骨性关节炎生物学标志物研究进展

骨性关节炎(oseteoarthritis,OA)是一种随着年龄增长发病率明显升高的退行性变,常累及脊柱、髋、膝等人体负重关节,以关节缓慢发展的疼痛、肿胀,伴功能障碍为临床表现,主要有滑膜增生、软骨破坏、软骨下骨骨化及骨赘形成等一系列病理表现。OA对人类的健康和生活质量影响很大,随着老龄化社会的到来,本病的发病率日趋升高,其研究已成为医学领域中的重要课题。目前,OA的早期诊断、病变监测和有效防治仍是骨科领域亟待解决的疑难问题。随着分子生物学的发展和研究手段的提高,许多研究者都在试图寻找用于临床评价OA的生物学标志物。本文将就OA研究中所使用的主要标志物进行综述,为深入研究OA提供方便。     

Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis

Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5.% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecul sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker β and α collagen chains. The molecular weight of this collagen was estimated to be 150000. Based on incorporation of [¹⁴C]proline, its ratio of hydroxy[¹⁴C]proline to total ¹⁴C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3,4°C, 16 h) of degenerative cartilage samples.Since the total collagen content (μg hydroxyproline/mg cartilage), hydroxy[¹⁴C]proline/mg cartilage, specific radioactivity of hydroxy[¹⁴C]proline (cpm/μg), in the whole cartilage, and the specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.     

Application of mesenchymal stem cell therapy for the treatment of osteoarthritis of the knee: A concise review

Osteoarthritis(OA) refers to a chronic joint disease characterized by degenerative changes of articular cartilage and secondary bone hyperplasia. Since articular cartilage has a special structure, namely the absence of blood vessels as well as the low conversion rate of chondrocytes in the cartilage matrix, the treatment faces numerous clinical challenges. Traditional OA treatment(e.g., arthroscopic debridement, microfracture, autologous or allogeneic cartilage transplantation,chondrocyte transplantation) is primarily symptomatic treatment and pain management, which cannot contribute to regenerating degenerated cartilage or reducing joint inflammation. Also, the generated mixed fibrous cartilage tissue is not the same as natural hyaline cartilage. Mesenchymal stem cells(MSCs) have turned into the most extensively explored new therapeutic drugs in cell-based OA treatment as a result of their ability to differentiate into chondrocytes and their immunomodulatory properties. In this study, the preliminary results of preclinical(OA animal model)/clinical trials regarding the effects of MSCs on cartilage repair of knee joints are briefly summarized, which lay a solid application basis for more and deeper clinical studies on cell-based OA treatment.     

Proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (Nampt) involve regulation of insulin signaling pathway and Nampt enzymatic activity

Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E(2) (PGE(2)) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)(3)) inhibitor diminished visfatin-induced PGE(2) release in chondrocytes. Moreover, visfatin-induced IGF-1R(-/-) chondrocytes released higher concentration of PGE(2) than IGF-1R(+/+) cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE(2) release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE(2) release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity.     

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

关节镜下髁间窝成形术治疗髁间窝撞击综合征病人膝关节屈伸功能受限

目的:对髁间窝撞击综合征致膝关节屈伸功能受限患者关节镜下髁间窝成形手术疗效的探讨。方法:2008年2月-2009年7月选择关节镜下确诊骨性关节炎伴髁间窝撞击综合征病人45例,对所有患者行关节镜下髁间窝扩大成形术。结果:术后患者膝关节屈伸功能明显改进,对其中42例患者进行术后6个月随访,发现患者膝关节疼痛明显缓解或消失,关节屈伸功能较术前得到明显改善。术前Lysholm评分平均(48.7±13.1)分(差),术后平均(87.0±7.3()良),关节功能均获明显改善(P<0.001)。结论:髁间窝撞击综合征是导致该病患者膝关节疼痛及屈伸功能受限的主要原因,关节镜下髁间窝扩大成形术对该病有明显的疗效。     

Cyclooxygenase inhibition by diclofenac formulated in bioadhesive carriers

Adverse effects and gastrointestinal toxicity limit the use of Diclofenac, a frequently-used NSAID for treatments of rheumatic disorders and other chronic inflammatory diseases. Diclofenac-carrier formulations may alleviate adverse effects, increase efficacy and allow local administration. We report here our first step, biophysical and biochemical investigations of Diclofenac formulated in our previously-developed bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) on their surface. Both liposome types encapsulated Diclofenac at high efficiency, encapsulated doses reaching 13mg drug/ml, and performed as sustained-release Diclofenac depots, half-lives of drug release (under fastest conditions) ranging from 1 to 3days. Therapeutic activity of liposomal Diclofenac was evaluated in CT-26 cells that possess the CD44 hyaluronan receptors and integrins, and are a bench-mark for intracellular COX enzymes. HA-BAL and COL-BAL showed high cellular-affinity that was 40 fold and 6 fold over that of regular liposomes. Free, and liposome-encapsulated, Diclofenac showed similar activities. For example: 2-3nM Diclofenac given to intact cells generated COX-inhibition levels in the range of 60-70% for free drug and for encapsulated drug in COL-BAL and in HA-BAL. We propose these novel Diclofenac formulations possess key physicochemical and biochemical attributes for task performance, meriting the next step into in vivo studies.     

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF‐κB signalling pathway

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF‐κB signalling and reduced the production of pro‐inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF‐κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti‐inflammatory effect of PAB and rescue the activation of NF‐κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF‐κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.     

Assessment of biomechanical properties of the extracellular and pericellular matrix and their interconnection throughout the course of osteoarthritis

During osteoarthritis (OA)-triggered cartilage degeneration, the chondrocytes spatially rearrange from single to double strings, and then to small and finally big clusters. Both the extracellular matrix (ECM) and the pericellular matrix (PCM) progressively degrade in osteoarthritis, changing the overall mechanical properties of the cartilage. We investigated the mechanical properties particularly elasticity of the ECM and PCM and their interconnection as a function of chondrocyte spatial organisation.Human articular cartilage samples from 30 patients were categorised according to their cellular pattern. Elasticity of the ECM and PCM was assessed by means of atomic force microscopy (AFM). Significant decreases were observed in the elasticity of both the ECM and the PCM with each change of cellular pattern, except from single to double strings in the ECM (p = 0.072). Spatial reorganisation strongly correlated with the elasticity of the ECM (r = −0.768, p < 0.001) and of the PCM (r = −0.729, p < 0.001). The ECM/PCM ratio remained unchanged (r = −0.099, p = 0.281).This study is the first to describe and quantify the differences in the elastic moduli of the ECM in relation to the PCM on the basis of chondrocyte spatial arrangement. This study shows that the elastic changes of the ECM and the PCM occur simultaneously, unidirectionally, and to a comparable degree.     

Discovery of potent,selective, bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model,Part I: Transformation of selective pyrazolodiazepinone phosphodiesterase 4 (PDE4) inhibitors into selective PDE2 inhibitors

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition.