The Emerging Role And Mechanisms Of Paeoniflorin In Osteoarthritis

Osteoarthritis(osteoarthritis,OA)is a common orthopedic and frequently-occurring disease.The cause of OA is still unknown.The most typical pathological features of OA are apoptosis of chondrocyte,destruction of cartilage matrix and ossification of subchondral bone.In the progression of OA,the imbalance between catabolic and anabolic mechanism of cartilage remodeling caused erosion of articular cartilage and extracellular matrix degradation.A number of drugs including non-steroidal anti-inflammatory drugs(NSAIDs),glucosamine,steroids and hyaluronic acid(HA)were used for the treatment of early stage of OA.However,these methods can only alleviate the pain and easily to bring the side effects of digestive and cardiovascular system.Paeoniflorin,a pinane monoterpene glucoside found,was first isolated from the Ranunculaceae plant.Paeoniflorin has been demonstrated to possess various pharmacological effects,such as widen blood vessels,analgesia,anti-inflammatory,anti-ulcer,anti-free radical oxidative damage,antipyretic and antispasmodic in clinical application.A recent research indicated that a novel ester derivative of paeoniflorin,called CP-25 can decrease the production of pro-inflammatory cytokines(IL-1?,IL-6,IL-17 and TNF-a)as well as up-regulate the expression of anti-inflammatory cytokine TGF-?1 in the serum in adjuvant-induced arthritis model.Paeoniflorin was also demonstrated to inhibit the apoptosis of nucleus pulposus cells and the activation of Caspase-3 and Caspase-9 through the regulation of Bcl-2 family protein expression.To date,nothing is known about the role of paeoniflorin in OA.In the current study,we will assess the effects of PF in the pathophysiology of OA and find out related mechanisms.The present study included 3 parts.Firstly,we investigated the effects of PF the expression of MMP-1,MMP-3,MMP-13 and TIMP-1 in IL-1?-induced rat chondrocytes.We found that PF down-regulated both the mRNA and protein expressions of MMP-1,MMP-3 and MMP-13,as well as increased the level of TIMP-1 in a dose-dependent manner.Furthermore,paeoniflorin suppressed the nuclear factor kappa B(NF-?B)signaling pathway by inhibiting the degradation of I?B-a.Secondly,we investigated the effects of different concentration of PF on the expression of Bcl-2,Bax and Caspase3 activity in IL-1?-induced rat chondrocytes.We found that PF exerted an anti-apoptosis role in IL-1?-induced rat chondrocytes by inhibiting the expression of Bax and reducing the Caspase-3 activity as well as increasing the expression of Bcl-2.We also initiated that PF inhibited IL-1?-induced apoptosis partially by Akt signaling pathway.Finally,we investigated the in vivo effects of PF in in rabbit model of OA induced by anterior cruciate ligament transection(ACLT).We found PF reduced the IL-1?-induced catabolic factors expression and proteoglycan(PG)degeneration.All the results demonstrate that PF could protect articular cartilage by anti-degeneration and anti-apoptosis in the pathophysiology of OAPart1 The effects of PF on MMPs and TIMP-1 expression in IL-1?-induced rat chondrocytesObjective:To investigate the effects of PF on the expression of MMP-1,MMP-3,MMP-13 and TIMP-1 in IL-1?-induced rat chondrocytes and its molecular mechanismsMethods:Rat articular chondrocytes for primary culture were isolated from the knee joints of 4-week-old SD rats.Rat chondrocytes viability and the cytotoxicity were examined by MTT after treated with different concentrations of PF(12.5-200?M)for 48 hours.Chondrocytes were pretreated with various concentrations of PF for 3 h and then treatment with IL-1?(10ng/ml)for 24 h.Expression levels of MMP-1,MMP-3,MMP-13 and TIMP-1 were examined by real-time PCR and western blot.The NF-?B p65 and inhibitor of nuclear factor kappa B-a(I?B-?)from rat chondrocytes were examined by Western blot.Results:PF has no significant cytotoxic effect at 12.5-200?M following incubation for 48 hours.PF decreased both the mRNA and protein expressions of MMP-1,MMP-3 and MMP-13,as well as increased the level of TIMP-1 in a dose-dependent manner.PF also suppressed the nuclear factor kappa B(NF-?B)signaling pathway by inhibiting the degradation of I?B-?.Conclusions:PF has no cytotoxic effect on chondrocytes at 12.5-200?M level.PF plays anti-catabolic effect in chondrocyte metabolism by suppressing the NF-?B signaling pathway.Part 2 The effects of PF on cell apoptosis in IL-1?-induced rat chondrocytesObjective:To investigate the effects of PF on apoptosis of rat chondrocytes and its potential molecular mechanismsMethods: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 PF for 3 h and then treatment with IL-1?(10ng/ml)for 24 h.The LDH release rate of cells was determined by LDH release assay kit.Annexin V-FITC and PI staining were used to detect the early and advanced apoptotic cell by flow-cytometry analysis.The activity of Caspase-3 in chondrocytes was determined using Caspase-3 activity assay kits.Expression levels of Bcl-2 and Bax were examined by real-time PCR and western blot.The Akt and p-Akt from rat chondrocytes were examined by western blot.Results:PF(25 and 50?M)significantly decreased the release of LDH and ratio of apoptotic cell in a dose-dependent manner IL-1?-induced rat chondrocytes.PF decreased both the mRNA and protein expressions of Bax,as well as increased the level of Bcl-2.PF also reduced the activity of Caspase-3 in chondrocytes.Furthermore,PF can regulate the Akt signaling pathway by increasing the phosphorylation of Akt.Conclusions:PF exert its potential protective role by anti-apoptosis in IL-1?-induced rat chondrocytes.Part 3 The in vivo effects of PF in experimental osteoarthritis in rabbitsObjective:To investigate the protective effects of PF in a rabbit experimental model of osteoarthritis(OA).Methods:New Zealand rabbits underwent anterior cruciate ligament transection(ACLT)on knee joint to induce OA.All the rabbits were randomly divided into four groups:(1)Normal group:health rabbits without operation;(2)Control group:intra-articular injection with 0.3 ml DMSO;(3)Low-dose PF group:intra-articular injection with 0.3 ml25?M PF;(4)High-dose PF group:intra-articular injection with 0.3 ml 50?M PF.The injection was carried out weekly four weeks after surgery.All rabbits in each group were sacrificed 9 weeks after the injection.The articular cartilages were evaluated by Safranin O-fast green staining.The gene expression of MMP-1,MMP-3,MMP-13 and TIMP-1 were analyzed by real-time PCR.Results:Assessed by the modified Mankin score and Safranin O-fast green staining,PF slightly decreased cartilage degradation compared with control group in vivo.PF decreased the mRNA expressions of MMP-1,MMP-3 and MMP-13,as well as increased the level of TIMP-1.Conclusions:PF inhibited articular cartilage degeneration in vivo,which was consistent with our previous study in vitro.