| Osteoarthritis(OA)is a chronic degenerative joint disease which affects the pain and function of patients all over the world.With the increase of age,the incidence of osteoarthritis is increasing,which is a serious burden on medical care,economy and society in China.The pathological features of osteoarthritis include:chondrocyte apoptosis,cartilage matrix destruction,subchondral ossification,osteophyte formation and synovial inflammation.The pathogenesis of osteoarthritis is not very clear yet,so the treatment of osteoarthritis is quite limited.Non-surgical treatment of early osteoarthritis mainly includes oral non-steroidal anti-inflammatory drug(Nonsteroidal Antiinflammatory Drugs,NSAIDs)and intra-articular injection of sodium hyaluronate and so on.However,NSAIDs drugs have some toxic and side effects on gastrointestinal and cardiovascular diseases,which is a danger to the patient's health.Therefore,it affects the application in the treatment of osteoarthritis.Quercetin(QUE)is one kind of pentahydroxyflavone,which exists widely in leaves,stems,and root masses of many plants with highly medicinal value.Recently,quercetin has been found to possess the effect of anti-inflammatory,anti-oxidation,anti-apoptosis,scavenging oxygen free radicals,inhibiting osteoclast absorption and immune regulation.At present,quercetin has been widely studied in the field of tumor,but the influence of quercetin on chondrocytes has not been widely reported.Therefore,we want to make sure whether quercetin can inhibit the apoptosis of chondrocytes and affect the expression level of cytokines in the pathological process of osteoarthritis,so that quercetin would play an important role in the treatment of osteoarthritis.This study was divided into two parts:in vitro and in vivo.firstly,the chondrocytes of SD rats were cultured in vitro,and 50 ?g/ml IL-1 ?was added to the chondrocytes to induce apoptosis.Then,different concentrations of quercetin were added to the experimental group to detect the apoptosis rate of chondrocytes and the difference of the expression of Bcl-2 and Bax,Caspase-9 genes in chondrocytes.Bcl-2 protein and Bax protein expression level as well as Caspase-9 and Caspase-3 protein level were further detected by the analysis of Westernblot.Our study showed that quercetin could inhibit the apoptosis of chondrocytes induced by IL-1 ?.By the mechanism of up-regulating the expression of anti-apoptotic Bcl-2 gene and Bcl-2 protein,inhibiting the expression of pro-apoptotic Bax gene and Bax protein,Caspase-9 protein and Caspase-3 protein,we made sure that quercetin can protect the cartilage.Secondly,in the OA animal model induced by intraarticular injection of papain,the swelling degree of knee joint(diameter of knee joint)was measured 4 weeks after intragastrically administered with different concentrations of quercetin suspension regularly and quantitatively every day.Histological features and serum levels of TNF-?and IL-1 ? were measured to investigate the effect of quercetin on osteoarthritis.The results suggest that quercetin can reduce articular cartilage injury,relieve joint swelling and inhibit the expression of TNF-? and IL-1 ? in serum,which may play a role in the treatment of osteoarthritis.Therefore,we conclude that a certain concentration of quercetin can play a protective role in articular cartilage by inhibiting inflammation and anti-apoptosis mechanism.Part 1 The effect of quercetin on interleukin-1? induced apoptosis of rat chondrocyteObjective:To observe the effect of quercetin on the apoptosis of chondrocytes induced by interleukin-1 ?(IL-1-?)and its molecular mechanism.Methods:Chondrocytes were divided into 5 groups randomly,including control group(no intervention),IL-1 p group(IL-1 ? 50 ?g/mL,for 24 h)and three different concentrations of quercetin group(100 ?M,200 ?M,400 ?M quercetin with IL-1 p together for 24 h).Chondrocytes of each group were incubated in the orifice plate and repeated for three times.The apoptosis rate of each group was calculated by flow cytometry.The relative expression levels of Bcl-2,Bax and Caspase-9 were gained by PCR and Westernblot methods,while Caspase-3 expression level was analyzed by Westernblot.Results:Compared with the control group,the apoptosis rate of chondrocytes in the other 4 groups was significantly increased(P<0 05).The expression of Bax gene and Caspase-9 gene in the other 4 groups were significantly higher than those in the control group,while the expression of anti-apoptotic Bcl-2 gene was significantly decreased(P<0 05).Western blot analysis also showed that the expression of Bax protein and Caspase-9 protein were significantly increased,while the expression of Bcl-2 protein was significantly decreased.(P<0.05).Compared with the IL-1? group,the apoptosis rate of chondrocytes in the high concentration quercetin group(400 ?M)was significantly lower than that in the control group(P<0.05).The expression of Bax gene and Caspase-9 gene were significantly x lower in the high concentration quercetin group than in the control group,while the expression of anti-apoptotic Bcl-2 gene was significantly increased(P<0.05).Western blot analysis showed that the expression of Bax protein,Caspase-9 protein and Caspase-3 cleavage protein decreased significantly,but the expression of Bcl-2 protein increased significantly(P<0.05).Conclusion:Quercetin can decrease the expression of Bax,increase the expression of Bcl-2,and decrease the expression of Caspase-9 and Caspase-3,which would contribute inhibiting the apoptosis of chondrocytes induced by IL-1 ?.Part 2 The in vivo effect of quercetin on rat osteoarthritis model Objective:To observe and verify the protective effect of quercetin in different concentrations on rat osteoarthritis model.Methods:48 SD rats were divided into 6 groups randomly,including blank group,model control group,aspirin positive control group,low-dose quercetin group,middle-dose quercetin group and high-dose quercetin group.With the exception of the blank group,the remaining 40 rats were injected with papain into the right knee joint to make the OA model.After 4 weeks of modeling,the 40 rats in the model group were intragastrically administered with 4 mL different drug solutions per day.The different drug solutions were as follows:model control group:0.9%saline 4mL per day,aspirin positive control group:5mg/mL aspirin solution gavage 4mL per day;In the low-dose quercetin group,the quercetin suspension containing 2.5mg/mL was given intragastrically for 4mL per day,the middle-dose quercetin group was given quercetin suspension with 5mg/mL for 4mL per day,and the high-dose quercetin group was given quercetin suspension containing 10mg/mL for 4mL per day.It lasted for 4 weeks.On the second day after the last gavage,the diameter of the right knee joint was measured,the tissue of the knee joint was taken for histological preparation,and the levels of serum inflammatory cytokines TNF-a and IL-1? were measured by ELISA method.Results:Compared with the blank group,the diameter of the right knee joint in the model group and the quercetin group was larger than that in the control group significantly(P<0.05).The levels of TNF-a and IL-1? in serum of the model group,the low-dose and middle-dose quercetin group were also higher significantly(P<0 05).The diameter of knee joint in the high-dose quercetin group and aspirin-positive control group was significantly decreased,and the histological score was significantly lower(P<0.05),compared with the model group.The levels of serum TNF-? and IL-1? in the high-dose quercetin group and aspirin positive control group were significantly lower than those in the model group by ELISA method,and the serum IL-1 ? level in the middle dose quercetin group was also significantly lower than that in the model group(P<0.05).Conclusion:Quercetin can down-regulate the expression of inflammatory factors and protect articular cartilage in OA animal model. |
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