Glabridin Inhibits Osteoarthritis Development Via Protecting Chondrocytes Against Oxidative Stress,apoptosis And Promoting MTOR Mediated Autophagy

Osteoarthritis(OA)is a degenerative joint disease with a high prevalence rate in the elderly population.The knee joint is the most common site of this disease.The pathogenesis of OA is still not well understood,and so far,the drugs approved by regulatory agencies have not shown convincing effects on the treatment of OA.When the disease is advanced,patients are left with costly joint replacements.Oxidative Stress(OS)is a negative effect produced by free radicals in the body.OS plays an important role in the occurrence and development of OA.Imbalance of the REDOX system can lead to intraarticular lesions and synovial inflammation.The accumulation of reactive oxygen species can interfere with the anabolic activities of chondrocytes,leading to dysfunction and degeneration of chondrocytes,and further leading to apoptosis and senescence.The excessive apoptosis of chondrocytes is a very important cause of cartilage degeneration.Some studies have suggested that antioxidants may have an anti-apoptotic effect on chondrocytes by reducing oxidative stress.In addition,autophagy inhibition is believed to be associated with cartilage degeneration and chondrocyte apoptosis in osteoarthritis.Autophagy of chondrocytes has protective and anti-apoptotic effects and has become one of the hot spots in osteoarthritis research.When OA occurs,cartilage tissue containing only a single chondrocyte has poor self-repair ability after injury due to insufficient blood supply and innervation.With the continuous degradation of cartilage matrix,the functional activity of chondrocytes is also continuously impaired,and the ability of chondrocytes to secrete type ? collagen,aggregated proteoglycan and other matrix components is decreased,which further increases the difficulty of self-repair of articular cartilage.Glabridin is a flavonoid extracted from the licorice plant,which has been used in clinical practice to treat many ailments,from common coughs to cancer.Glabridin has a strong scavenging effect on free radicals.As a natural antioxidant,it has the same antioxidant power as vitamin E.Therefore,it is used to prevent and treat some pathological changes related to free radical oxidation,such as atherosclerosis,cell aging and so on.As an important cell pathway,mammalian target of rapamycin(mTOR)signaling pathway is widely involved in cell proliferation,apoptosis and autophagy.This study aims to explore the feasibility of glabridin in the treatment of OA and its possible mechanism,in order to provide a possible method for the treatment of OAChapter ? Study on inhibiting osteoarthritis development in rats by intraarticular injection of glabridinObjective:To investigate the effect of glabridin on delaying OA progression in rats and its possible mechanism.Methods:Male SD rats with body weight range of 80-90g were selected.In this study,the model of osteoarthritis was established by anterior cruciate ligament transection(ACLT)of the right knee.The contralateral knee sham operation was performed without ligament amputation.They were randomly divided into six groups:normal group,sham operation group,normal saline group and glabridin intervention group(1,5,10mg/kg).Rats were injected with glabridin 100?l twice a week for 4 or 8 weeks.After 4 or 8 weeks,the rats were first evaluated for pain and inflammation by hot plate method and weight-bearing asymmetry test.Subsequently,the rats were sacrificed and the knee tissue samples were collected.The pathological changes of osteoarthritis cartilage were evaluated by Osteoarthritis Research Society International(OARSI)grading system,and the knee cartilage tissue of rats was evaluated by hematoxylin eosin(HE),saffronin O solid green and alcian blue staining.The expression levels of mTOR,LC3B,MMP13(matrix metalloprotein),Adamts5 and type ? collagen in knee cartilage were detected by immunohistochemical staining.The apoptotic level of knee cartilage was evaluated by TUNEL staining.Pathological observation was made on abnormal liver and kidney tissues,and the toxicity of the drug was preliminarily evaluatedResults:1.All SD rats recovered well after operation,without death,j oint infection and other adverse conditions.2.The results of hot plate test and weight-bearing asymmetry test showed that glabridin could effectively relieve OA pain in rats.3.OARSI pathological score showed that the degeneration of osteoarthritis cartilage could be effectively alleviated by glabridin.4.HE,sanguine O solid green and alcian blue staining showed that the degradation of cartilage extracellular matrix was effectively alleviated after glabridin intervention.5.Immunohistochemical staining results showed that the expression of mTOR was significantly decreased after glabridicin intervention,while the expression of LC3B was significantly increased,and the expression of two key enzymes Mmp13 and Adamts5 in the extracellular matrix of chondrocyte was significantly decreased.The expression of type ? collagen was significantly increased after glabridin intervention.6.TUNEL staining of knee cartilage showed a decrease in chondrocyte apoptosis in articular cartilage after glabridin intervention.7.No negative effects of the drug on histopathology of liver and kidney in rats were detected.Conclusion:1.After the rats were injected with different concentrations of glabridin in the knee joint,no death,joint infection,wound nonhealing and other adverse complications occurred in all rats.Moreover,no obvious toxic and side effects were observed in liver and kidney histopathology of rats after administration,suggesting that glabridin has certain safety in delaying OA progression in rats.2.The knee joint injection treatment with different concentrations of glabridin could significantly inhibit the erosion and degradation of articular cartilage and reduce the loss of proteoglycan at 4W and 8W.3.Glabridin could effectively inhibit the apoptosis of articular chondrocytes,and could increase the expression of LC3 while decrease the expression of mTOR,suggesting that inhibition of apoptosis,increased autophagy and mTOR signaling pathway are involved in the process of Glabridin retarding the progression of OA in rats.Chapter ? Effects of glabridin on proliferation activity,extracellular matrix generation and oxidative stress level of human OA chondrocytesObjective:To study the effects of glabridin on human OA chondrocyte proliferation activity,extracellular matrix generation and oxidative stress level.Methods:First,chondrocytes from human knee OA were extracted for in vitro experiment.In this experiment,alcian blue staining and type ? collagen immunofluorescence staining were used to identify chondrocytes from human knee OA.Chondrocytes were incubated with glabridin at different concentrations(0,0.01,0.1,1,5 and 10 ?M)for 24 h.CCK-8 assay was used to detect the effect of glabridin on the influence of chondrocytes vitality,alamar blue assay was used to detect the effect of glabridin on chondrocytes activity in different periods(1,3,5,7 d),western blot assay was used to detect different concentrations of glabridin(0,0.01,0.1,1,5 ?M)on type ? collagen expression of chondrocytes.Meanwhile,the expression levels of extracellular matrix related genes,type? collagen(Col2Al),Aggrecan(Acan),SRY-box 9(Sox9)and Proteglycan 4(Prg4)in chondrocytes were detected by reverse transcription and polymerase chain reaction(RT-PCR).Subsequently,the effects of glabridin on the expression levels of type ?collagen,ACAN and SOX9 were detected by immunofluorescence staining.Reactive oxygen species(ROS)levels in chondrocytes were detected by ROS detection kit Superoxide Dismutase(SOD)and Catalase(CAT)levels in chondrocytes were detected by ROS detection kit and Western-blot methodResults:1.The proteoglycan in the cytoplasm of chondrocytes was stained blue by alcian blue,and the primary human OA chondrocytes are spindle-shaped.Immunofluorescence staining showed that type ? collagen in the cytoplasm of human chondrocytes was red.Both staining methods showed that the cells extracted from articular cartilage were chondrocytes.2.Glabridin had no obvious cytotoxicity to human OA chondrocytes at a concentration of 0-1 ?M,but had obvious cytotoxicity at a concentration of 5?M or higher.Alamar blue method detected that 0-10?M glabridin has no obvious toxicity to human OA chondrocytes after 1,3,5,and 7 days of culture.3.In human OA chondrocytes,we found that the expression levels of extracellular matrix-related genes COL2A1,AC AN,SOX9 and PRG4 were up-regulated after glabridin treatment(0.01-5?M).4.The level of reactive oxygen species in chondrocytes was significantly reduced.In addition,the activities of antioxidant enzymes CAT and SOD increased significantly under the intervention of glabridin.Conclusion:1.Chondrocytes were extracted from human knee cartilage in vitro.2 Glabridin had no negative effect on the proliferation activity of chondrocytes in a certain concentration range.3.Glabridin could stimulate the production of extracellular matrix in chondrocytes.Glabridin could reduce the production of reactive oxygen species in chondrocytes and reduce the level of intracellular oxidative stressChapter ?:Glabridin inhibits apoptosis of human OA chondrocytes and activates mTOR mediated autophagy pathwayObjective:To investigate the effects of glabridin on apoptosis and mTOR mediated autophagy of human OA chondrocytes.Methods:Human OA chondrocytes were cultured in vitro.When the cell density reached between 60%-80%,glabridin was stimulated with different concentrations(0,0.01,0.1,1?M)for 24 h.The effect of glabridin on the apoptosis of chondrocytes was detected by flow cytometry,TDT-mediated DUTP Nick-End Labeling(TUNEL)and immunofluorescence staining.The expression levels of apoptotic genes,including poly ADP-ribose polymerase(PARP),cleaved PARP,Bax,and Bcl-2,were detected by Western-blot assay after glabridin treatment.The formation of autophagosomes in chondrocytes treated with glabridin was observed by transmission electron microscopy,and then LC3 immunofluorescence staining and Western-blot detection of autophagy related proteins(LC3,Atg5,Beclinl)were performed to observe the effect of glabridin on mTOR-mediated autophagy pathway.Results:1.After cell stimulation with different concentrations(0,0.01,0.1,1?M)of glabridin for 24 hours,flow cytometry showed that the apoptotic rate of chondrocytes was reduced,and the results of TUNEL staining also indicated that after the intervention of glabridin,the apoptosis rate of chondrocytes decreased.In addition,the immunofluorescence detection of cleaved-capase3 indicated that the expression of cleaved-capase3 in chondrocytes was significantly reduced after the intervention of glabridin.2.Transmission electron microscopy observed that autophagosomes in chondrocytes increased significantly after glabridin treatment.Immunofluorescence staining indicated that LC3 expression in chondrocytes increased after glabridin intervention.The results of the western blot experiment further showed that the expression of autophagy-related proteins LC3?/LC3?,Beclinl and Atg5 increased after the intervention of glabridin compared with the control group.3.The results of western-blot experiments suggest that the expression of phosphorylated mTOR is significantly reduced after the intervention of glabridin.Conclusion:1.Glabridin could inhibit the apoptosis of human OA chondrocytes.2.Glabridin could promote autophagy of human OA chondrocytes.3.Glabridin might inhibit chondrocyte apoptosis through mTOR-mediated autophagy pathway.