The Effects And Mechanisms Of Platelet-derived Growth Factor-BB On Osteoarthritis

Objective: Osteoarthritis is a common joint disease that mainly affects the elders.Treatments for osteoarthritis include physical treatment and rehabilitation,topical and oral drugs therapies,intra-articular drugs therapies and joint surgery.However,all the treatments mentioned above mainly aim to control the symptoms instead of improving or reversing the joint condition.Platelet-derived growth factor(PDGF)-BB,consisting of two PDGF-B monomers,is the one of the effective constituents in platelet-rich plasma.Previous studies have found that PDGF-BB can promote cell proliferation,cell chemotaxis,tissue remodeling,angiogenesis and other functions,so as to promote the healing of injured tissues.The purpose of this study was to investigate the effect and mechanism of PDGF-BB in the rat osteoarthritis model induced by sodium iodoacetate.Methods:(1)Cell culture and treatment: the knee cartilage of seven-day old SpragueDawley rats was used to culture chondrocytes in vitro.In vitro chondrocytes were grouped and treated with different concentrations of PDGF-BB for inflammation induction.PDGF receptor inhibitors,PKA inhibitors,and SOX-9 si RNA intervention were added according to the grouping.(2)Intervention of animal models: Sprague-Dawley rats aged 8-12 weeks were injected with MIA into the unilateral joint cavity for osteoarthritis model induction.After the successful modeling was identified by radiography at 2-3 weeks,different concentrations of PDGFBB were used for treatment,and PDGF receptor inhibitors and PKA inhibitors were added for intervention according to the grouping.(3)Histological staining and scoring: Haematoxylin & eosin staining and toluidine blue staining were performed on isolated rat knee joints.The Osteoarthritis Research Society International scoring system and Mankin scoring system were used to score the stained tissue sections.(4)Cell activity and NO level detection: CCK-8 kit was used to detect the activity of cells in vitro.At the same time,Griess reaction was used to detect NO levels in cell medium in vitro.(5)Detection of inflammatory factors and metabolic factors: Enzyme linked immunosorbent assay was used to detect inflammatory cytokines in chondrocytes in vitro and in vivo.Meanwhile,real-time quantitative reverse transcription polymerase chain reaction was used to detect inflammatory cytokines metabolic factors.In addition,the metabolic factors of cartilage in vitro and in vivo were detected by immunohistochemistry and immunofluorescence staining.(6)Detection of apoptosis and cell cycle of chondrocytes: Flow cytometry was used to detect the apoptosis level and cell cycle of chondrocytes in vitro.Meanwhile,Td T-mediated d UTP Nick End Labeling was used to detect the apoptosis level of chondrocytes in vivo.(7)Detection of signaling molecules in chondrocytes: Western blot was used to detect the signal molecules.The signaling molecules mentioned above were also detected by immunofluorescence staining of chondrocyte slides and knee tissue slices.Results: PDGF-BB was found to exhibit anti-inflammatory effect,proliferation promoting effect,anti-apoptosis effect,promoting anabolism,and inhibiting catabolism in osteoarthritis cartilage.Besides,PDGF-BB was found to inhibiting joint cartilage degradation,decreasing cartilage hyperplasia and osteophyte formation,and promoting the angiogenesis in subchondral bone in osteoarthritis rat model.As for the mechanisms,PDGFBB was found to increase the phosphorylation of Erk1/2 and decrease the phosphorylation of p38 resulting in the upregulating of Bax downregulation of caspase-3 to decrease the apoptosis of chondrocyte.Meanwhile,PDGF-BB could decrease the activation of JAK2/STAT3,PI3K/AKT,and p38/Run X-2 and increase the activation of protein kinase A/SOX-9 pathway.Besides,the crosstalk was also discovered between transcription factor SOX-9 and Run X-2.One of the possible downstream of these signal pathways was found to be the regulation of CCAAT enhancer binding protein δ and β.Conclusion: PDGF-BB upregulated Erk phosphorylation and PKA/SOX-9 signaling pathway,inhibited caspase-3-dependent apoptosis,and inhibited JAK2/STAT3,PI3K/AKT,and p38/Run X-2 signaling pathways to exhibit chondroprotective effects in osteoarthritis chondrocytes.PDGF-BB articular injection has the potential to be a conservative treatment for osteoarthritis.