Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion:In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

Polymorphism of MMP-3 gene and imbalance expression of MMP-3 / TIMP-1 in articular cartilage are associated with an endemic osteochondropathy, Kashin- Beck disease

Background The etiology of Kashin-Beck disease (KBD), an endemic osteochondropathy, is largely unknown. Matrix metalloproteinase-3 (MMP-3) plays a central role in the initiation and progression of cartilage destruction, however, no study has reported on the relationship between KBD and MMP-3. The objective of this study was to explore the polymorphism of MMP-3 gene and expression of MMP-3 / TIMP-1(Tissue inhibitors of matrixmetalloproteinases-1) in the pathogenesis of KBD. Methods Single nucleotide polymorphism (SNP) genotyping was conducted in 274 KBD cases and 248 healthy controls for eight SNPs in MMP-3 using the Sequenom MassARRAY system. Additionally, the expression of MMP-3、TIMP-1 in different layers of the articular cartilage was analyzed by immunohistochemistry for 22 KBD patients, 15 osteoarthritis (OA) patients and 21 controls. Results The results showed that six SNPs (rs520540、rs591058、rs679620、rs602128、rs639752 and rs678815) in MMP-3 were associated with the increased risk of KBD, however, after Bonferroni correction, only the SNP rs679620 in the recessive model remained significant difference (OR = 2.31, 95%CI = 1.29–4.14, P = 0.0039), homozygous for “T” allele have a risk for KBD than “C” allele carriers. Moreover, the percentages of cells expressing MMP-3 in articular cartilage were significantly higher in the KBD and OA groups than in the controls (t = 5.37 and 4.19, P<0.01). While the KBD and OA groups had lower levels of TIMP-1 positive staining compared with the controls (t = 5.23and 5.06, P<0.01). And there was no significant different between KBD and OA for the levels of MMP-3 and TIMP-1 positive staining (t = 0.05and 0.28, P>0.05). Conclusions MMP-3 is associated with the susceptibility of KBD, and the imbalance expression of MMPs / TIMPs leading to cartilage degradation may play an important role in cartilage degradation and osteoarthritis formation in OA and KBD.

An insight into anti-arthritic property of phytochemicals against Rheumatoid arthritis using molecular modelling and docking approach

Arthritis literally refers “joint inflammation”, it is a condition where one or more joints are inflamed. More than 100 different types of Arthritis were identified, most common types are rheumatoid arthritis and osteoarthritis. The present study mainly focuses on the development of the novel phytochemical inhibitors against rheumatoid arthritis and osteoarthritis using an integrative cheminformatics drug discovery platform. In this study, we identified potential 405 phytochemical drug candidates, screened against eight selected targets of rheumatoid arthritis and osteoarthritis using molecular docking tool AutoDock. Three phytochemicals Withanolide, Diosgenin and bamyrin exhibited promising binding towards multiple drug targets selected for this study. When comparing with the binding between reference drugs, withanolide showed highest activity against Interleukin-23, Matrix metalloproteinase-3 and Interleukin 8 with binding energies -11.6, -9.4 and -8.3 kcal/mol respectively. Diosgenin also exhibited best activity against three targets that were Interleukin-23, JNK alpha and MMP-3 with -11.3, -10.4, -9.5 kcal/mol binding energies respectively. This study may be important contributing factor to develop new therapeutic drugs for rheumatoid arthritis and osteoarthritis.

Aspirin Exerts Neuroprotective Effects by Reversing Lipopolysaccharide-Induced Secondary Brain Injury and Inhibiting Matrix Metalloproteinase-3 Gene Expression

Objective. This study is aimed at exploring the possible neuroprotective mechanism of aspirin and the effect of bacterial endotoxin lipopolysaccharide (LPS) during cerebral ischaemia-reperfusion (CIRP) injury. Methods. We established three animal models: the CIRP, LPS, and CIRP+LPS models. Mortality, the injured brain area, and the beam walking test were used to estimate the degree of cerebral injury among the rats. Immunohistochemistry and immunofluorescence were used to detect activated microglia, matrix metalloproteinase-3 (MMP-3), and osteopontin (OPN). Results. The injured brain area and mortality were dramatically reduced ( p < 0.01 ), and the beam walking test scores were elevated ( p < 0.01 ) in the acetylsalicylic acid (ASA) group compared to the control group. The number of microglia-, MMP-3-, and OPN-positive cells also increased. Furthermore, the number of GSI-B4, OPN, and MMP-3 cells decreased in the ASA group compared to the control group. After LPS stimulation, the number of microglia reached a peak at 24 h; at 7 d, these cells disappeared. In the ASA group, the number of microglia was significantly smaller ( p < 0.05 ), especially at 24 h ( p < 0.01 ), compared to the LPS group. Moreover, the injured brain area and the mortality were dramatically increased and the beam walking test scores were reduced ( p < 0.01 ) after LPS simulation following CIRP. The degree of injury in the ASA group resembled that in the control group. However, the number of MMP-3-immunoreactive neurons or microglia was significantly larger than that of the control group ( p < 0.05 ). In the ASA group, the MMP-3 expression was also considerably decreased ( p < 0.05 ). Conclusions. After CIRP, microglia were rapidly activated and the expression of MMP-3 and OPN significantly increased. For rats injected with LPS at reperfusion, the injured brain area and mortality also dramatically increased and the neurologic impairment worsened. However, ASA exhibited a neuroprotective effect during CIRP injury. Furthermore, ASA can reverse LPS-induced cerebral injury and inhibit the inflammatory reaction after CIRP injury.

Dissecting the Molecular Mechanism of Wang-Bi Capsule in the Treatment of Experimental Rheumatoid Arthritis Based on Synovial Tissue Proteomic Analysis

Wang-Bi capsule (WB) is a traditional Chinese medicine formula and has been applied for rheumatoid arthritis (RA) treatment for many years. However, its underlying molecular mechanisms still remain unclear. In this study, collagen-induced arthritis (CIA) rats were used to observe the therapeutic effect of WB used at different time points, and the proteomic analysis of synovial tissue was applied to reveal its basic molecular mechanisms. The results demonstrated that WB not only effectively ameliorated the symptoms and synovitis, but also downregulated the serum levels of inflammatory cytokines/chemokines in CIA rats. Furthermore, the proteomic analysis of synovial tissue showed that WB could regulate several signaling pathways associated with inflammation or cell migration, such as “IL-1 signaling,” “IL-8 signaling,” and “CXCR4 signaling.” The expression levels of proteins including matrix metalloproteinase 3 (MMP3), MMP19, lipopolysaccharide-binding protein (LBP), serine/threonine kinase interleukin-1 receptor-associated kinase 4 (IRAK4), and actin-related protein 2/3 complex subunit 5 (ARPC5) in these pathways were downregulated significantly by WB when compared with the model group. In sum, this study indicated that WB had obvious inhibitory effects on synovitis of CIA rats, and the mechanisms of which may be involved in downregulating the expression levels of several key proteins including MMP3, MMP19, LBP, IRAK4, and ARPC5.

Adenosine Inhibits TNFα-Induced MMP-3 Production in MH7A RA Synoviocytes via A2A Receptor Signaling

Adenosine is the effector molecule; however, the contributions of synoviocyte adenosine receptors (AdoRs) are unknown, and matrix metalloproteinase 3 (MMP-3) is released by fibroblast-like synoviocytes in response to inflammatory signaling. To elucidate the therapeutic mechanisms of methotrexate, we investigated the effects of A2A AdoR activation and inhibition on tumor necrosis factor-alpha (TNFa)-induced MMP-3 release by MH7A human rheumatoid synovial cells. MH7A cells constitutively expressed membrane-associated A2A AdoRs, and HENECA enhanced intracellular cAMP. Stimulation with TNFa markedly enhanced release of MMP-3 from MH7A cells, whereas HENECA partially and dose-dependently inhibited TNFa-evoked MMP-3 release. Similarly, dbcAMP partially inhibited TNFa-induced MMP-3 release. Pretreatment with ZM241385 reversed the inhibitory effects of HENECA. Further, TNFa induced p38 MAPK and ATF-2 phosphorylation, whereas HENECA suppressed p38 MAPK and ATF-2 phosphorylation. We concluded that adenosine signaling via A2A AdoRs, adenylyl cyclase, and cAMP reduces TNFa-induced MMP-3 production by interfering with p38 MAPK/ATF-2 activity. Activation of A2A AdoR pathway and suppression of MMP-3 release may explain the antirheumatic effects of methotrexate.

Comparative effectiveness of two methods for inducing osteoarthritis in a novel animal model, the Diannan small-ear pig

Background Varieties of animals were used to study osteoarthritis pathogenesis. The Diannan small-ear pig, which is native to Yunnan, China, is thought to have an articular anatomy similar to that of humans and is more likely to be a source of pathological tissues than other animals. The aim of this study was to determine whether this animal can serve as a more effective osteoarthritis model and explore the role of SDF-1/CXCR4 signaling pathway in the development of Osteoarthritis in animals. Methods Twenty-seven adult pigs were randomly divided into three groups and underwent the Hulth procedure, papain articular injection, and conventional breeding. After 4, 8, and 12 weeks, cartilage tissues from knee joint were extracted for general and histological observation, immunofluorescence, and biochemical analysis. Synovium was taken out for stromal cell-derived factor-1 analysis. Results Histopathological observation showed obvious cartilage loss in two experimental groups, this cartilage loss was more severe in the chemical groups. Synovial stromal cell-derived factor1 levels increased over time in all groups. mRNA and protein levels of matrix metalloproteinase-3 were much higher in the chemical groups than in the other groups, whereas levels of collagen type II and aggrecan were significantly lower in the chemical groups than in the other groups. Immunofluorescence assays of collagen type II revealed an apparent reduction in this marker in the chemical groups compared with the other groups. Conclusions These results indicated that the Diannan small-ear pig can be used as an effective osteoarthritis model. In addition, it is much more convenient and much faster to induce osteoarthritis by intra-articular injection of papain, which is a method worthy of being promoted.