Effects Of Bee Venom On Autophagy And Apoptosis Balance Of Chondrocytes In Arthritic Rats Induced By Sodium Iodoacetate

ObjectiveOsteoarthritis is the most common chronic degenerative joint disease,which is commonly seen in the elderly.With the development of social economy and the improvement of the quality of people’s life,there are more and more obese people in China.The prevalence of osteoarthritis is increasing in China since obesity is an important cause for the development and progression of osteoarthritis.According to statistics,the prevalence rate of osteoarthritis in China is about 15%.The large number of people affected places a heavy burden on families and countries.The prevention and treatment of osteoarthritis has become a major challenge in China’s medical and health care.However,the pathogenesis of osteoarthritis has not been elucidated so far,and there is no specific drug that can completely cure osteoarthritis.Therefore,revealing the pathogenesis of osteoarthritis and finding new safe and effective therapeutic drugs have become the top priority in the prevention and treatment of osteoarthritis.Apitherapy as a traditional Chinese medicine characteristic therapy,simple and cheap,has a significant curative effect for rheumatism rheumatoid arthritis and other joint diseases.And apitherapy is getting more and more attention in clinical practice now.However,at present,there are few basic researches on apitherapy or bee venom,and the principle and mechanism of apitherapy for joint diseases have not been clarified.In this study,the rat model induced by sodium iodoacetate was elected as a purpose to duplicate the pathogenesis of osteoarthritis in human.This research attempts to observe the effects of bee venom on autophagy and apoptosis balance of chondrocytes in knee joint in rat model induced by sodium iodoacetate through animal experiments with apitherapy as the main intervention method,which will reveal the mechanism of bee venom in treating osteoarthritis.MethodsEighty female SD rats were randomly divided into blank control group,model control group,high-dose bee venom group,medium-dose bee venom group,low-dose bee venom group,acupuncture group,glucosamine hydrochloride group,NF-κB antagonist group,10 rats in each group.Except for the normal group,the rats in the other groups were induced to establish the rat osteoarthritis(OA)model by injecting sodium iodoacetate into the right knee cavity.When sodium iodoacetate was injected,the entry point was at the lower side of the patella of the right knee of the rats,and the needle tip was tilted upward to puncture into the articular cavity,where the sodium iodoacetate solution was slowly injected in.After successful modeling,rats in the normal group and model group were not treated.The rats in the low-dose,medium-dose and high-dose bee venom groups were all treated with live bee stings,once every 3 days.The rats in the low-dose group consumed 1 live bee each time per rat,and the stinging point was the inner side of the lower edge of the patella of the right knee joint.The rats in the medium-dose group consumed 2 live bees each time per rat,and the stinging points were medial and lateral to the lower margin of the patella of the right knee joint.The rats in the high-dose group consumed 4 live bees each time per rat,the stinging points were medial and lateral to the lower edge of the patella as well as the medial and lateral to the upper edge of the patella of the right knee.The rats in the acupuncture group were treated with acupuncture,each rat was given 2 needles per treatment,once every 3 days,and the acupuncture points were medial and lateral to the lower margin of the patella of the right knee joint.The rats in glucosamine hydrochloride group were treated with intragastric administration twice a day,with dose of 67.5mg/kg each time.Rats in the NF-κB antagonist group were injected with NF-κB antagonist in the lateral thigh muscle of the right lower limb,with dose of 10mg/kg,once every 3 days.All above groups were treated for 6 weeks consecutively,and all rats were killed the next day after the last intervention at the end of 6 weeks.During intervention,changes in weight,arthritis index and mechanical pain threshold of each rat were recorded once a week.At the end of the experiment,all rats were anesthetized and sacrificed in order to collected the arterial blood from the abdominal cavity of the rats,and extracted the joint fluid of the right knee joint of the rats.And then,amputated the right knee joint of each rat and fixed with 4%paraformaldehyde.Detect the concentrations of inflammatory cytokines TNF-α and IL-1β in serum and joint fluid of each rat respectively by elisa.The right knee joint of rats fixed with 4% paraformaldehyde were prepared for paraffin sections,the cartilage tissue and synovial tissue of the joint treated with HE staining respectively,and observed the pathological manifestations of the cartilage and synovial for the arthritis score.In addition,paraffin sections of cartilage tissue were also taken to detect the expression of autophagy and apoptosis-related proteins in cartilage tissue with immunohistochemistry method in order to evaluate the level of autophagy and apoptosis in chondrocytes,and detected the levels of NF-κB nuclear transport by immunofluorescence assay.ResultsAfter 2 weeks of modeling induced by the injection of sodium iodoacetate into the joint cavity,the right knee joint of the rats presented varying degrees of redness,swelling and pain,and the mechanical pain threshold of the rats decreased.Sacrificed two rats randomly,and opened the right knee cavity.Slight visible corrosion of articular cartilage was found,which means the RAT KOA model was successfully established.After 6 weeks of experimental intervention by different means,there was no significant difference in the weight change of rats in each group,and the mechanical pain threshold of rats in each treatment group increased in different degrees,especially in the high-dose group of bee venom,while the mechanical pain threshold of the model control group continued to decrease.The arthritis symptoms of the rats in different treatment groups were relieved to different degrees after 6 weeks.According to the arthritis score results,the arthritis symptoms of rats in the NF-κB antagonist group,acupuncture group and glucosamine hydrochloride group were not significantly reduced compared with the model control group(P>0.05).In addition,among three bee venom groups,only the scores of rats in the high-dose group were significantly lower than those in the model control group(P<0.05),and the arthritis symptoms of rats in the medium-dose and low-dose groups were not significantly reduced(P>0.05).The results of ELISA showed that there was no significant difference among the experimental groups of inflammatory cytokines IL-1β and TNF-α in the joint fluid or serum(P>0.05),while the levels of IL-1β in serum were significantly higher than those in the joint fluid(P<0.05).HE staining results showed obvious degradation of cartilage matrix and cartilage tissue in model rats induced by sodium iodoacetate.After 6 weeks of treatment,the bee venom significantly reduced the loss of chondrocytes and cartilage matrix,especially in the high-dose bee venom group.Immunohistochemical results showed that the expression of Bax in the cartilage of experimental rats was significantly increased compared with that of normal rats,especially in model control rats.The expression of Bax in bee venom groups were less than that in the model control group,and the medium-dose and high-dose bee venom group were less than low-dose bee venom group.Bcl-2 was highly expressed in normal cartilage tissue,but it was significantly reduced after sodium iodoacetate injection.After treatment,the expression of Bcl-2 was increased in all experimental groups.Interestingly,caspase3 was not significantly expressed in either the normal group or the experimental group.Beclin1,LC3-b and p62 were all highly expressed in normal cartilage.The expressions of Beclin1,LC3-b and p62 in all experimental groups were significantly lower than those in blank control group.The expressions of Beclin1,LC3-b and p62 in bee venom groups were higher than those in model control group,and the medium-dose and high-dose bee venom group were higher than low-dose bee venom group.Immunofluorescence results showed that the NF-κB signaling pathway was activated in rat chondrocytes of all the experimental groups.ConclusionBee-sting therapy has obvious anti-inflammatory and analgesic effects,can significantly reduce the joint inflammation and synovitis in KOA rats,reduce the arthritis index,improve the injury of articular cartilage.Bee venom can promote autophagy of chondrocytes and inhibit apoptosis of chondrocytes,thereby alleviating the disease progression of KOA,which is related to the activation of NF-κB signaling pathway in chondrocytes and synovial cells.The efficacy of bee-sting therapy was better than that of glucosamine hydrochloride,NF-κB antagonists and acupuncture,and showed dose-dependent effect.The efficacy of bee venom high dose group was significantly better than that of medium dose group and low dose group.In addition,the experimental results showed that acupuncture did not show significant efficacy,suggesting that bee venom plays the main role in the treatment of KOA.