Study On The Protective Effect And Mechanism Of Garlic-derived S-allylmercaptocysteine(SAMC)in Osteoarthritis

BackgroundOsteoarthritis(OA)is a chronic and progressive joint disorder,which has a higher rate of morbidity in female than male.It leads to severe chronic pain,loss of mobility and disability,which affecting millions of people worldwide.It characterized by structural damage to one or more joints,the mainly pathogenic sites are finger joints,knee,hip,and spine.OA is manifested as non-inflammatory degeneration of articular cartilage and formation of joint marginal osteophyte,which usually results from aging and other reasons such as trauma,congenital anomaly,and deformity of joint.The treatment for OA including lifestyle change to decrease weight-bearing,pharmacological interventions(corticosteroids and non-steroidal anti-inflammatory drugs(NSAIDs),and surgical intervention(osteotomy or joint prosthesis).These therapeutic approaches may give relief to patient,but the treatment effects are limited.For this reason,it is necessary to explore useful medicine or drugs for OA treatment.Garlic and its derivatives have widely been used in many therapies,such as liver injury,renal damage,cardio-protection,cancer chemoprevention and osteoarthritis.Organosulfur compounds(OSCs)are the major active components in garlic,which including the oil-soluble compounds,such as diallyl sulfide(DAS),diallyl disulfide(DADS),diallyl trisulfide(DATS)as well as water-soluble fractions containing S-allylcysteine(SAC)and S-allylmercaptocysteine(SAMC).Increasing evidence reports that garlic plays a pivotal role in relieving symptoms of OA patients in clinic.For instance,OSCs protects the injured chondrocytes through inhibiting nuclear factor(NF)-κB activation.Also,DAS downregulates COX-2 and PGE2 expression on joint inflammation induced by urate crystal and IL-1β,which suggests its potential protective role in OA treatments.SAMC is the major water-soluble fraction of garlic,which has been showed potential role in anti-oxidative stress,anti-inflammation,anti-tumorigenesis,et al.However,there are no reported studies devoted to explore the association of SAMC with application of OA treatment.Because of the continuous existence of inflammation and oxidative stress in osteoarthritis,the degradation of cartilage extracellular matrix and cartilage damage are caused.Therefore,the main treatment for OA is to reduce the inflammation and oxidative stress of joints.Transcription factor E2 related factor 2(Nrf2)plays a key role in the prevention of a series of pathological damages(including reactive oxygen species and toxic substances).Nrf2 is a member of the basic leucine zipper(bZIP)transcription factor subfamily.Nrf2 is usually associated with the cytosolic actin binding protein 1(Keapl)in the cytoplasm,which makes Nrf2 ubiquitinate continuously and degrade by proteasome.Therefore,Nrf2 continued to degrade at a low level under basic conditions;when cells were exposed to stress,such as mild oxidative,electrophilic stress or chemical inducers,Nrf2 separated from Keapl,and transferred to the nucleus.In the nucleus,Nrf2 interacts with the antioxidant response element(ARE)to increase the transcription of many antioxidant and other cell protective genes,such as antioxidant glutathione transferase(GST)and NAD(P)H:quinone oxidoreductase-1(NQO1).In recent years,Nrf2,as a new target therapy for human diseases,especially those with potential oxidative and inflammatory stress factors,has shown a good prospect.In this study,we focused on the therapeutic effects of SAMC in osteoarthritis models,and evaluate the mechanism of SAMC in OA model both in vivo and in vitro.Changes of matrix metalloprotease(MMPs)expression in extracellular matrix(ECM)synthesis and degradation,inflammatory cytokines and oxidative status were examined to evaluate the role of SAMC in OA model.Furthermore,the potential role and mechanism of SAMC on OA was investigated both in vivo and in vitro,and rovide clues and basis for the development of clinical targeted drugs.Objectives1.To explore the role of SAMC in osteoarthritis both in vivo and in vitro.2.The protective effect of SAMC in osteoarthritis which we focus on is by regulating inflammation(IL-1β、TNF-α 和 IL-6)and oxidative stress(iNOS、NOX4,4HNE).3、Nrf2 acts as a critical target of SAMC in osteoarthritis,the signaling pathway that is involved in through Nox4/NF-κB.Our study will identify a potential,novel target for osteoarthritis treatment.Materials and methods1.Set up osteoarthritis(OA)animal surgical model in vivo.2.Isolate and culture primary chondrocytes.3.Measure cell proliferation rate by MTT.4.Set up IL-1β-induced chondrocytes osteoarthritis(OA)model in vitro.5.X ray to test the morphological change of rat knee articular in OA.6.Histology and Immunochemistry to examine the structure change.7.Adenovirus Preparation and virus infection.8.Inflammatory cytokines and degradation production of collagen expression tested by ELISA.9.Detection of oxidant-antioxidant Biomarkers.10.Protein expression detected by Western blot.11.Gene expression detected by Q-PCR.12.Statistical differences were analyzed by one-way ANOVA followed by Bonferroni test for multiple comparisons using SPSS 21.0 software.Results1.Improvement of osteoarthritis by SAMCFour weeks after osteoarthritis operation,the knee joint space was significantly enlarged compared with the control group,while the morphological changes were alleviated by intra-articular injection of SAMC.The articular surface of sham operation group was smoother than that of osteoarthritis group,and the preservation of SAMC group was better than that of osteoarthritis group.The most important pathological manifestation of osteoarthritis is articular cartilage degeneration.H&E and SO(Safranin o-fast green)staining showed that the cartilage surface of SAMC group was more complete than that of arthritis group,and the formation of cartilage injury was reduced.The expression of type Ⅱ collagen(Col Ⅱ)in the cartilage of the SAMC group was elevated than that the OA group.Accordingly,SAMC downregulated the expression of Col Ⅱ degradative products including C2C epitope,CTX-Ⅱ,and COMP in osteoarthritis ratsThe proteolysis of col Ⅱ involves a variety of proteases,which are mainly regulated by the balance of matrix metalloproteinases(MMPs)and their endogenous inhibitors,tissue inhibitors of metalloproteinases(TIMPs).The expression of MMP-2,MMP-9 and MMP13 increased,while the expression of TIMP-1 decreased in osteoarthritis rats.However,SAMC can improve OA by reducing the activity of MMPs and inhibiting the degradation of type Ⅱ collagen,therefore stabilize ECM.2.SAMC reduces inflammatory response in osteoarthritisA variety of inflammatory cytokines are involved in the pathogenesis of osteoarthritis.SAMC treatment reduced the expression of pro-inflammatory cytokines TNF-α,IL-1β,IL-6 in osteoarthritis rats and chondrocytes,to protect the articular cartilage from inflammation.COX-2 and iNOS are involved in the induction of osteoarthritis and tissue damage,while the increase of COX-2 and iNOS in the articular cartilage can be down-regulated by SAMC.The regulation of inflammatory mediators by SAMC is related to the inhibition of NF-κB signaling pathway.The nuclear translocation of NF-κB p65 in articular cartilage and chondrocytes of OA is up-regulated,and the degradation of IκBα is increased,indicating the activation of NF-κB.However,SAMC can reverse the activation of NF-κB.3.SAMC regulates the redox homeostasis of osteoarthritisIn the process of oxidative stress,lipid peroxidation of chondrocytes leads to collagen degradation,cartilage damage and bone joint damage.ROS,as a promoter of oxidative stress,is regulated by members of the NOX4 and NAD(P)H oxidase(NOXs)families.Compared with the sham operated group,the expression of NOX4 in osteoarthritis model was enhanced.Therefore,4-hydroxyadrenaline(4HNE),a common byproduct of lipid peroxidation,showed the same changes in articular cartilage of osteoarthritis rats.However,both changes were attenuated by SAMC treatment.Serum T-AOC,SOD,CAT,GSH-px and GSH were down-regulated in osteoarthritis rats,but these changes could be reversed by SAMC treatment.At GSSG level,oxidative GSH,malondialdehyde(MDA)were up-regulated in osteoarthritis rats,but SAMC treatment could alleviate oxidative stress in a dose-dependent manner.4.SAMC participates in the oxidative response and inflammation of osteoarthritis Chondrocytes by regulating Nrf2Oxidative stress has been proved to be involved in the inflammatory pathway under the condition of OA.The key regulators of redox-oxidative regulator Nrf2 have been evaluated in the OA rats treated with SAMC.Nrf2 is an antioxidant transcription factor,which can inhibit the degradation of cartilage collagen induced by ROS.SAMC could promote Nrf2 expression under osteoarthritis status,thereby reverse the changes in response to inflammation.In addition,Nrf2 and its downstream gene NQO-1 were activated in the SAMC treatment group,accompanied by the inhibition of NOX4 expression,and the decrease of lipid peroxidation by-product(4HNE)in articular cartilage.In order to elucidate the key role of Nrf2 in the regulation of osteoarthritis protection by SAMC,we further used adenovirus to knockdown Nrf2 gene.In primary rat chondrocytes,IL-1β was used to simulate osteoarthritis.The loss of Nrf2 function led to the aggravation of oxidative state,with the up-regulation of NOX4 expression and the increase of 4HNE.Nrf2 gene knockdown diminshed the role of SAMC in IL-1β induced osteoarthritis chondrocytes.In Nrf2 deficient cells,SAMC can not reverse the imbalance of MMPs/TIMP-1 ratio caused by osteoarthritis,which leads to collagen degradation.Furthermore,the destruction of chondrocyte matrix even aggravated in the absence of Nrf2.In order to determine the signaling pathway regulated by SAMC,we also detected the inflammatory response of chondrocytes induced by IL-1β.The expression of COX-2 and iNOS was up-regulated after IL-1β stimulation,but decreased after SAMC treatment;however,the response to SAMC in Nrf2 deficient chondrocytes was disappeared.In addition,Nrf2 can inhibit the activation of NF-κB,which is involved in the pathogenesis of inflammatory diseases and the expression of inflammatory mediators,indicating that SAMC may target Nrf2 thereby regulate the NF-κB signal pathway,to participate in the protective effect of osteoarthritis chondrocytes.Conclusions1.To confirm the protective effect of the natural product SAMC in the osteoarthritis model in vivo and in vitro:to inhibit the inflammatory response,maintain the redox homeostasis,and prevent the destruction of cartilage in the process of osteoarthritis.2.The protective effect of SAMC on OA may be to reduce the production of inflammatory cytokines(TNF-α,IL-1β and IL-6)and lipid peroxidation products(4HNE),so as to reduce the production of MMPs to regulate the type II collagen damage caused by the imbalance of MMPs/TIMP-1 ratio,and finally improve the collagen damage induced by OA.3.The specific mechanism of SAMC may target Nrf2,thus influence the NF-κB signaling activation.4.SAMC can be used as a new natural drug for the treatment of osteoarthritis.To the elucidation of its mechanism will also provide new theoretical basis for clinical targeted treatment of osteoarthritis.