Experimental Study On Effect Of Chondroprotective Agents Against Osteoarthritis

Osteoarthritis(OA) is the most common disorder of articular cartilage turnover in middle aged and older people with a trend to progressive impairment.More than half of people over 50 years complain of OA-related symptoms,such as joint pain, stiffness,loss of physical function,increasing immobility,and muscle weakness.Such signs and symptoms of the disease often culminate in reductions of life quality and burden to social health care system.More and more attentions have been paid to prevention and treatment of the disease.Contemporary management of OA aims to 1) to relieve the symptoms of the disease,2) to control the progression of the disease process,and 3) to avoid adverse effects while alleviating pain and disability. Pharmacologic therapy for osteoarthritis mainly consists of analgesics and nonsteroidal antiflammartory drugs(NSAIDs).Although these can improve OA symptoms,they may cause serious gastrointestinal and cardiovascular adverse events and do not affect the underlying structural cartilage damage.A disease-modifying therapy would be more beneficial.Under the circumstance,chondroprotective agents, especially,glucosamine sulfate(GS) and chondroitin sulfate(CS) have been employed to prevent and treat OA widely.As cartilage constituents,GS and CS have been regarded as inhibitors of cartilage degeneration,via affecting the synthesis of collagen and proteoglycan(PG) in extracelluar matrix(ECM),and modulation of inflammatory pathways.In Europe GS and CS can be medically prescribed while in the USA they are marketed only as nutraceuticals,either separately or in combination.The diversities of GS and CS in usage and management may due to pendulous results from more than 20 years research.In randomized,double-blind,placebo controlled clinical trials, respectable levels of evidence in terms of pain relief and functional improvement of GS and CS have been achieved.However,in practice it is always difficult to distinguish responders from non-responders to such therapy,which assessment endpoints rely on self-claim of patients.In vitro tests,both substances have been demonstrated the ability of beneficial influence on a complex cartilage biometablic factors,including proteolytic enzymes,cytokines and their receptors related to cartilage structure and inflammatory pathway.But concentrations of GS and CS applied in vitro tests are much higher than usual oral and absorbable dosages in clinical or food supply intentions.It should be cautious when result of vitro is extrapolated to vivo.Currently,effective evidences of GS and CS on OA animal models are limited,while research on animal remains valuable as a result of low sensitivity diagnostic tools in clinical trials and low availability of diseased tissues in vitro tests.For functional evaluation on symptom and structure-modifying compounds,it is crucial to find and understand which animal model and species is the most relevant for human OA.In order to produce more valid information in these fields,animal tests in inflammatory, pain and spontaneous OA models were carded out respectively in the research to study the effects of GS,CS and combined treatment on OA symptom relief and structure maintenance.In anti-inflammatory tests,after a 30-day-gavage treatment with dosages of 1g/kg.BW GS in GS group,0.5g/kg.BW CS in CS group,1g/kg.BW GS+ 0.5g/kg.BW CS in combined group and same volume distilled water in control group separately.Not only GS or CS group,but combined group was found to rapidly decrease ear edema in mice induced by ethyl phenylpropiolate after 4h dosing and hind paw edema in rats induced by yeast after 1h,6h,24h and 72h dosing compared with control(P＜0.001). It is suggested that GS and CS could inhibit acute and subacute phase of inflammation in animal,which may via modulate inflammatory pathways.In analgesic tests,with the same treatment managed in anti-inflammatory tests,GS, CS and combined group provoked a significant time delay of hind paw lick on a 55℃hot plate and elicited inhibitory activity on acetic acid-induced writhing response within 15 min in mice.All of three test groups possess analgesic effects equally.It is postulated that the analgesic activity of GS and CS is caused by the inhibition of inflammatory reaction.Hartley outbreed strain guinea pig,a strain of spontaneous onset of progressive degenerative changes in the knee joints that closely resemble the development of OA in humans was selected to serve as an OA model.Before using the model,the variation disciplinarian of pontaneous onset of OA in Hartley ginuea pigs was studied. The changes of histological lesion including structure and component in knee cartilage measured by H.E,Alcain Blue,PAS and Mallory stain and serum level of PG measured by ELISA in Hartley guinea pigs aged from 0.5 month old to 6 months old were observed.At 0.5 to 1 month of age,the structure and content of PG and collagen in female guinea pigs cartilage were well-balanced;at 2 months of age,female guinea pigs had the earliest or slight histopathological signs of OA,while moderate and severe histophaological evidences of OA were observed in 3-4 months and 5-6 months aged female guinea pigs respectively.These histopathologic features included chondrocyte death/loss and PG loss with fibrillation extending into the upper,middle and deep zone in various grade.Even then,the relative progressive changes in cartilage correlated with age were not observed in male guinea pigs.From 0.5 to 6 months age,male guinea pigs only had minimal to mild histopathological changes of OA.The overall serum levels of PG in male guinea pigs were higher than those in female,but the continuous decreasing tendency of serum levels of PG with age had no difference by sex.Moreover,the serum levels of PG in guinea pigs at 1 month age declined dramatically compared with other ages(P＜0.001).It is demonstrated that spontaneous OA doesn't occur in male,but in female guinea pigs within 6 months age. It is similar to human,women develop knee OA more frequently and younger than men.Gender may influence knee OA via multiple routes including hormonal influences on cartilage metabolism.2-month age female guinea pigs are under the critical period of OA and suitable for intervention study.The drop of PG level in serum indicating the change related to OA which is earlier and more sensitive than histopathological signs in cartilage can be served as an early index of OA.GS,CS and combined therapy had been administered to 2-month age female guinea pigs for 5 months via ad limbitum with the same dosage treated in anti-inflammatory and analgesic tests.The features of histological lesion in knee cartilage and serum level of PG were examined initially and monthly after initial treatment.Initial average histopathological scores of cartilage in all groups were the same of 4 pionts.After 5-month intervention,the average histopathological scores in GS,CS,combined and control groups were(11.6±2.88 points),(3.2±1.48points),(1.8±1.64points) and (14.8±1.10points) in turn.Average serum levels of PG decreased from(122.6±29.6 ng/ml) initially to(57.7±15.0ng/ml) after 5 months later in GS group;from (117.0±23.1ng/ml) to(46.5±6.3ng/ml) in CS group;from(114.2±25.9ng/ml) to (50.4±12.7ng/ml) in combined group and from(122.5±23.3ng/ml) to(30.9±5.8ng/ml) in control group。Compared to self group at initial time,GS group hadn't moderate histopathological change of OA until 4 months later;CS group had mild change of OA;Combined group only had minimal change after 4-month treatment,and had no histopathological evidence of OA at the end of the intervention,while control group had severe histopathological evidence of OA at 3 months after initial dosage.The serum levels of PG in all three test groups were all decreased slowly after 4 -month treatment compared to those in the control group,with a significant difference (P＜0.05).GS and CS can postpone and inhibit the pathological changes of articular cartilage,as well as serum PG levels decrease in female Hartley guinea pigs.The combination of GS and CS results in a greater chondroprotective effect than either compound alone,which suggests a cartilage-regenerating effect.After all,aged cartilage may alter chondrocyte function,collagen and PG properties in ECM and responds differently to cytokines,growth factors and mechanical factors. GS and CS can promote cartilage matrix synthesis and slow the progression of OA via affect and influence the expression or activity of many mediators of OA,including a reduction in PG degradation and inhibition of synthesis and activity of degradative enzymes and inflammatory mediators such as aggrecanases,MMPs,nitric oxide(NO), and PGE₂.GS and CS possess both symptom-modifying and structure-modifying effects on OA.Time of onset of experimental or clinical improvement with chondroprotective agent may slower than NSAIDs,but its lasting effect and safety bring chondroprotective agent a widespread use in OA prevention and treatment.