Protective Effect Of Chitosan Oligosaccharides On Cartilage Of Rat Osteoarthritis

Osteoarthritis(OA)is a common degenerative disease in old age that mainly feature by joint pain and dysfunction due to articular cartilage degradation,chondrocyte apoptosis,subchondral bone remodeling,and inflammation/synovitis,and osteophyte formation.The latest literature estimates that more than 2.5 billion people are suffering from osteoarthritis disease problems around the world.Over 60 years of age OA incidence of up to 60%in our population.Especially at present,OA greatly affects people's quality of life and the prevalence is speedy increasing,and it is predicted that OA is a major cause of functional disability in the coming decades.And it has become an important subject in the field of orthopedic research.Thus,OA should be considered as a serious social problem,worthy of widespread attention.Although the physiological and pathological characteristics of articular cartilage and the metabolic function of articular cartilage cells have a certain degree of understanding,but the pathogenesis of OA is still not very clear.The goal of the OA study is to find new treatment strategies that can prevent,delay,or discontinue disease progression,but unfortunately,due to the multifactorial and complex nature of the disease,the current advances in these interventions are highly challenging The So far,the joint efforts of academia and the medical community have failed to apply the relieving anti-OA drugs with compelling efficacy and high safety to daily clinical practice.At present,in addition to analgesic treatment and end-stage surgery,there is no more effective treatment,therefore,for the etiology and treatment of OA is still a worldwide problem.The study found that articular cartilage itself does not have blood supply,so cartilage degeneration in the event of its own regeneration capacity is scarce.This requires us to develop a class of ability to inhibit cartilage degeneration,promote cartilage repair,innovation and reconstruction of joint structure of the drug in order to fundamentally solve the bottleneck of OA treatment.Therefore,the joint structure of improved drug therapy OA research more and more popular.In recent years,the representative of the joint structure improvement drugs is chitin and its derivatives,chitosan(Chitosan)is a primary derivative of chitin,is currently the most studied,the most widely used representative.Chitosan oligosaccharides is an enzymatic product of chitosan,and has good water solubility in addition to the general properties of chitosan.Little attention about the preventative and therapeutic effect of COS on osteoarthritis.The experimental study on the molecular level of the chondrocytes and the animal model is still lacking.In this study,we first evaluated the cytotoxicity of chitooligosaccharides to chondrocytes.The effects of in vitro and in vivo on rat OA chondrocytes were further studied.The aim of this study was to investigate the protective effect of COS on OA articular cartilage.To provide a new way of thinking for the prevention and treatment of OA,and further study of its biological function of the possible molecular mechanisms.Part I The toxicity experiment of chitosan oligosaccharides on chondrocytes in vitroObjective To investigate chondrocytes cytotoxicity of chitosan oligosaccharides in vitro,and evaluate the biological safety.Method Experiments were conducted using 4-to 6-day-old Sprague-Dawley neonatal rats provided by the Center of Experimental Animals of Wuhan University.Articular chondrocytes were isolated from the knee joints of the rats and cultured in vitro via an enzymatic digestion method.Chondrocytes were identified by collagen type II using immunohistochemical staining.The second or third generation chondrocytes were used in the experiment.Chitosan oligosaccharides were dissolved in the medium,and its final concentration was adjusted to 50,100,and 200 ?g/ml.The chondrocyte cytotoxicity was determined CCK-8 assay after 24 h,48 h and 72 h co-culture.Results The original generation of articular chondrocyte began to attach wall after 4 to 6 hours culture,and grew well after 72 hours with confluent growth and paving stone shaped or polygon exterior form.The immunohistochemical staining showed the cultured cells could secrete collagen type ?,that were identified as chondrocytes.The CCK-8 test showed that the concentration of COS ranged from 50 to 200 ?g/ml showed no significant cytotoxicity against normal chondrocytes for 24 h,48 h and 72 h(P>0.05).Conclusion Chitosan oligosaccharides have no cytotoxicity against normal chondrocytes in vitro and good biological safety performance.All of these can provide the basis for further in vitro and in vivo studies.Part ? The anti-apoptotic effect of COS on chondrocytes in vitroObjective To investigate the protective effect of COS on IL-1?-induced apoptosis in vitro.Methods Experiments were conducted using 4-to 6-day-old Sprague-Dawley neonatal rats provided by the Center of Experimental Animals of Wuhan University.Articular chondrocytes were isolated from the knee joints of the rats and cultured in vitro via an enzymatic digestion method.The second or third generation chondrocytes were used,and attached chondrocytes were stimulated with COS(50 ?g/ml,100 ?g/ml and 200 ?g/ml)for 2 hours before 10 ng/mL IL-1? co-treatment for 24 h.Specific experiments included:1.CCK-8 proliferation inhibition test;2.Flow cytometry analysis of chondrocytes early and late apoptosis;3.Hoechst 33342 apoptotic cell nucleus fluorescence staining of chondrocytes;4.Rhodamine-123 staining of mitochondrial membrane potential of chondrocytes;5.Western blot assay of chondrocytes intracellular protein Bcl-2,Bax,p38,p-p38 and caspase-3 expression level;6.Real-time PCR detect the expression of iNOS,MMP-13 and TIMP-1 mRNA.Results CCK-8 results showed that COS elicited a good protective effect on cell viability.The results of FCM showed that COS could decrease the early and late apoptosis rate of chondrocytes induced by IL-1?(P<0.05).The morphological analysis of Hoechst 33342-stained nuclei revealed similar results.COS treatment decreased the strong staining intensity of the nuclear chromatin via IL-1? challenge and ameliorated the nuclear damage after COS co-treatment was administered(P<0.05).The results of Rhodamine-123 demonstrated that COS greatly prevented the IL-1?-induced collapse of the mitochondrial membrane potential(P<0.05).Western blot analysis showed that COS significantly increased the proportion of Bcl-2/Bax protein in chondrocytes.At the same time,the expression of p-p3 8 and caspase-3 protein was decreased in a dose-dependent manner(P<0.05).Real-time PCR results showed that COS reduced the expression of iNOS mRNA and MMP-13 mRNA in chondrocytes in a dose-dependent manner,and increased the expression of TMMP-1 mRNA in a dose-dependent manner(P<0.05).Conclusion The mechanism of COS against chondrocyte apoptosis is mainly through the activation of p38 MAPK signaling pathway to effectively inhibit IL-1?-induced chondrocyte apoptosis.COS have the potential as a unique biological therapy for the prevention and treatment of OA.Part ? The protective effect of COS on articular cartilage in a rat OA modelObjective To establish a reasonable OA model in rats for evaluation and research COS treatment of osteoarthritis and to find a new effective treatment for osteoarthritis.Method Thirty-six adult male SD rats were randomly divided into 3 groups:single model group(OA group),chitosan oligosaccharide treatment group(COS group)and sham operated group(CON group).For the CON group,the wounds were sutured after exposing the knee joint cartilage surface.The rats in OA and COS groups were operated by anterior cruciate ligament transection combined with part medial menisci resection in the right knee joint.From the 4th week after surgery,the CON group and OA group received an injection of 50 ul normal saline into the right knee joint.Meanwhile,the COS group received an injection of 50 ul of COS(1 mg/mL),repeated weekly for 5 weeks.All animals were not sacrificed until the 11th week after surgery.The general changes in each group within the articular cartilage of the femoral condyle.The pathological changes of cartilage were examined by HE and Safran O staining,and the cartilage modified Mankin's histological score was performed.At the same time,the morphological changes of femoral condylar cartilage were observed by scanning electron microscopy.Meanwhile,Westem blot and Real-Time PCR were used to detect Bax,Bcl-2,p38,p-p38,Caspase-3 and iNOS proteins and mRNA expression level in articular cartilage.Results General observation can be seen OA group right knee articular cartilage color dull,uneven surface with osteophyte formation,suggesting that modeling more successful.At the same time,the right knee articular cartilage surface of COS group was good,no obvious osteophyte formation,articular cartilage degeneration of the pathological degree and the severity of osteophyte was significantly lighter than the OA group.The modified Mankin scores of three groups were 1.5±0.4 in the CON group,9.6±1.6 in the OA group and 3.8±1.8 in the OA group.The Mankin's score in COS group was significantly lower than that in OA group,and there was significant difference between the two groups.Scanning electron microscopy further confirmed the changes in the degree of cartilage degeneration.In terms of the related proteins and mRNA expression in articular cartilage,Bax,p-p38,Caspase-3 and iNOS proteins and mRNA expressions were lower,and Bcl-2 was higher in the COS group than that in the OA group(P<0.05).Conclusion Intra-knee injections of COS can reduce the degree of cartilage degeneration.All of these can provide the experimental foundation and theoretical basis for acting therapeutic drug for OA.