The Role And Corresponding Molecular Mechanisms Of Vaspin In Osteoarthritis

Osteoarthritis (OA) is a chronic cartilage and joint disease among elderly individuals, primarily characterized by articular cartilage breakdown, osteophyte formation, subchondral sclerosis and synovium alterations. Age, gender, injury and obesity are considered to be major risk factors for the progression of OA. Accumulating evidence demonstrated that obesity exert pro-inflammatory. In the past, adipose tissue was considered as energy storage, while this tissue was known as endocrine organ recently. Obesity-related factors especial these adipokines play a role of cartilage matrix degradation and subchondral bone remodeling by inducing pro-inflammatory and catabolic factors during the pathophysiology of OA. In the present study, we investigate the role of specific adipokine in the OA progression, and also the corresponding molecular mechanisms, which can provide potential therapeutic target for OA.Many researchers found that the (Synovial fluid, SF) levels of most adipokines raised in obesity patients and related to the OA progress. And these adipokines could significantly increase the levels of pro-inflammatory factor-1β (interleukin-1 beta, IL-1β) and also the catabolic factors including matrix metalloproteinases (MMPs) and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS), which cause cartilage degeneration. However, the newly found adipokine visceral adipose tissue-derived serine protease inhibitor (vaspin) played an opposite role with other adipokines. Recently, researchers found that vaspin could play inhibitory roles on inflammatory states of vascular smooth muscle cells. Another study demonstrated vaspin downregulated MMP-9 and cathepsin K expression in the osteoclast precursors. Besides, vaspin could also play a anti-inflammatory role in rat adipose tissue by inhibiting the expressions of resistin, leptin and tumor necrosis factor-a (TNF-a). The role and corresponding molecular mechanisms of vaspin in OA was still absent. In the current study, we will assess the effects of vaspin in the pathophysiology of OA and find out related mechanisms.There are four parts in the present study. Firstly, we investigated the expression of vaspin in the OA joint tissues, and also the distribution between paired serum in OA patients and in healthy controls. We found that all the joint tissues from OA patients expressed vaspin gene, and the expression of vaspin protein was more abundant in the degeneration area. Furthermore, serum vaspin was reduced in OA patients compared to healthy controls, which suggested that vaspin was related to the OA progress. Secondly, we investigated the effects of different concentration of vaspin on the expression of MMPs, ADAMTS, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS) in IL-1β-induced rat chondrocytes. We found that vaspin inhibited IL-1β-induced production of catabolic factors MMP-2, MMP-9 and ADAMTS-5, and inflammatory mediators COX-2, PGE2 and iNOS in rat chondrocytes. Thirdly, we investigated the corresponding molecular mechanisms of vaspin in rat chondrocytes. We found that vaspin inhibited IL-1β-induced inflammatory partially by suppressing the nuclear factor kappa B (NF-κB) signaling pathway. Finally, we investigated the in vivo effects of vaspin in the rat articular cartilage. We found vaspin reduced the IL-1β-induced catabolic factors expression and proteoglycan (PG) degeneration. All the results demonstrate that vaspin could protect articular cartilgae and inhibit OA progress.Part 1 Expression of vaspin in OA patients and healthy controlsObjective:To investigate the expression of vaspin in the knee joint, and to investigate different expression of vaspin in OA patients and healthy controls.Methods:Specimens were obtained from patients underwent total knee arthroplasty (TKA). The gene expression of vaspin was measured by quantitative real-time polymerase chain reaction (qPCR) in the OA joint tissues including cartilage, synovium, meniscus, infrapatellar fat pad and osteophyte. The vaspin protein expression in the cartilage, synovium and osteophyte were detected by immunohistochemistry (IHC). Levels of vaspin in serum were analyzed by enzyme-linked immunosorbent assay (ELISA) in 23 healthy controls and 26 OA patients.Results:All the joint tissues including cartilage, synovium, meniscus, infrapatellar fat pad and osteophyte from OA patients expressed vaspin messenger ribonucleicacid (mRNA), and the expression of vaspin protein was observed in OA cartilage, synovium and osteophyte. Furthermore, serum vaspin was reduced in OA patients compared to healthy controls.Conclusions:These results suggest that all the joint tissues expressed vaspin gene, and vaspin may be involved in the pathophysiology of OA and may have local effects in the joint during the process of OA.Part 2 Vapsin play anti-inflammatory and anti-catabolic in rat chondrocytesObjective:To research the cell viability and the cytotoxicity of vaspin in chondorcytes. To investigate the anti-inflammatory and anti-catabolic role of vaspin in IL-1β-induced rat chondrocytes.Methods:Rat articular chondrocytes for primary culture were isolated from the knee joints of 4-week-old Sprague-Dawley (SD) rats, and the cultured chondrocytes at passage 3 were used to avoid loss of phenotype. Rat chondrocytes viability and the cytotoxicity were examined by CCK-8 after treated with different concentrations of vaspin (10-500 ng/ml) for 24 and 48 hours. Rat condrocytes were treated with different concentrations of vaspin in the absence or presence of IL-1β. The gene expression of MMP-2, MMP-9, ADAMTS-4 and ADAMTS-5 were examined by qPCR, as well as the protein production of COX-2, PGE2 and iNOS were examined by ELISA.Results:Vaspin was not able to stimulate the proliferation of chondrocytes and no significant cytotoxic effect at 10-500 ng/ml following coincubation for 24 and 48 hours. Vaspin inhibited IL-1β-induced the gene expression of MMP-2, MMP-9 and ADAMTS-5 and also the protein production of COX-2, PGE2 and iNOS in rat chondrocytes.Conclusions:Vaspin has no cytotoxic effect on chondrocytes at 500 ng/ml level. Vaspin plays anti-inflammatory and anti-catabolic effect in chondrocyte metabolism.Part 3 Vapsin inhibit IL-1β-induced inflammatory and catabolic responses in rat chondrocytes and related mechanismsObjective:To investigate the role and corresponding molecular mechanisms of vaspin in IL-1β-induced inflammatory and catabolic responses in rat chondrocytes.Methods:Rat articular chondrocytes for primary culture were isolated from the knee joints of 4-week-old SD rats. Chondrocytes were pretreated with various concentrations of vaspin for 1 h prior to treatment with IL-1β (10 ng/ml) for 24 h. The p-NF-κB and inhibitor of nuclear factor kappa B-α (IκB-α) from rat chondrocytes were examined by Western blot. The effects of inhibitor of NF-κB BAY 11-7082 on COX-2, PGE2 and iNOS were investigated by ELISA.Results:Vaspin inhibited IL-1βp-induced p-NF-κB and degeneration of IκB-α in the rat chondrocytes. The inhibitor of NF-κB BAY11-7082 inhibited the IL-1β-induced production of COX-2, PGE2 and iNOS in the culture medium.Conclusions:Vaspin inhibited IL-1β-induced inflammatory and catabolic effect in chondrocyte metabolism, which partially by suppressing the NF-κB signaling pathway.Part 4 Vapsin inhibit IL-1β-induced catabolic role on rat articular cartilageObjective:To investigate the role of vaspin on the IL-1β-induced catabolic in rat articular cartilage.Methods:Forty 3 months male SD rats (250 g) were used in this study. Randomly divided into 4 groups, which were intra-articular injected with sterile saline as controls group, 50 μg IL-1β group,50 μg vaspin group and 50 μg IL-1β+50 μg vaspin group. All rats were sacrificed 48 h after intra-articular injection. The gene expression of MMP-2, MMP-9, ADAMTS-4 and ADAMTS-5 was analyzed by qPCR. The tibial plateaus were also collected and the PG content in articular cartilage were evaluated by Safranin O-fast green stainingResults:Vaspin inhibited IL-1β-induced the gene expression of MMP-2, MMP-9 and ADAMTS-5 in rat articular cartilage. Vaspin alone had no effect on the metabolism of PG, while the PG were degraded by IL-1β in full thickness. Vaspin inhibited IL-1β-induced PG depletion in the articular cartilage.Conclusions:Vaspin inhibited IL-1β-induced articular cartilage degeneration in vivo, which will provide new ideas and approaches to osteoarthritis prevention and treatment.