochemically for syndecan-2,MMP-2,MMP-13 and TIMP-2 at 16 weeks.RESULTS:The model group had more malignant nodules than the SSd group;all model-group HCC cells were grade III;SSd-group HCC cells were grades I-II.Controls showed normal hepatic cell phenotypes and no syndecan-2 + staining.Syndecan-2 + staining was greater in the model group(35.2%,P≤0.001) than in controls or the SSd group(16.5%,P ≤ 0.001).The model group had more intense MMP-2 + staining than controls(0.37 vs 0.27,P≤0.01) or the SSd group(0.31 vs 0.37,P≤0.05);and higher MMP-13 + staining(72.55%) than in controls(12.55%,P≤0.001) and SSd group(20.18%,P≤0.01).The model group also had more TIMP-2 + staining(57.2%) than controls(20.9%,P≤0.001) and SSd group(22.7%,P≤0.001).Controls and SSd group showed no difference in TIMP-2 + rates.CONCLUSION:SSd inhibited HCC development,and downregulated expression of syndecan-2,MMP-2,MMP-13 and TIMP-2 in rat HCC liver tissue.

Effects of Biejia Ruangan Tablet(复方鳖甲软肝片)-Containing Serum on Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 Expression in Cultured Renal Interstitial Fibroblasts

Objective： To investigate the effects of Biejia Ruangan Tablet （复方鳖甲软肝片, BRT）- containing serum on the expression of matrix metalloproteinase （MMP-9） and tissue inhibitor of metalloproteinase （TIMP-1） in cultured renal interstitial fibroblasts. Methods： Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose （7 g/kg）, mid-dose （3.5 g/kg）, and low-dose （1.75 g/kg） BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 （recombined human TGF-β 1）, and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1. Results： The high dose BRT-containing serum could decrease the type Ⅰ and Ⅲ procollagen gene expression which boosted by TGF- 13 1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF- β1 （P〈0.05）. Treatment of cells with recombined human TGF-β 1 had no significant effect on MMP-9 expression and BRT- containing serum also had no effect on MMP-9 expression. Conclusions： High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type Ⅰ and Ⅲ procollagen and TIMP-1 expression.

Effects of sodium arsenite on the expression of microRNA-191 and tissue inhibitor of metalloproteinase 3 in L-02 cells

Objective To investigate the effects of sodium arsenite（NaAsO2）on the expression of microRNA-191（miR-191）and tissue inhibitor of metalloproteinase 3（TIMP-3）in human normal hepatic cells（L-02 cells）.Methods L-02 cells were exposed to different doses of Na2As O2[0（control group）,5,25,50 and 75μmol/L]

Tissue Inhibitor of Metalloprotease-1(TIMP-1)Regulates Adipogenesis of Adipose-derived Stem Cells(ASCs)via the Wnt Signaling Pathway in an MMP-independent Manner

Tissue inhibitor of m etalloprotease-1(TIM P-1)is a tissue inhibitor o f matrix metalloproteinases(MMPs).It however exerts multiple effects on biological processes,such as cell growth,proliferation,differentiation and apoptosis,in an MMP-independent manner.This study aimed to examine the role of TIMP-1 in adipogenesis of adipose-derived stem cells(ASCs)and the underlying mechanism.We knocked down the TIMP-1 gene in ASCs through lentiviral vectors encoding TIMP-1 small interfering RNA(siRNA),and then found that the knockdown of TIMP-1 in ASCs promoted the adipogenic differentiation of stem cells and inhibited the Wnt/β-catenin signaling pathway in ASCs.We also noted that mutant TIMP-1 without the inhibitory activity on MMPs promoted the activation of Wnt/β-catenin pathway as well as the recombinant wild type TIMP-1 did,which indicated that the effect of TIMP-1 on Wnt/β-catenin pathway was MMPindependent.Our study suggested that TIMP-1 negatively regulated the adipogenesis of ASCs via the Wnt/β-catenin signaling pathway in an MMP-independent manner.

Occurrence of Two Distinct types of Tissue Inhibitors of Metallo-proteinases-2 in Fugu rubripes

In this study, genes of two distinct tissue inhibitors of metalloproteinases-2 (TIMP-2) from Japanese puffer fishFugu rubripes, Fugu TIMP-2a and TIMP-2b, were cloned. The open reading frames of Fugu TIMP-2a and TIMP-2b cDNAsare composed of 660 and 657 nucleotides and 220 and 219 amino acids, respectively. Both Fugu TIMP-2s contain 12 cysteineresidues, which might form six disulfide bonds as in other animals’ TIMP-2s. Reverse-transcribed polymerase chain reactionanalysis showed the mRNAs of Fugu TIMP-2a and TIMP-2b to be expressed in some tissues examined with different expres-sion patterns. These findings suggest that the two distinct Fugu TIMP-2s might perform different functions in Fugu tissues.

Effect of quercetin on expression of matrix metallo - proteinases and tissue inhibi tor of matalloproteinase -1 in cultured rat hepatic stellate cells

Objective: To study the effects of quercetin (QU) on matrix metallo-proteinases (MMPs) , the tissue inhibitor of matalloproteinase-1 (TIMP-1) , procollagen I and 2 proteoglycans (decorin and biglycan) mRNA expression in cultured rat hepatic stellate cell line HSC-T6 cells. Methods: Cells were treated with different concentrations of QU (12. 5, 25, 50 μmol/L) or drug solvent (0. 1 % Me2SO) for 24 h. mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR). Results: QU (12.5 - 50 μmol/L) enhanced collagenase (rat MMP-13) and membrane typel-MMP (MMP-14) mRNA expression, decreased procollagen I mRNA expression in a concentration-dependent manner, but did not affect gelatinase-A (MMP-2) , TIMP-1, decorin and biglycan expression. Conclusion: QU may decrease matrix deposition and increase matrix degradation, which might be beneficial to liver fibrosis.

Diabetes-related intestinal region-specific thickening of ganglionic basement membrane and regionally decreased matrix metalloproteinase 9 expression in myenteric ganglia

BACKGROUND The importance of the neuronal microenvironment has been recently highlighted in gut region-specific diabetic enteric neuropathy. Regionally distinct thickening of endothelial basement membrane(BM) of intestinal capillaries supplying the myenteric ganglia coincide with neuronal damage in different intestinal segments. Accelerated synthesis of matrix molecules and reduced degradation of matrix components may also contribute to the imbalance of extracellular matrix dynamics resulting in BM thickening. Among the matrix degrading proteinases, matrix metalloproteinase 9(MMP9) and its tissue inhibitor(TIMP1) are essential in regulating extracellular matrix remodelling.AIM To evaluate the intestinal segment-specific effects of diabetes and insulin replacement on ganglionic BM thickness, MMP9 and TIMP1 expression.METHODS Ten weeks after the onset of hyperglycaemia gut segments were taken from the duodenum and ileum of streptozotocin-induced diabetic, insulin-treated diabetic and sex-and age-matched control rats. The thickness of BM surrounding myenteric ganglia was measured by electron microscopic morphometry. Wholemount preparations of myenteric plexus were prepared from the different gut regions for MMP9/TIMP1 double-labelling fluorescent immunohistochemistry. Post-embedding immunogold electron microscopy was applied on ultrathin sections to evaluate the MMP9 and TIMP1 expression in myenteric ganglia and their microenvironment from different gut segments and conditions. The MMP9 and TIMP1 messenger ribonucleic acid(m RNA) level was measured by quantitative polymerase chain reaction.RESULTS Ten weeks after the onset of hyperglycaemia, the ganglionic BM was significantly thickened in the diabetic ileum, while it remained intact in the duodenum. The immediate insulin treatment prevented the diabetes-related thickening of the BM surrounding the ileal myenteric ganglia. Quantification of particle density showed an increasing tendency for MMP9 and a decreasing tendency for TIMP1 from the proximal to the distal small intestine under control conditions. In the diabetic ileum, the number of MMP9-indicating gold particles decreased in myenteric ganglia, endothelial cells of capillaries and intestinal smooth muscle cells, however, it remained unchanged in all duodenal compartments. The MMP9/TIMP1 ratio was also decreased in ileal ganglia only. However, a marked segment-specific induction was revealed in MMP9 and TIMP1 at the m RNA levels.CONCLUSION These findings support that the regional decrease in MMP9 expression in myenteric ganglia and their microenvironment may contribute to extracellular matrix accumulation, resulting in a region-specific thickening of ganglionic BM.

EFFECTS OF GENISTEIN ON INVASION AND MATRIX METALLOPROTEINASE ACTIVITIES OF HT1080 HUMAN FIBROSARCOMA CELLS

Effects of genistein on invasion and matrix metalloproteinase activities were investigated in HT1080 human sarcoma cells.Invasion of HT1080 cells through reconstituted basement membrane was inhibited when the cells were treated with 100 μ mol/L and 200 μ mol/L genistein.At the same concentrations,genistein not only suppressed latent forms of matrix metalloprotinese 2 and 9(MMP 2 and MMP 9) to convert into active forms,but also increase dramatically the tissue inhibitor of metalloproteinase(TIMP 1) mRNA contents and reverse the imbalance of MMPs and TIMPs.However,expressions of MMP 2 and MMP 9 were not significantly affected.Suppression of MMP activation and increase of TIMP 1 expression will decrease matrix degradation by MMPs,and consequently inhibit invasions of the cells.These results emphasized the existence of the imbalance between MMPs and TIMPs in tumor invasion and metastasis formation.The value of genistein as a drug for antiinvasion and anti metastasis chemotherapy was suggested.

Prevention of peritoneal adhesions:A promising role for gene therapy

Adhesions are the most frequent complication of abdominopelvic surgery,yet the extent of the problem,and its serious consequences,has not been adequately recognized.Adhesions evolved as a life-saving mecha-nism to limit the spread of intraperitoneal inflammatory conditions.Three different pathophysiological mechanisms can independently trigger adhesion formation.Mesothelial cell injury and loss during operations,tissue hypoxia and inflammation each promotes adhesion formation separately,and potentiate the effect of each other.Studies have repeatedly demonstrated that interruption of a single pathway does not completely prevent adhesion formation.This review summarizes the pathogenesis of adhesion formation and the results of single gene therapy interventions.It explores the prom-ising role of combinatorial gene therapy and vector modif ications for the prevention of adhesion formation in order to stimulate new ideas and encourage rapid advancements in this field.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Doxycycline blocks gastric ulcer by regulating matrix metalloproteinase-2 activity and oxidative stress

AIM: To examine the effect of doxycycline on the activity of matrix metalloproteinases (MMPs) and oxidative stress in gastric tissues of rats following gastric injury.METHODS: Gastric ulcers were generated in rats by administration of 70% ethanol,and activity of doxycycline was tested by administration 30 min prior to ethanol.Similarly,the effect of doxycycline was tested in an indomethacin-induced gastric ulcer model.The activities and expression of MMPs were examined by zymography and Western blot analysis.RESULTS: Gastric injury in rats as judged by elevated ulcer indices following exposure to ulcerogen,either indomethacin or ethanol,was reversed significantly by doxycycline.Indomethacin-induced ulcerated gastric tissues exhibited about 12-fold higher proMMP-9 activity and about 5-fold higher proMMP-3 activity as compared to control tissues.Similarly,ethanol induced about 22-fold and about 6-fold higher proMMP-9 and proMMP-3 activities,respectively,in rat gastric tissues.Both proMMP-9 and MMP-3 activities were markedly decreased by doxycycline in ulcerogen treated rat gastric tissues.In contrast,the reduced MMP-2 activity in ulcerated tissues was increased by doxycycline during ulcer prevention.On the other hand,doxycycline inhibited significantly proMMP-9,-2 and -3 activities in vitro.In addition,doxycycline reduced oxidative load in gastric tissues and scavenged H2O2 in vitro.Our results suggest a novel regulatory role of doxycycline on MMP-2 activity in addition to inhibitory action on MMP-9 and MMP-3 during prevention of gastric ulcers.CONCLUSION: This is the first demonstration of dual action of doxycycline,that is,regulation of MMP activity and reduction of oxidative stress in arresting gastric injury.

SIGNIFICANCE OF MMP-2 AND TIMP-2 mRNA EXPRESSIONS ON GLOMERULAR CELLS IN THE DEVELOPMENT OF GLOMERULOSCLEROSIS

Objective To study the expressions of MMP-2 and TIMP-2 mRNA on cultured rat mesangial cells (MsC) and in human diseased glomeruli, and to explore their significance in the development of glomerulosclerosis. Methods The expressions of MMP-2, TIMP-2, and Col ⅣmRNA on cultured rat MsC stimulated by IL-1 or/and TGF-β1were investigated through Northern blot analysis. The levels of MMP-2 and TIMP-2 mRNA expressions and immunoreacti-vity of PCNA and Col Ⅳin human diseased glomeruli from renal biopsies of lupus nephritis (LN) patients were examined by insituhybridization and immunohistochemistry, respectively. Results The levels of MMP-2, TIMP-2, and Col ⅣmRNA expressions were markedly increased on cultured rat MsC stimulated by IL-1 or/and TGF-β1. Meanwhile, upregulation of MMP-2 and TIMP-2 mRNA expressions was confirmed in diseased glomeruli from patients with various subtypes of LN, and was closely related to the positive cell number of PCNA presentation and deposition of Col Ⅳin glomeruli. Conclusion The results suggest that the over-expressions of MMP-2 and TIMP-2 mRNA on glomerular cells might play a critical role in the development of glomerulosclerosis.

Effects of Calcium Dobesilate on Glomerulus TIMP1 and Collagen Ⅳ of Diabetic Rats

Summary ： To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 （TIMP1）, collagen Ⅳ , and ultrastrueture of glomerular basement mem- brane in diabetic rats, rats model of diabetes was established by unilateral nephreetomy and intraperitoneal injection of 1% STZ （55 mg/kg）, and rats were administered calcium dobesilate 100 mg/ kg （DD group） or distilled water （DM group） respectively. 12 weeks later, the changes in the renal uhrastrueture and ereatinine clearance rate （Cer） were examined in each group. The expression of glomerular TIMP1 and collagen Ⅳ were studied by immunohistoehemieal staining. Our results showed that after 12 weeks, the Cer in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane （GBM） in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance betweeh the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen Ⅳ in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the overaccumulation of collagen Ⅳ and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.

miR-181b promotes the oncogenesis of renal cell carcinoma by targeting TIMP3

Renal cell carcinoma(RCC)has a poor prognosis due to limited diagnosis and treatment.Thus,it is necessary to find novel prognostic biomarkers and therapeutic targets.The aberrant expression of microRNAs plays an important role in RCC oncogenesis.Tissue inhibitors of metalloproteinase 3(TIMP3)acts as a downstream target of miR-181b.The aim of this study was to understand the role and molecular mechanism of miR-181b in RCC oncogenesis.The results showed that miR-181b expression was significantly higher in RCC tumour tissues,especially in those with significant invasion or metastasis.miR-181b overexpression promoted proliferation and migration of the RCC cell line 786-O,while miR-181b knockdown had the opposite effect.In addition,miR-181b was inversely correlated with TIMP3 expression in RCC tumour tissues.miR-181b overexpression reduced TIMP3 expression in RCC cell line 786-O or OS-RC-2,while miR-181b knockdown had the inverse effect.Mechanistically,a luciferase reporter assay confirmed the binding sites of miR-181b on the 3’-UTR of TIMP3,confirming the targeting effect of miR-181b on TIMP3.Overall,miR-181b promotes the development and progression of RCC by targeting TIMP3 expression,indicating the potential use of miR-181b in the diagnosis and treatment of RCC.

Modified Xiaochaihu Decoction(加味小柴胡汤) Promotes Collagen Degradation and Inhibits Pancreatic Fibrosis in Chronic Pancreatitis Rats

Objective:To investigate the effect of Modified Xiaochaihu Decoction(MXD,加味小柴胡汤)on collagen degradation in rats with chronic pancreatitis(CP).Methods:Rats were injected dibutyltin dichloride(DBTC,7 mg/kg of body weight)into the right caudal vein to induce CP model.Thirty heallhy male Wistar rats were randomly divided into three groups by a random number table:the control,the model and the treatment groups.Rats of treatment group were administered MXD(10 g/kg of body weight)orally once daily starting from the day post-model establishment.Pancreatic tissues were harvested after 28-day feeding and fibrosis was evaluated by picro-sirius red staining.The contents of collagen typeⅠandⅢwere detected using enzymelinked immunosorbent assay(ELISA),the expression of matrix metalloproteinase 13(MMP13)and tissue inhibitor of metalloproteinase 1(TIMP1)was analyzed by Western blot and real-time polymerase chain reaction(PCR).Results:The fibrosis scoring of pancreatic tissues,the concentrations of collagen typeⅠandⅢ,the expression levels of MMP13 and TIMP1 proteins and mRNA in the model group were all increased compared with the control group(P<0.05).After treatment with MXD,the fibrosis scoring of pancreatic tissues,the concentrations of collagen typeⅠandⅢ,the expression levels of MMP13 proteins and m RNA in the teatment group were all decreased compared with the model group(P<0.05),but there were no significant differences in the expression levels of TIMP1 proteins and m RNA(P>0.05).Conclusion:MXD could promote collagen degradation and reverse pancreatic fibrosis in CP rats via a mechanism involve up-regulation of MMP13 expression.