| Osteoarthritis(OA)is the most common chronic joint disease and is becoming more prevalent worldwide with increasing life expectancy and aging of populations.It is thought to be a multifactorial degenerative joint disease,resulting in excessive morbidity,physical disability,reduced life quality,and extra health costs.Existing studies believe that OA is a total joint disease characterized by chronic progressive degeneration of cartilage as the main pathological manifestations,accompanied by abnormal remodeling of adjacent bone tissue and osteophyte formation,and involving meniscus,synovium,fat and other tissues at the same time.Among them,articular cartilage,as an important component of joints,plays important functions such as supporting and dispersing charge load,and its structural integrity is seriously damaged in OA.As the only cell component in cartilage,chondrocytes have important function in the phenotype of cartilage matrix secretion and maintenance of cartilage.A variety of factors including age-related senescence as well as the various stress events(e.g.,trauma and radiation,etc.)can cause chondrocyte senescence by activation of inflammation,reactive oxygen species(ROS)and other pathways,and accompanied by a series of age-related changes,such as hypertrophy,weakness and even loss of proliferation differentiation ability.These alterations will hinder the process of cartilage repair,gradually lead to cartilage degradation,and eventually evolved into OA.Thus,the senescence of chondrocytes plays an important role in the occurrence and development of OA.Studies have shown that the main manifestations of cell senescence are stagnation of growth cycle,metabolic changes and loss of proliferation ability.Markers associated with cell senescence include senescence-associated?-galactosidase(SA-?-Gal),senescence related heterogeneous chromosomes,up-regulated expression of p53,P21 and P16.Cell senescence can be caused by the aging of the body,but also can promote the aging of tissues.In the post-traumatic OA,p16INK4apositive senescent chondrocytes gradually accumulate in the cartilage tissue,as well as excessively secrete various cytokines(such as interleukin 1 and 6),growth factors(such as epidermal growth factor and transforming growth factor)and matrix-degrading enzymes(such as matrix metalloproteinases(MMPs),etc.),the above characteristics,also known as the senescence-associated secretory phenotype(SASP).With the continuous secretion of SASP,intra-articular senescent microenvironment(IASM)will be gradually formed,which can accelerate the aging of surrounding normal chondrocytes,significantly inhibit the proliferation and differentiation of progenitor chondrocytes and mesenchymal stem cells,and intensify the development of OA.Other studies have found that senescent cells have stronger ability to resist external adverse stimuli than normal cells,and their own survival mechanism of anti-apoptotic(such as up-regulated expression of anti-apoptotic protein BCL-2 and BCL-xl)enables them to resist the influence of apoptosis and SASP,and continue to survive in inflammatory and senescent microenvironment.It has been found that targeted clearance of p16INK4a positive senescent cells in the knee joint of premature aging mice by transgenic means can reduce intra-articular inflammation and delay the progress of post-traumatic OA.However,the operation of this transgenic method is relatively complex and involves ethical issues.Recently,a new class of small molecule drugs named"Senolytics"has been found to selectively remove senescent cells,achieving similar effects as genetically modified methods.Navitoclax(also known as ABT263)is a new kind of senolytic drug discovered in recent years.It can specifically inhibit BCL-2,BCL-xl and BCL-w proteins in BCL family,activate the Caspase signaling pathway and thus induce cell apoptosis.ABT263 was able to selectively eliminate the aging hematopoietic stem cells in the hematopoietic system of premature aging mice induced by total body irradiation(TBI),so as to enhance the function of stem cells in the body of premature aging mice and restore tissue vitality for mice with progeria.It has been reported that ABT263 can induce apoptosis of cancer cells and is used in the treatment of lymphocytic leukemia and small cell lung cancer,but its application in osteoarthritis has not been reported.In order to solve this problem,this study mainly investigate whether ABT263 can selectively remove senescent cells in OA articular cartilage and its influence on inflammation in OA.We first verified that ABT263 can effectively eliminate the senescent rat chondrocytes induced by ionizing radiation(IR).Then we investigate the effect of ABT263 on SASP expression in monolayer and pellet cultured OA chondrocytes,proving that it can improve the inflammatory microenvironment by inducing apoptosis of senescent cells.In addition,we also demonstrated that intra-articular injection of ABT263 can reduce the cartilage and subchondral bone injury in Sprague Dawley(SD)rat knee during post-traumatic OA.Purpose:To investigate the protective effect of Navitoclax(ABT263)on reducing inflammation and cartilage destruction by clearing senescent osteoarthritic chondrocytes in osteoarthritis and its possible cellular and molecular mechanisms.Methods:Part ? Effect of ABT263 on the viability of normal chondrocytes and its clearance efficiency on senescent chondrocytes1.Isolation,culture and expansion of primary chondrocytesFirstly,human OA articular cartilage was obtained from total knee arthroplasty,and primary human OA chondrocytes were extracted according to methods on previous literature.Primary chondrocytes of SD rats were extracted and isolated from the rat articular cartilage by referring to previous methods.Both types of primary chondrocytes were cultured and expanded in vitro,and the chondrocytes were identified morphologically by special staining,and the primary and P1 cells were used in subsequent experiments in vitro.2.The establishment of ionization irradiation(IR)induced senescent rat chondrocytesIn order to establish the model of senescent chondrocytes in vitro,the senescent rat chondrocytes were induced by X-ray irradiation.Primary chondrocytes were irradiated under certain conditions.After irradiation,the cells were cultured for 3 days and then passaged at1:3,and then cultured for 7 days.The senescence phenotypes were then verified by SA-?-Gal staining,crystal violet staining and q PCR.3.The effect of ABT263 on normal rat chondrocytes viabilityIn order to explore whether ABT263 has cytotoxic effect on normal chondrocytes,CCK-8 was used to detect the cell viability of normal chondrocytes under different concentration of ABT263.Chondrocytes were treated with ABT263 for 24 hours,48 hours and 72 hours.Two groups of different concentration gradients were set to determine the detailed concentration range of cytotoxicity of ABT263.4.The clearance efficiency of ABT263 on IR-induced senescent rat chondrocytesIn order to investigate the removal efficiency of ABT263 on IR-induced senescence chondrocytes,cell counting kit-8(CCK-8)was used to detect the cell proliferation-toxicity effect of ABT263 at 24,48 and 72 hours after treatment.Phase contrast microscope was used to observe and compare the growth status of senescent chondrocytes under the influence of different concentrations of ABT263.Flow cytometry was used to detect apoptosis and the proportion of apoptotic cells was statistically analyzed.Part ? Study on ABT263 improving inflammatory microenvironment of human OA chondrocytes and maintaining chondrocyte phenotype1.To study the senescent cells in human OA cartilage tissue and primary OA chondrocytesThe OA cartilage blocks were obtained from total knee arthroplasty.The sections of the severely degenerated cartilage and the similar normal appearance of the non-weight-bearing cartilage were selected,and the primary OA cartilage cells were extracted.Subsequently,the senescent cells in OA cartilage blocks and primary chondrocytes were evaluated using SA-?-Gal staining and HMGB1 immunohistochemical staining.2.Effects of ABT263 of different concentrations on OA chondrocytes in micromass cultureTo explore the appropriate concentration of ABT263 on OA chondrocytes,OA chondrocytes were cultured in micromass system for 21 days,and then treated with ABT263at different concentrations for 3 days and 10 days respectively.The senescent cells were cultured in growth medium for another week after 3 days of treatment,and then detected by SA-?-Gal staining.3.Effects of ABT263 on inflammatory microenvironment of OA chondrocytes in monolayer culture and pellet cultureOA chondrocytes were evaluated immediately 3 days after treatment with ABT263,and the other group was cultured for another week and evaluated at 10th day.The senescent cells and cell apoptosis were detected by SA-?-Gal staining,flow cytometry and immunofluorescence.q PCR and western blot were used to detect the expression of inflammatory factors.Bone marrow Mesenchymal stem cells(BMSCs)pellet culture was evaluated by immunohistochemical staining after 21 days culture,and the m RNA and protein levels of senescent-associated genes and inflammatory factors in the pellet were quantitatively detected by q PCR and western blot.4.Effects of ABT263 on chondrocyte phenotypes in monolayer culture and pellet cultureThe m RNA level and protein expression of COLII,ACAN and SOX9 were detected by q PCR and western blot respectively at day 3 and day 10 after treatment of ABT263.and the evaluation methods above were applied in OA chondrocytes and BMSC in pellet culture system for 21 days,so as to explore the effects of ABT263 on chondrogenic phenotypes and chondrogenic differentiation of mesenchymal stem cells.Part ? Intra-articular injection of ABT263 alleviated the cartilage and subchondral bone damage and reduced inflammation in post-traumatic OA of rat knee1.Establishment of post-traumatic OA model in rat knee by DMM surgeryThe OA model induced by Destabilization of the Medial Meniscus(DMM)surgery was achieved through cutting the tibial ligament of the medial meniscus in male SD rats aged 4-6weeks.The sham operation group used a medial incision to expose the knee cavity and then closed the incision with sutures.The intra-articular injection was performed 4 weeks after the operation.2.Intra-articular injection of ABT263 and histological evaluation of articular cartilageTo investigate the appropriate concentration of ABT263 in the knee joint of rats,different concentrations(0.25,1 and 5 M)of ABT263 were injected into the joint cavity of rats 4weeks after DMM operation.Two injections were administered weekly for two weeks,followed by H&E and Safranin O/Fast Green staining to evaluate the articular cartilage in the experimental group and the control group and the OARSI histological score was performed.3.Micro CT scanning of isolated rat knee joint,immunohistochemical staining and RT-q PCR analysisAfter 4 times of intra-articular injection,the experimental rats were sacrificed and knee samples were fixed.The total knee joints of rats were performed micro-CT scanning and three-dimensional reconstruction to evaluate the subchondral bone injury.Immunohistochemical staining and q PCR were used to qualitatively and quantitatively analyze the expression of senescence related genes,inflammatory factors and cartilage matrix proteins in articular cartilage,and to explore the biological effects of ABT263 in the articular cavity of rats.Result:Part ? Effect of ABT263 on the viability of normal chondrocytes and its clearance efficiency on senescent chondrocytes1.ABT263 had no cytotoxic effect on normal rat chondrocytes in a certain concentration rangeThe results of CCK-8 showed that ABT263 within concentration of 2.5 u M had no cytotoxic effect on normal rat chondrocytes.For OA chondrocytes,ABT263 showed no significant cytotoxicity at concentrations less than 3?M.2.ABT263 exhibited a dose-dependent manner clearance effect in IR-induced Sn Cs After X-ray irradiation,rat chondrocytes showed the characteristics of cell senescence,a large number of SA-?-Gal positive staining appeared in the cytoplasm,the proliferation ability was significantly reduced,and the expression of senescence-associated genes was significantly increased.CCK-8 results showed that ABT263 significantly inhibited the cell viability of senescent rat chondrocytes,and the inhibitory effect was enhanced with the increase of ABT263 concentration.The results of phase contrast microscope and flow cytometry showed that ABT263 could eliminate Sn Cs by promoting apoptosis of senescent chondrocytes,and the elimination effect enhanced with the increase of concentration.Part ? Study on ABT263 improving inflammatory microenvironment of human OA chondrocytes and maintaining chondrocyte phenotype1.A certain proportion of SA-?-gal positive Sn Cs was found accumulation in human OA articular cartilageThe SA-?-gal staining indicated the existence of SA-?-gal positive senescent cells in advanced OA cartilage tissue and primary OA chondrocytes,and the histochemical staining results suggested that HMGB1 expression was weakened in advanced OA cartilage tissue and partially expressed outside the chondrocytes nucleus.2.ABT263 of 2.5?M and 5?M had a potent clearance effect on Sn Cs in micromass-cultured OA chondrocyte.OA chondrocytes were treated with different concentrations of ABT263 for 3 days and10 days after 21-day micromass culture.The results of SA-?-gal staining indicated that only ABT263 of 2.5 and 5 u M could effectively eliminate SA-?-gal positive Sn Cs,while the lower concentration of ABT263 had no obvious killing effect on SA-?-gal positive Sn Cs.3.ABT263 promoted apoptosis of senescent chondrocytes and improved inflammatory microenvironment in OA in monolayer and pellet cultureThe results of SA-?-gal staining and HMGB1 fluorescence showed that the proportion of Sn Cs in OA chondrocytes was significantly reduced by ABT263,and the number of Sn Cs was continuously inhibited after one-week culture.Flow cytometry indicated that the cell apoptotic ratio increased after 3-day treatment with ABT263,and the expression of C-Caspase-3 protein increased by Western blot,suggesting that ABT263 mediated apoptosis of senescence chondrocytes through Caspase-3 signaling pathway.In monolayer culture,the expression of SASP was inhibited after one week without ABT263.Immunohistochemical staining results showed that ATB263 could significantly reduce SASP protein expression in the pellet culture of OA chondrocytes.q PCR and western blot quantitative detection results were consistent with the immunohistochemical staining results above,suggesting that ABT263 could improve OA inflammatory microenvironment to a certain extent by promoting apoptosis of senescent chondrocytes.4.ABT263 was beneficial to chondrocyte phenotype maintenance in monolayer culture and pellet cultureUnder monolayer culture,q PCR and western blot results indicated that m RNA and protein expression of ACAN were significantly increased on day 10,indicating that proteoglycan synthesis in cartilage matrix was significantly enhanced.For pellet culture,the results of Safranine O staining indicated increased proteoglycan synthesis in OA chondrocytes and HBMSC,and western blot showed significantly increased COLII and ACAN protein expression,suggesting enhanced cartilage matrix synthesis in pellet culture.Part ? Intra-articular injection of ABT263 alleviated the cartilage and subchondral bone damage and reduced inflammation in post-traumatic OA of rat knee1.Intra-articular injection of ABT263 alleviated cartilage and subchondral bone injury caused by post-traumatic OA in DMM ratsH&E and Safranin O/Fast Green staining results suggested that the surface wear of tibial cartilage was relatively severer in the solvent injection group,while the surface damage of ABT263 injection group was lighter and showed relatively intact cartilage structure,indicating that intra-articular injection of ABT263 could attenuate the cartilage damage caused by traumatic OA.Compared with the solvent injection group,the lower OARSI score of the ABT263 injection group also suggested that the cartilage damage was relatively mild,indicating that ABT263 had a protective effect on cartilage in post-traumatic OA.Micro-CT scan and three-dimensional reconstruction results exhibited that the subchondral bone structure of the solvent injection group collapsed and the rough surface,while the subchondral bone of the ABT263 injection group displayed a relatively smooth and flat surface.2.Intra-articular injection of ABT263 decreased the number of p16-positive Sn Cs and reduced inflammation in rat articular cartilageImmunohistochemical results suggested that the number of p16-positive Sn Cs decreased,and the expression of HMGB1 increased in chondrocytes,while the expression of MMP13 was down-regulated.Quantitative analysis results from q PCR indicated that the gene expressions of P16,P21,IL1,IL6,MMP13 and ADAMTS5 were significantly down-regulated,while the expressions of COLII and ACAN were up-regulated,suggesting that the inflammatory response in articular cartilage of rats was attenuated and the catabolic effect of cartilage matrix was weakened,accompanied by enhanced matrix deposition.Conclusions:1.Sn Cs exist and accumulate in human OA cartilage tissue and primary OA chondrocytes;2.The appropriate concentration of ABT263 can effectively remove senescent chondrocytes without affecting the viability of normal chondrocytes;3.ABT263 induce apoptosis of senescent chondrocytes through Caspase-3 signaling pathway,thereby improving the inflammatory microenvironment of OA chondrocytes and contributing to maintaining the chondrogenic phenotype;4.Intra-articular injection ABT263 could protect cartilage and subchondral bone from damage in post-traumatic OA,and at the same time alleviates the level of inflammation in the articular cartilage,inhibits the catabolism of cartilage,and promotes the synthesis of cartilage matrix.Significance:This study provides a new strategy for the clinical treatment of OA,proving that targeting and eliminating Sn Cs by senolytics might be a potential means to mitigate the progression of OA from the source of the disease.Moreover,this research discovered a new application field of drug ABT263 and provides a theoretical basis for the treatment of OA with senolytics.Furthermore,it reveals the potential possibility of OA as a new indication of ABT263. |
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