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.

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 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.