Study On Screening Of Chondroitin Sulfate E Oligosaccharide Targeting Complement Protein And Its Treatment Of Osteoarthritis

Osteoarthritis(OA)is a very common joint degenerative disease with a variety of pathological changes,involving articular cartilage,synovium,and periarticular muscles,etc.Patients eventually lose the ability to move after experiencing severe pain.According to statistics,more than 300 million people worldwide are suffering from OA,and this number continues to grow due to the increasing problems such as population aging and obesity.However,the current clinical efficacy for OA is very limited,so it is imperative to develop potential drugs for OA treatment.The pathogenesis of OA is complex,involving mechanical,metabolic,inflammatory,and other factors.Cartilage injury leads to the release of chondrocyte extracellular matrix components,thus activating the complement system,promoting the release of inflammatory factors and activating related signaling pathways.In this process,the complement system plays a central role.Chondroitin sulfate(CS)is a linear anionic glycosaminoglycan composed of Dglucuronic acid and N-acetyl-D-galactosamine,which can be divided into CS-A,CS-E and other types according to their chemical structures.Studies have shown that CS can reduce the pain of OA patients and delay the course,which has become one of the options in the clinical practice.Its activity in the treatment of OA may be related to the complement system,but the functional unit and mechanism of action have not been clarified.In this study,CS-E(marine source)oligosaccharides were prepared first,andΔUA-diSE was founded as the smallest functional unit with the best anti-complement activity,and then the main target of ΔUA-diSE was confirmed as C5.Through in vitro and in vivo studies,we reveal the key role of ΔUA-diSE in the regulation of complement system and the mechanism of treating OA,including the interaction between ΔUAdiSE and C5,inhibiting the formation of membrane attack complex(MAC,also known as C5b-9),down-regulating the expression of MAC-induced related proteins,as well as inhibiting the activation of downstream signaling pathways,such as MAPK and NFκB.In addition,we constructed C5 gene knockout(C5-/-)mice models to study the activity and mechanism of ΔUA-diSE.We found that the deletion of C5 can reduce the inflammatory response and reduce the efficacy of ΔUA-diSE.This further confirms the key role of C5 in OA and the central role of targeting C5 in the efficacy of ΔUA-diSE.By identifying ΔUA-diSE as a complement inhibitor with anti-complement activity and exploring its efficacy and mechanism in alleviating OA,this study not only provides a new idea for the development of OA treatment,but also may provide a promising C5 targeted therapy for other related diseases.Compared with other CS,the sulfation site of CS-E is different,it is more complicated in preparation and more difficult in separation.Currently,local inj ection is commonly applied in clinical treatment of OA,while ΔUA-diSE has a low molecular weight(Mr=605)and a fast metabolic rate,and conventional injection requires large dose and high cost.Therefore,this study preliminarily explored the administration mode and dosage form of ΔUA-diSE in order to prolong its action time,reduce the cost and improve its efficacy.At present,various forms of anti-OA drug delivery systems with long-term activity have been reported,such as liposomes,nanoparticles,particles,etc.Although they extend the release time,there are still some deficiencies,such as poor targeting and low release efficiency.Pluronic F127(PF127)is an FDA-approved safe,temperature-sensitive biomaterial that can be converted from solution to hydrogel at body temperature(37℃),enabling in situ sustained release of loaded drugs.In addition,hyaluronic acid(HA)is a class of highly hydrophilic linear glycosaminoglycans that occur naturally in joints and have been shown to maintain synobial lubrication,promote cartilage repair,and improve j oint function.Therefore,this study developed a novel PF-HA-diSE hydrogel,which loaded ΔUA-diSE into PF127 containing HA,as if stored in a natural function tank.The hydrogel has good gelling performance and can inhibit the formation of C5b9 by binding with complement,thus inhibiting the expression of MMP13 and other factors.In vivo experiments showed that PF-HA-diSE hydrogel had a good therapeutic effect on OA model constructed by DMM surgery,alleviating the disease,and reducing the expression of C5b-9 and related inflammatory proteins at the joint site.At the same time,this study also reported the binding between PF127 and complement,and the role of this active behavior in the treatment of OA for the first time.Besides being a temperature-sensitive sustained-release vector,the complement binding ability of PF127 is also worthy of attention and application.This study is a preliminary application and attempt of the PF-HA-diSE hydrogel composite system in OA,which provides a reference for further research and has broad application prospects in bone tissue engineering.In conclusion,ΔUA-diSE,the smallest functional unit of CS-E oligosaccharides with the best anti-complement activity,was prepared and screened out,its main target C5 was confirmed,and the interaction between the two was explored.The mechanism of ΔUA-diSE by regulating complement activity was further elucidated through in vivo and in vitro experiments.In addition,a new temperature-sensitive multifunctional PFHA-diSE hydrogel was developed to improve the efficacy of ΔUA-diSE.This study provides a new idea and research basis for the development of CS drugs targeting complement system,which is expected to overcome the bottleneck of the lack of effective treatment in the current clinical practice of OA.