The Relieving Effects Of Marine Type ? Collagen On Osteoarthritis And The Underlying Mechanisms

Objects:It provides a new prospect and potential strategy for the treatment of degenerative osteoarthritis(OA)to develop a novel and multifunctional bio-material with combined anti-inflammatory effects,cartilage-repairing function,and high bio-safety.Till now,there are no studies on such bio-material experimentally and clinically.Accordingly,type II collagen isolated from terrestrial animals could promote normal chondrocytes to secrete cartilage matrix and participate in the repair of hyaline cartilage.However,it is still a great challenge for their use under the condition of OA.It is prone to cause abnormal immunological responses from the type II collagen isolated from terrestrial animals,for the shared immunodominant epitope with human beings.So,it is unreasonable for the use of type II collagen isolated from terrestrial animals in the treatment of OA.Besides,our previous study has discovered that collagen components have preventive effects on inflammation in some extent.Nevertheless,it is still unclear whether the type II collagen with triple superhelical structure carries anti-inflammatory functions.Herein,we use the cartilage from marine squid,and aim to develop a novel marine type II collagen(MCII)that is cost-effective,non-immunogenic,and might have an anti-inflammatory effect on OA.In this study,we managed to illustrate the inhibitory effects and the underlying mechanisms of MCII on the cellular apoptosis and hypertrophic changes of chondrocytes.Besides,we also firstly demonstrated that MCII could promote the cartilage-repairing via immunological regulation of macrophages.Above all,rat models with surgery-induced OA were used to evaluate the OA-relieving effect of MCII,which might be a promising biomaterial for future use in OA treatment.Material and methods:(1)MCII was acquired with pepsin digestion.The characterization of MCII was detected with amino acid analysis,molecular weight and purity detection,and et al.(2)In order to clarify the immunogenicity of MCII,both in vitro and in vivo experiments were undertaken.Responses for MCII in humoral and cellular immunity were detected compared with responses for bovine type II collagen.(3)Transwell assays were performed to investigate the chemotactic behavior of M0macrophages under the effect of MCII.Besides,inflammatory M1macrophages model was constructed to evaluate the anti-inflammatory effects of MCII on M1 macrophages and the underlying mechanisms.(4)Primary murine articular chondrocytes(MAC)were extracted.The effects of MCII on the apoptosis condition of MAC and the underlying mechanisms were investigated.(5)Two experimental models based on direct interaction and conditioned cultures were conducted.We investigated the direct effects of MCII on the hypertrophic changes of MACs.Besides,the indirect effects on the hypertrophic changes of MACs were investigated by conditioned culture with the inflammatory macrophages under the treatment with MCII.What's more,the effects of MCII on the alterations of M2 macrophages were also investigated.The indirect influences from MCII involved M2macrophages on the matrix secretion of MACs were detected subsequently.(6)We established a rat model of OA and examined the effect of MCII treatment on injured articular cartilage.The structural integrity and the GAG content of the cartilage tissue,and the cellular apoptosis and hypertrophic changes of chondrocytes were observed histopathologically.Besides,the inflammation condition for synovial membrane and the alteration of M2macrophages were also observed.The secretory levels of inflammatory cytokines,MMP-13 and the production of pro-chondrogenic cytokines were detected bio-chemically.Above all,the OA-relieving effect of MCII and the underlying mechanisms were managed to be demonstrated.Results:(1)The MCII consisted of 18 different kinds of amino acids.The SDS-PAGE showed that there was only one kind of?1-chain with a molecule weight of 110 k Da in MCII.(2)Different from BCII,MCII presented no obvious immunogenicity.(3)MCII markedly impeded chemotaxis of macrophages.MCII significantly reduced the production of pro-inflammatory cytokines by enhancing the activity of TCPTP and subsequently prompting the dephosphorylation of p-STAT1 in M1 macrophages.(4)MCII activated the glycine receptors(Gly R)distributed on the cellular surface of MACs,resulting in a reduction in intracellular calcium concentration(Ca²⁺)in inflammatory cells.Consequently,MCII up-regulated the expression of anti-apoptotic gene Bcl-2,and inhibited the expression of pro-apoptotic gene Bax.In addition,MCII also suppressed Cyt C release induced by NO from mitochondria to cytoplasm in chondrocytes,which further attenuated Caspase-3 activation.The apoptotic level was obviously attenuated by MCII for MACs stimulated by SNP(50?M).(5)MCII directly inhibited the hypertrophic changes of MACs via inhibiting the activation of p65 and down-regulating the expression of MMP-13.Besides,the hypertrophic changes of MACs and the secretory of MMP-13 were suppressed by decreased inflammatory responses from M1 macrophages.MCII could enhance the secretory ability of MACs for Col II and GAG directly.Besides,we also observed that the expression of pro-chondrogenic cytokines TGF-?and IGF were obviously elevated from the MCII-induced M2-like macrophages,which enhanced ability of MACs to secrete extracellular matrix components indirectly.(6)MCII greatly maintained the structural integrity and the content of proteoglycan in surgery-induced OA lesions of rat models.Microscopically,MCII greatly inhibited the cellular apoptosis and hypertrophic changes of chondrocytes.Meanwhile,ameliorated synovial hyperplasia was observed and decreased pro-inflammatory cytokines were detected.MCII immunomodulated a phenotype shift of macrophages from M0 to M2,and increased level of TGF-?and decreased levels of IL-1?and TNF-?were detected in synovial fluid.Above all,the MCII exhibited therapeutic effects on damaged cartilage in OA rats.Conclusions:(1)This study,for the first time,successfully isolated a novel type II collagen without immunogenicity,exhibiting better biosafety than type II collagen isolated from terrestrial animals.(2)MCII could impede the chemotactic behavior of macrophages.Besides,we also illustrated that MCII promoted the dephosphorylation of p-STAT1 by enhancing TCPTP activity in M1 macrophages,thus effectively lowering the local pro-inflammatory cytokine levels.(3)MCII activated the glycine receptors(Gly R)distributed on the cellular surface of MACs,resulting in a reduction in intracellular calcium concentration(Ca²⁺),which inhibited the cellular apoptosis and hypertrophy of chondrocytes.These data indicated that MCII greatly alleviated the injuries of chondrocytes and promoted the cartilage-repairing.(4)Pro-chondrogenic cytokines TGF-?and IGF released from the MCII-induced M2-like macrophages enhanced ability of MACs to secrete extracellular matrix components.(5)MCII greatly alleviated the OA via three manners.Firstly,MCII could directly exert anti-inflammatory effect on M1 macrophages,accompanied by down-regulating the expression of pro-inflammatory cytokines.Secondly,MCII inhibited the cellular apoptosis and hypertrophic changes of chondrocytes via down-regulating the expression of MMP-13.Thirdly,MCII induced the alteration of M2macrophages and its expression of pro-chondrogenic cytokines TGF-?,which improved the GAG content in articular cartilage.Above all,this study provided validated evidence for the potential use of MCII in the treatment of OA.