| Osteoarthritis(OA),also known as degenerative osteoarthropathy,is one of the common age-related diseases.With the accelerated aging process in China,the prevalence rate of OA is gradually increasing.OA has become a major problem affecting the health and quality of life of elderly people in China,causing enormous economic burden to patients,families and society.At present,the main clinical treatment for OA is nonsteroidal anti-inflammatory drugs and joint replacement surgery,both of which are mainly to relieve the clinical symptoms and unable to change the course of this disease.There is no effective etiological treatment so far.Apoptosis is an active physiological process in which the body eliminates excess or abnormal cells through gene regulation.Chondrocytes are the only cellular components in normal articular cartilage.The decrease of chondrocyte abundance and degradation of extracellular matrix caused by excessive apoptosis of chondrocytes are considered as the main causes of OA.Autophagy,as a mechanism to maintain normal cell function and homeostasis,has been found to be closely related to apoptosis recently.Some studies have shown that autophagy plays an anti-apoptotic role in chondrocytes,and enhancement of autophagy can delay the progress of OA.Therefore,the drugs that can activate autophagy may be an effective method to treat OA in the future.Inflammatory response plays an important role in the development of OA.Proinflammatory cytokines are one of the key mediators causing chondrocyte apoptosis,among which interleukin-1?(IL-1?)and Tumor necrosis factor ?(TNF?)are generally X considered as the main proinflammatory cytokines causing degradation of articular cartilage matrix.In addition,other pro-inflammatory cytokines such as interleukin-18(IL-18)have also been found to play a key role in the development of OA recently.Previous studies have shown that the concentration of IL-18 in synovial fluid of OA patients is significantly increased,and IL-18 can induce the inflammatory response of synovial cells and chondrocytes.What's,IL-18 can induce apoptosis in human articular chondrocytes.However,the effect of IL-18 on autophagy regulation of chondrocytes are still unclear.Therefore,the purpose of this study was to investigate the effects of IL-18 on autophagy regulation of chondrocytes and the possible mechanisms involved,and to explore the potential etiological treatment options.This study is consisted of four parts.Firstly,the effect of IL-18 on rat chondrocyte apoptosis was studied.We found that IL-18 could induce apoptosis and degeneration of chondrocytes in vitro,and the degeneration of articular cartilage was observed in rat knee joint cavity injection model in vivo.Secondly,the effect of IL-18 on rat chondrocyte autophagy was studied.Results showed that IL-18 could induce autophagy deficiency of chondrocytes in vitro.Thirdly,we investigated the specific molecular mechanism of IL-18-induced autophagy deficiency in chondrocytes and its relationship with chondrocyte degeneration.Results showed that autophagy deficiency of chondrocytes caused by IL-18 was achieved via the PI3K-Akt-m TOR signaling pathway,and it was closely related to chondrocyte degeneration.Finally,we observed the effect of Rapamycin(Rapa),a common autophagy agonist,on IL-18-induced autophagy deficiency and apoptosis of chondrocytes in vitro and in vivo.We found that rapamycin could improve IL-18-induced autophagy deficiency and protect chondrocytes from IL-18-induced apoptosis.All the results demonstrated that IL-18-mediated autophagy deficiency can promote chondrocyte apoptosis and plays an important role in the development of OA.Moreover,rapamycin can activate autophagy to protect cartilage from IL-18-induced chondrocyte apoptosis,providing a potential etiological treatment for OA.Chapter ?The effect of IL-18 on chondrocyte apoptosis.Objective:To investigate the effect of IL-18 on chondrocyte apoptosis.Methods:Chondrocytes for primary culture in vitro were isolated from the hip joints of 4-week-old Sprague-Dawley(SD)rats,and the passage times were controlled within 4 generations.The expression of Col2,Sox9 and Aggrecan genes at m RNA and protein levels were examined by real-time Polymerase chain reaction(RT-PCR)and Western-blot respectively after treated with different concentrations of IL-18(0-100ng/m L)for 24 h.The expression of apoptosis-related factors including apoptosis suppressor B-cell lymphoma 2(Bcl-2),apoptosis promoter Bcl-2 associated X protein(Bax)and Caspase3/9 at protein levels were detected by western-blot.Twenty 6-week-old male SD rats(150-200g)were randomly divided into two groups,10 in each group.50?l saline were injected intra-articularly in rats twice a week in the control group,while saline containing 100ng/m L IL-18 at the same dose were injected intra-articularly in rats twice a week in the IL-18 group.All animals were sacrificed after 8 weeks since first injection,and the knees samples were stained with SO.Tissue immunohistochemical analysis was used to detect the expression of Aggrecan,Matrix Metallopeptidase 13(MMP13)and Caspase3.Results:The results of in vitro experiments showed that IL-18 significantly reduced the expression of chondrocyte phenotype genes Col2,Sox9 and Aggrecan at m RNA and protein levels,and IL-18 increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2.Besides,the activation of Caspase3/9 was observed.The results of in vivo experiments showed that the degree of cartilage degeneration in the IL-18 group was more severe than that in the control group,and the expression of Aggrecan and MMP13 was decreased while the expression of XII Caspase3 was increased.Conclusion:IL-18 could promote chondrocyte apoptosis and lead to degeneration of articular cartilage.Chapter ? The effect of IL-18 on chondrocyte autophagy.Objective:To investigate the effect of IL-18 on chondrocyte autophagy.Methods:Chondrocytes for primary culture in vitro were isolated from the hip joints of 4-week-old SD rats,and the passage times were controlled within 4 generations.The expression levels of autophagy markers such as Atg5,Atg7,Beclin1,LC3 B and P62 proteins were detected by Western-blot after treated with IL-18(100ng/m L)for different periods(0-24h).The change with IL-18 treatment time in the number of autophagosomes was detected by LC3 B immunofluorescence.Results:Western-blot results showed that the protein levels of Atg5,Atg7,Beclin1,and the LC3 B II/LC3 B I radio were significantly up-regulated after treating with IL-18(100 ng/m L)for 2h,and then decreased gradually over time,which dropped nearly to the baseline level after 24 h of IL-18 treatment.The autophagy substrate P62 protein gradually accumulated in chondrocytes.Immunofluorescence results showed that LC3 B fluorescence intensity increased in chondrocytes after treating with IL-18(100 ng/m L)for 6h,and then decreased.Conclusion:IL-18 could induce a strong autophagy response in chondrocytes at the initial stage of stimulation,but as the treatment time increased,the formation of autophagosomes in chondrocytes was suppressed.Chronic stimulation of IL-18 could induce autophagy deficiency in chondrocytes.Chapter ? Studies on the specific molecular mechanism of IL-18-induced autophagy deficiency and its correlation with chondrocyte degradation.Objective:To explore the specific molecular mechanism of IL-18-induced autophagy deficiency and its correlation with chondrocyte degradation.Methods:Chondrocytes for primary culture in vitro were isolated from the hip joints of 4-week-old SD rats,and the passage times were controlled within 4 generations.The phosphorylation levels of key proteins in PI3K-Akt-m TOR pathway including PI3 K,Akt and m TOR were detected by Western-blot after treated with different concentrations of IL-18(100ng/m L)for 24 h.The change with IL-18 treatment time in the number of autophagosomes was detected by LC3 B immunofluorescence.In addition,we designed a series of pathway inhibition and activation tests.Chondrocytes were pretreated with 740Y-P(a PI3 K activator)or SC79(an Akt activator)or 3BDO(an m TOR activator)or LY294002(a PI3 K inhibitor)for 1h,and then treated with IL-18(100ng/m L)for 24 h.Expression of chondrocyte phenotypic genes such as Col2,Sox9 and Aggrecan at protein levels was detected by Western-blot.XIV Results:IL-18 increased the phosphorylation levels of PI3 K,Akt and m TOR proteins,especially at concentrations of 10ng/m L and 100ng/m L.Inhibition and activation tests of the PI3K-Akt-m TOR pathway showed that the chondrocyte-specific gene degradation caused by IL-18 stimulation was further aggravated by 740Y-P or SC79 or 3BDO treatment,while it was improved by LY294002 treatment.Conclusion:IL-18 induced autophagy deficiency by activation of PI3K-Akt-m TOR pathway,and this signaling pathway was closely related to chondrocyte degradation.Chapter ? The effect of rapamycin on IL-18-induced autophagy deficiency and apoptosis in chondrocytes.Objective:To investigate the effect of rapamycin,a common autophagy agonist,on IL-18-induced autophagy deficiency and apoptosis in chondrocytes.Methods:Chondrocytes for primary culture in vitro were isolated from the hip joints of 4-week-old SD rats,and the passage times were controlled within 4 generations.The P3 of chondrocytes cultured in vitro were divided into four groups: chondrocytes of the control group was untreated;chondrocytes of the IL-18 treatment group were treated with IL-18(100ng/m L)for 24h;chondrocytes of the rapamycin treatment group were treated with rapamycin for 24h;chondrocytes of the IL-18+rapamycin treatment group were pretreated with rapamycin(10n M)for 1h,followed with 24 h IL-18 stimulation(100 ng/m L).The protein levels of autophagy markers including Atg5,Atg7,Beclin1,LC3 B,P62,and apoptosis-related factors such as Bax,Bcl-2 and Caspase3/9 were evaluated by Westernblot.Forty 6-week-old male SD rats(150-200g)were randomly divided into 4 groups,10 in each group.50?l saline were injected intra-articularly in rats twice a week in the control group;saline containing 100ng/m L IL-18 at the same dose were injected intra-articularly in rats twice a week in the IL-18 group;saline containing 10 n M rapamycin at the same dose were injected intra-articularly in rats twice a week in the rapamycin group;while saline containing 100ng/m L IL-18 and 10 n M rapamycin at the same dose were injected intra-articularly in rats twice a week in the IL-18+rapamycin group.All animals were sacrificed after 8 weeks since first injection,and the knees samples were stained with SO.Tissue immunohistochemical analysis was used to detect the expression of Atg5 and Caspase3.Results:The results of in vitro experiments showed that rapamycin significantly alleviated IL-18-induced autophagy deficiency(marked as reduced Atg7,reduced LC3 B II/LC3 B I radio and the accumulation of P62)and chondrocyte apoptosis(marked as reduced Bcl-2,increased Bax and Caspase3/9).The results of in vivo experiments showed that the degree of cartilage degeneration in the IL-18+rapamycin group was less severe than that in the IL-18 group.Besides,compared with the IL-18 group,the expression of Caspase3 was decreased and the expression of Atg5 was increased in the IL-18+rapamycin group.Conclusion:Rapamycin could alleviate IL-18-induced autophagy deficiency il-18-mediated autophagy deficiency and play the anti-chondrocyte apoptosis and cartilage protection role. |
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