Inflammation-associated proteinase functions are key determinants of inflammatory stromal tissues deconstruction. As a specialized inflammatory pathological process, dental internal resorption (IR) includes both soft and hard tissues deconstruction within the dentin-pulp complex, which has been one of the main reasons for inflammatory tooth loss. Mechanisms of inflammatory matrix degradation and tissue resorption in IR are largely unclear. In this study, we used a combination of Cre-loxP reporter, flow cytometry, cell transplantation, and enzyme activities assay to mechanistically investigate the role of regenerative cells, odontoblasts (ODs), in inflammatory mineral resorption and matrices degradation. We report that inflamed ODs have strong capabilities of matrix degradation and tissue resorption. Traditionally, ODs are regarded as hard-tissue regenerative cells; however, our data unexpectedly present ODs as a crucial population that participates in IR-associated tissue deconstruction. Specifically, we uncovered that nuclear factor-kappa b (NF-κB) signaling orchestrated Tumor necrosis factor α (TNF-α)-induced matrix metalloproteinases (Mmps) and Cathepsin K (Ctsk) functions in ODs to enhance matrix degradation and tissue resorption. Furthermore, TNF-α increases Rankl/Opg ratio in ODs via NF-κB signaling by impairing Opg expression but increasing Rankl level, which utterly makes ODs cell line 17IIA11 (A11) become Trap⁺ and Ctsk⁺ multinucleated cells to perform resorptive actions. Blocking of NF-κB signaling significantly rescues matrix degradation and resorptive functions of inflamed ODs via repressing vital inflammatory proteinases Mmps and Ctsk. Utterly, via utilizing NF-κB specific small molecule inhibitors we satisfactorily attenuated inflammatory ODs-associated human dental IR in vivo. Our data reveal the underlying mechanisms of inflammatory matrix degradation and resorption via proteinase activities in IR-related pathological conditions.
Humans ; Matrix Metalloproteinases/metabolism* ; Minerals/metabolism* ; NF-kappa B/metabolism* ; Odontoblasts/metabolism* ; Osteoclasts/metabolism* ; RANK Ligand/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

The clinical treatment of joint contracture due to immobilization remains difficult. The pathological changes of muscle tissue caused by immobilization-induced joint contracture include disuse skeletal muscle atrophy and skeletal muscle tissue fibrosis. The proteolytic pathways involved in disuse muscle atrophy include the ubiquitin-proteasome-dependent pathway, caspase system pathway, matrix metalloproteinase pathway, Ca-dependent pathway and autophagy-lysosomal pathway. The important biological processes involved in skeletal muscle fibrosis include intermuscular connective tissue thickening caused by transforming growth factor-β1 and an anaerobic environment within the skeletal muscle leading to the induction of hypoxia-inducible factor-1α. This article reviews the progress made in understanding the pathological processes involved in immobilization-induced muscle contracture and the currently available treatments. Understanding the mechanisms involved in immobilization-induced contracture of muscle tissue should facilitate the development of more effective treatment measures for the different mechanisms in the future.
Atrophy ; Autophagy ; Calcium ; metabolism ; Caspases ; metabolism ; Connective Tissue ; metabolism ; pathology ; Contracture ; etiology ; metabolism ; pathology ; therapy ; Fibrosis ; Humans ; Immobilization ; adverse effects ; Joints ; Lysosomes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Muscle, Skeletal ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Proteolysis ; Signal Transduction ; physiology ; Transforming Growth Factor beta1 ; metabolism ; Ubiquitin ; metabolism

Adult ; Diabetes, Gestational ; metabolism ; Female ; Humans ; Matrix Metalloproteinases ; metabolism ; Placenta ; enzymology ; Pregnancy

BackgroundCorneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.

MethodsADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.

Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).

Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

Adipose Tissue ; cytology ; Animals ; Cell Proliferation ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Exosomes ; metabolism ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rabbits

The suitable experimental animal model is important in research of pathogenesis and therapeutic strategies of diabetic foot ulcer, and the murine model is the most commonly used one at present. It can be divided into two types: the animal model simulating pathological conditions and the model simulating clinical symptoms. This article reviews the current research progress on the mechanisms of diabetic ulcer pathogenesis, and relevant treatment strategies, including the inhibition of matrix metalloproteinases (MMPs) expression, promotion of angiogenesis and anti-inflammatory therapy.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Diabetic Foot ; etiology ; genetics ; therapy ; Disease Models, Animal ; Humans ; Matrix Metalloproteinase Inhibitors ; therapeutic use ; Matrix Metalloproteinases ; genetics ; metabolism ; Mice ; Neovascularization, Physiologic ; physiology

BACKGROUND: N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. METHODS: Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10⁻¹², 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. RESULTS: Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. CONCLUSIONS: Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression.
Aggrecans ; Cell Death ; Collagen ; Collagen Type I ; Collagen Type II ; Diphosphonates ; DNA ; Down-Regulation ; Humans* ; In Vitro Techniques ; Intervertebral Disc* ; Matrix Metalloproteinases* ; Metabolism ; Osteoclasts ; Phenotype ; Proteoglycans ; Risedronate Sodium ; RNA, Messenger ; Starvation ; Tumor Necrosis Factor-alpha* ; Up-Regulation

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1β (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Chondrocytes ; cytology ; enzymology ; Gene Silencing ; Lentivirus ; genetics ; Matrix Metalloproteinases ; metabolism ; Rabbits ; Urokinase-Type Plasminogen Activator ; genetics

Chronic inflammation has been known to be a risk for many kinds of cancers, including pancreatic and biliary tract cancer. Recently, inflammatory process has emerged as a key mediator of cancer development and progression. Many efforts with experimental results have been given to identify the underlying mechanisms that contribute to inflammation-induced tumorigenesis. Diverse inflammatory pathways have been investigated and inhibitors for inflammation-related signaling pathways have been developed for cancer treatment. This review will summarize recent outcomes about this distinctive process in pancreatic and biliary tract cancer. Taking this evidence into consideration, modulation of inflammatory process will provide useful options for pancreatic and biliary tract cancer treatment.
Biliary Tract Neoplasms/*etiology/metabolism ; Cell Transformation, Neoplastic ; Cyclooxygenase 2/metabolism ; Cytokines/metabolism ; Humans ; *Inflammation ; Matrix Metalloproteinases/metabolism ; NF-kappa B/metabolism ; Pancreatic Neoplasms/*etiology/metabolism ; Receptor, Epidermal Growth Factor/metabolism

OBJECTIVETo investigate the effects of adenovirus-delivered angiopoietin-1 siRNA (Ad. Ang-1siRNA) on the expression of matrix metalloproteinase-2, 9 (MMP-2, 9) and tissue inhibitor of metallopro-teinase-1 (TIMP-1) in rats with acute lung injury (ALI) induced by phosgene (Psg).

METHODSWe first established a rat model of Psg-induced acute lung injury (ALI). The rats were randomly divided into 6 groups: air control group with exposure to air, air+adenovirus (air+Ad) group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after air exposure, air+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after air exposure, Psg group with exposure to 8.33 mg/L Psg (purity 100%, of the same volume as the inhaled air in the air control group) for 5 min, Psg+Ad group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after exposure to the same dose of Psg, and Psg+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after exposure to the same dose of Psg. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 36 h after exposure. The protein expression of Ang-1, MMP-2, 9, and TIMP-1 in serum and BALF was determined by double-antibody sandwich ELISA. RT-PCR was used to determine the mRNA levels of Ang-1, MMP-2, 9, and TIMP-1 in lung tissue. The protein expression of MMP-2, 9 and TIMP-1 in lung tissue was determined by Western blot.

RESULTSA rat model of Psg-induced ALI was successfully established. The levels of MMP-2, 9 in serum, BALF, and lung tissue were significantly increased in the Psg group and Psg+Ad/Ang1 group as compared with the control group (P<0.01); no significant change was observed in serum TIMP-1 protein expression (P>0.05); interestingly, TIMP-1 protein expression in BALF and lung tissue was significantly increased (P<0.01). Compared with the Psg group, the Psg+Ad/Ang1 group showed a significant decrease in MMP-2, 9 expression in BALF, serum, and lung tissue (P<0.05), but no significant change in protein expression of TIMP-1 was discovered (P>0.05).

CONCLUSIONAd.Ang-1siRNA has a potential beneficial effect in rats with Psg-induced ALI through inhibition of MMP-2, 9 expression, but has no significant effect on the expression of TIMP-1.

Acute Lung Injury ; chemically induced ; metabolism ; Adenoviridae ; genetics ; Angiopoietin-1 ; physiology ; Animals ; Bronchoalveolar Lavage Fluid ; Chemical Warfare Agents ; toxicity ; Disease Models, Animal ; Lung ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; Matrix Metalloproteinases ; metabolism ; Phosgene ; toxicity ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism