the main characteristic feature of ra is swelling of the joints , and it is also sometimes manifested in other organ systems in severe disease states . some of the common physical symptoms of ra are swelling of joint areas , painful and stiff joints , fatigue , malaise , weight , loss and sometimes even depression . risk factors such as genetic predisposition and environmental factors like cigarette smoking , microbial infections , and blood transfusions   will lead to the activation of cd 4 t helper cells , which will lead to the stimulation of various cells in the joints that produce cytokines and inflammatory mediators that play an important role in the development of ra . inflammatory mediators like matrix metalloproteinases ( mmp ) and cell adhesion molecules ( cams ) , together with cytokines like tumor necrosis factor - alpha ( tnf- ) and interleukin-1 ( il-1 ) , lead to joint destruction in ra patients [ 2 , 6 ] . at present , there are few treatment options available for ra . nonsteroidal anti - inflammatory drugs ( nsaids ) , cyclooxygenase-2-selective nsaids , glucocorticoids , biological response modifying agents , and disease modifying antirheumatic drugs ( dmards ) are prescribed by physicians currently as single therapy or combined multiple drug therapy . it has beenshown to slow the disease progression in some cases , but itis not shown to completely reverse symptoms of ra and prevent the disease onset . it is naturally found in mucous secretions , connective tissue , skin , tendons , and ligaments , and it is found in highest concentration in cartilage . with age , the production of glucosamine by the body will decrease ; hence , it needs to be replenished by the use of oral glucosamine supplements . currently glucosamine is used in the therapy for osteoarthritis ( oa ) to synthesize glycosaminoglycans ( gags ) . studies have also shown that glucosamine supplementation can repair the cartilage to some extent in oa . recent studies have also shown that glucosamine has immunosuppressive properties , which can successfully suppress the activity of t - lymphocytes and dendritic cells   which are involved in adaptive immune responses . the present study investigated the efficacy of glucosamine against collagen - induced arthritis in female dark agouti ( da ) rats . glucosamine hydrochloride ( d-(+)-glucosamine hydrochloride ; g4875 ; c6h13no5hcl ; mol wt:215.63  g / mol ; sigma ) , complete freund 's adjuvant ( cfa ) , type ii collagen , and all other analytical chemicals used for the experiment were obtained from sigma aldrich ( st . the dose received by the treatment group in this study was 300  mg / kg / rat . female dark agouti ( da ) rats   of 610 weeks old were obtained from the institute of medical research , kuala lumpur , malaysia . rats were housed in standard laboratory conditions with 12:12 light - dark cycle with free access to food and water throughout the study . there were three groups of animals in this study , each group containing 10 rats . group 1control without any treatment , group 2arthritis only group , and group 3arthritis with glucosamine hydrochloride ( 300  mg / kg / rat ) . all the experimental procedures were in accordance with the guidelines for the care and use of laboratory animals , and approval from the institutional research and ethics committee was taken prior to the experiments . type ii collagen from chicken tracheal cartilage obtained from sigma aldrich ( usa ) was dissolved in 0.1  m of cold acetic acid . the dissolved collagen was then emulsified with an equal volume of complete freund 's adjuvant ( cfa ) ( sigma aldrich , usa ) . each rat was injected intradermally with 4  mg / kg of collagen suspension at multiple joint areas   and also at the base of the tail . a booster dose injection was given after a week following primary immunization , and a second booster was administered 15 days after first booster injection . glucosamine hydrochloride was administered orally using a rodent feeding tube with a dose of 300  mg / kg / rat . the treatment was started 21 days after the primary immunization and continued until day 45 . the severity of arthritis in the animals was evaluated by quantifying the changes in paw thickness in the front and hind paws . thickness was measured using a digital caliper once every five days ( there was not any significant change in the paw thickness during the 5-day period in the earlier studies done in our laboratory ) until day 45 . the body weight changes of the rats were also recorded once every five days during the study period . at the end of the experimental period , animals were anesthetized with sodium pentobarbital ( 40  mg / kg body weight ) and blood samples were collected by cardiac puncture . a volume of 3 - 4  ml of blood was collected in a vacutainer tube which contained edta . rats were sacrificed with an overdose of anesthesia , and limbs were harvested for joint histopathology . the c - reactive protein ( crp ) and tumour necrosis factor - alpha ( tnf- ) concentration level in the plasma were quantified using a commercial kit according to the standard protocol recommended by the manufacturer ( assay pro , usa , and ebioscience , usa ) . in the c - reactive protein assay , 25  l of the standards was added to the relevant wells , and 25  l of the diluted samples were added in duplicates . biotinylated rat crp ( 25  l ) was added to all wells , gently mixed , and kept for incubation for 2  h. after incubation , the plate was washed using wash buffer and 50  l of diluted streptavidin peroxidase conjugate was added and incubated for 30  min . the plate was washed and filled with 50  l chromogen substrate and left for 10  min for color development . a stop solution was added , and the absorbance produced was immediately read at 450  nm . the crp concentration of each of the samples was calculated in g / ml based on the standard curve obtained . the concentration of tnf- in the plasma was quantified using a commercial rat tnf- elisa kit . the previous night , the plate was coated with diluted capture antibody as described in the study protocol provided with this kit . then , 100  l of the standards were added to the standard wells and 100  l of samples was added to the sample 's wells in duplicates . the plate was incubated at room temperature for 2  h. after washing with buffer , 100  l of the diluted detection antibody solution was added to all wells , and the plate was incubated for 1  h at room temperature . afterwards , 100  l of the diluted avidin hrp was added to all the wells and incubated at room temperature for 30  min . following this , finally , 50  l of 2nh2so4 was added and the absorbance was read at 450  nm . the flesh from the joints was cleared away , and the joint sample was stored in 10% formalin solution for three weeks in specimen bottles . the decalcified samples were processed using leica tp 1020 tissue processor machine and embedded in paraffin blocks using a leica eg 1160 tissue embedding center . the haematoxylin and eosin stained slides were visualized and analyzed using a nikon eclipse 80i microscope . the joints were evaluated and analyzed according to a grading system adopted from a previous study   ( table 1 ) . statistical analysis was carried out by one - way analysis of variance ( anova ) using the spss . the histology of the joints and the plasma levels of c - reactive protein and tnf- were also statistically analyzed for nonparametric measures using the mann - whitney u test . in the arthritis only group , there was a significant decrease in the body weight ( p < 0.05 ) . arthritis rats that had oral supplementation of glucosamine showed a steady increase in the body weight initially , but there was a significant decrease in the weight gain after 15 days until day 25 , but weight increased significantly after day 25 in these rats . even though there was an increase in the body weight with glucosamine treatment , the weight change did not increase significantly compared to control rats ( figure 1 ) . a significant increase ( p < 0.05 ) in the hind paw thickness was observed in the arthritic rats from day 1 to day 45 when compared to control rats . glucosamine treatment depicted a significant reduction ( p < 0.05 ) in the paw thickness compared to arthritis alone at the 45th day . there was severe swelling of the joints in the hind paw in the arthritic rats . the group of rats that were started on oral supplementation of glucosamine showed only mild to moderate signs and symptoms of arthritis , when compared to the arthritis alone group . the swelling of the ankle joints in the hind paw was significantly less in the treated group than in the arthritis only group ( figures 2 and 3 ) . a significant increase ( p < 0.05 ) in c - reactive protein level was seen in arthritis only rats until 45 days compared to control rats . the level of c - reactive protein concentration was significantly reduced ( p < 0.05 ) after oral supplementation of glucosamine ( figure 4 ) . a significant increase ( p < 0.05 ) in the tnf- level was seen in the arthritis alone rats compared to controls . there was a significant decrease in tnf- levels in the plasma of the glucosamine treated group ( p < 0.05 ) than the untreated arthritis only control group . glucosamine has reduced the concentration of this inflammatory marker significantly in the treated group ( figure 5 ) . to further evaluate the treatment aspect of glucosamine hydrochloride against rheumatoid arthritis , histopathological analysis was done as described in table 1 , which is a method adapted from the previous study . arthritis only group showed a severity of grade three , while the glucosamine treated arthritic group had pathological characteristics of grade 10 severities ( figure 5 ) . synovial hyperplasia is observed in the untreated arthritis only group , which appeared to have developed extensive oedema causing the joint space to narrow . the group treated with glucosamine showed the best results , that is , the reversal of synovial hyperplasia as in these rats and the joint appeared to be similar to a normal joint ( figure 6 ) . there were healthy regenerative synovium and areas of fibrosis , angiogenesis , and fibroblasts proliferation with accumulation of collagen , suggesting good wound healing . in the untreated arthritis rats , the joints had extensive oedema with narrowing of the space . the surface of the joint margins showed degenerative changes . in the glucosamine treated arthritic groups , the synovial space was adequate with focal areas of degenerative changes ( figure 6 ) . in the untreated arthritic group , the synovium showed hyperplasia and increased angiogenesis and fibrosis . the group treated with glucosamine showed no synovial congestion compared to the other two groups ( figure 6 ) . there was an extensive inflammation with pannus formation in several places among untreated arthritic rats . the pannus was composed of a granulomatous accumulation of chronic inflammatory cells like lymphocytes , plasma cells , and macrophages and multinucleate giant cells seen in the untreated arthritic group . collage - induced arthritis ( cia ) was induced when type ii collagen was emulsified with complete freund 's adjuvant ( cfa ) and injected into the base of the rat 's tail . in this animal model of arthritis study , complete freund 's adjuvant injection will induce an immunological hypersensitivity reaction to collagen in the rats , leading to the development of systemic effects of chronic rheumatoid arthritis ; therefore , the collagen - induced arthritis da model shows most similarities to the human rheumatoid arthritis . dark agouti rats are especially susceptible to rheumatoid arthritis . these rats develop a chronic and severe form of autoimmune arthritis , which leads to permanent bone destruction and ankylosis of affected joints . arthritis - induced joint destruction in da rats is believed to be the consequence of cell mediated immune responses , which mimic the pathogenesis of human rheumatoid arthritis [ 1921 ] . the dose of glucosamine used in this study is 300  mg / kg body weight , which would give an approximate serum level of glucosamine ( 0.05  mm ) similar to that in humans [ 13 , 22 ] . because of the substantial amount of gastrointestinal loss in rats , a higher daily dose of glucosamine is usually selected in animal models of arthritis . glucosamine is a naturally occurring chemical component that is found in its highest concentration in joint areas [ 23 , 24 ] . our study showed a significant decrease in body weight around the 25th day in arthritis rats as compared to the normal control . glucosamine treatment ( 300  mg / kg ) showed significant recovery in the body weight during the last 10 days of the treatment when compared to their arthritic controls . glucosamine acts at the joints by being incorporated by the chondrocytes into the cartilage . here it will stimulate the synthesis of physiological proteoglycans and also will decrease the activity of matrix metalloproteinases , which are catabolic enzymes that attack the cartilage . glucosamine can also inhibit the degradation of cartilage , which is induced through il-1 [ 21 , 25 ] . thus , glucosamine acts as a substrate for the biosynthesis of mucopolysaccharides in bones and joints , which will aid in restoring damaged cartilage . several studies have implicated tnf- as a contributor to cellular damage in collagen - induced arthritis . the increased concentration of this cytokine in circulation can be interpreted as a progression of cartilage cell injury [ 26 , 27 ] . the anti - inflammatory action of glucosamine might have lowered circulating tnf- concentration and consequently reduced the joint damage . c - reactive protein is produced under conditions of inflammation in cartilage and bone and is a useful biomarker in the evaluation of disease progression and response to therapeutic intervention in a number of inflammatory disorders , including ra . higher concentration of c - reactive protein will point toward increased joint changes in arthritis . in our study , it is shown that glucosamine reversed inflammatory damage in joints when compared with untreated arthritic rats . histopathological changes in our study correlated with macroscopic observations , including changes in the paw thickness . a significant reduction in the joint changes was observed in glucosamine treated groups . in the treated rats , a reversal of many of the arthritis - induced histological changes was observed . in terms of the joint analysis grading system , the untreated arthritic rats mostly showed the grade 3 characteristics , which are of the most severity level , and the glucosamine treated group showed the least severity ( grade 1 - 2 ) . observed protective effects may be due to the inhibition of inflammatory mediators by glucosamine . glucosamine acts at the joints and is known to efficiently suppress the activity of t - lymphocytes and dendritic cells , two crucial cells involved in immune responses . tumour necrosis factor- ( tnf- ) and interleukin-1 ( il-1 ) are produced by macrophages and synovial lining cells and can be found in high concentrations in arthritis . both of these cytokines act synergistically in the production of matrix metalloproteinase ( mmp ) , expression of various cell adhesion molecules ( cams ) , and secretion of prostaglandins [ 6 , 29 ] . the observed joint destruction could be because of the accumulation of mmps , and prostaglandins and glucosamine treatment has suppressed the productions of these inflammatory mediators . by suppressing joint inflammatory responses , glucosamine prevents pannus formation . during arthritis , the pannus becomes fibrosed and will have minimal vascularization , and this will result in the destruction of the cartilage . histopathology revealed reduced pannus and bony ankylosis in da rats with glucosamine treatment confirming the protective effect of glucosamine against joint damage . this supports some of the other studies that have shown that glucosamine supplementation can repair cartilage damage to some extent in osteoarthritis patients [ 10 , 11 ] . thus , our study evaluated the effect of oral administration of glucosamine to dark agouti rats on gross , biochemical , and histopathological changes . glucosamine treatment not only suppressed joint swelling , it also reduced the inflammatory mediators like tnf- and c - reactive proteins . these observations support the view that glucosamine suppresses the chronic inflammatory phase in collagen - induced arthritis . a rapid and significant beneficial effect of glucosamine was observed against arthritis in da rats in this study . thus , the findings confirmed the joint modifying and anti - inflammatory properties of glucosamine in collagen - induced arthritis . in conclusion , the findings from this study demonstrate that glucosamine effectively reduces the degree of edema and inflammation caused by collagen - induced arthritis in female dark agouti rats . a dose - response study of glucosamine - induced anti - inflammatory effects and its molecular mechanisms are being investigated further .
the present study assessed the therapeutic efficacy of glucosamine hydrochloride against collagen - induced arthritis in female dark agouti rats ( da ) . 
 arthritis was induced by intradermaly injecting a collagen and complete freund 's adjuvant suspension at multiple sites in the rat at a dose of 4  mg / kg of body weight and thereafter followed by two more boosters of the same dose , after the 1st week and 2nd week of primary immunization . after 21 days from the day of primary immunization , 
 the arthritic group rats were given oral supplementation of glucosamine hydrochloride at a dose of 300  mg / kg of body weight until day 45 . 
 the arthritic group treated with glucosamine hydrochloride from day 21 to day 45 showed significant reduction in arthritic histopathological changes of the joints , reduction in paw thickness and also a significant decrease in c - reactive protein and tnf - alpha in the serum . 
 treatment with 300  mg / kg of glucosamine hydrochloride was able to reverse the arthritic changes , hence suggesting that glucosamine has a therapeutic effect against collagen - induced arthritis .