| Osteoarthritis is a complex disease mainly characterized by progressive cartilage loss.Aging is the critical risk factor for OA,while the mechanisms linking age and OA have not been completely understood.Metabolic disorders and inflammation of chondrocytes are major pathological changes in aging cells and osteoarthritis.Recent studies demonstrated age-related mitochondrial dysfunction may be a key contributing factor in the development of OA.Mitofusin 2(MFN2)is a key regulator of mitochondrial fusion,cell metabolism,autophagy and apoptosis.This study was performed to ascertain whether MFN2 was involved in the aging of chondrocytes and OA.Firstly,we studied the expression level of MFN2 in aging cells and OA.Then we used siRNA to study the effect of MFN2 on cellular metabolic changes.Further,we used lentivirus to study the effect of MFN2 on chondrocytes inflammatory reaction and OA progression,Our study gives new insights in mechanism of aging and OA,and provides potential therapeutic approaches.This study is consisted of four parts:1.The expression of MFN2 in the aging cartilage and the OA cartilage2.The effect of MFN2 on cellular metabolic changes3.The pro-inflammatory effect of MFN2 in osteoarthritis both in vitro and in vivo4.The regulatory mechanism of abnormal MFN2 expressionChapter I The expression of MFN2 in the aging cartilage and the OA cartilage Objective:To investigate the expression of MFN2 in the aging cartilage and the OA cartilage Method:Femoral heads were obtained from patients who underwent THA and TKA in the 2nd Affiliated Hospital,School of Medicine,Zhejiang University.Young group cartilages were obtained from 6 young patients who suffered osteonecrosis of the femoral head with severe clinical symptoms while slight imaging manifestations.Old group cartilages were obtained from 6 old patients who suffered femoral neck fractures and underwent THA within 3 days.OA group knee cartilages were obtained from 6 patients with osteoarthritis who underwent TKA.Articular cartilage was obtained from knees of 3-week-old,12-month-old Sprague Dawley(SD)rats.The expression of MFN2 was detected through Western-blot and immunofluorescence.Results:Western-blot showed a significant increase in expression level of MFN2 during aging(P<0.05),and a significant increase in expression level of MFN2 from medial zone(damaged zone)compared to lateral zone(intact zone)(P<0.05).Immunofluorescence showed a significant increase in expression level of MFN2 during aging,and a X significant increase in expression level of MFN2 from medial zone(damaged zone)compared to lateral zone(intact zone).Conclusion:MFN2 is highly expressed in the aging cartilage and the OA cartilage.Chapter II The effect of MFN2 on cellular metabolic changes Objective:To study the effect of MFN2 on cellular metabolic changes.Methods:Articular chondrocytes were obtained from knees of 3-week-old,5-month-old and 12-month-old SD rats.siRNA was used to transfect rat chondrocytes in vitro to interfere the expression of MFN2.MFN2 interference efficiency in chondrocytes was detected by Western-blot.Glucose uptake assay,measurement of chondrocytes oxygen consumption,ATP measurements,mitochondrial ROS analysis were performed to compare the metabolic changes between the rat chondrocytes from different ages,as well as the chondrocytes pretreated with siRNA or not.Results:Western-blot showed a high knockdown efficiency,and insignificant difference in the expression of P16,P21 as well as SOD2,NRF2 between siRNA-pretreated chondrocytes and control chondrocytes.Glucose uptake assay showed age-related decreases in glucose uptake from 5-and 12-month chondrocytes compared to which from 3-week chondrocytes,and a significant increase in glucose uptake with siRNA pretreated.The Cell Mito Stress Test showed significant increases in basal OCR and maximal OCR from 12-month chondrocytes compared to 3-week chondrocytes,and a significant decrease in basal and maximal OCR in siRNA-pretreated chondrocytes.ATP measurements showed a decrease in ATP levels in old chondrocytes,and a decrease in ATP levels in siRNA-pretreated chondrocytes.Mitochondrial ROS analysis showed an increase in mitochondrial ROS measured by Mito SOX Red in old chondrocytes,and a significant decrease in siRNA-pretreated chondrocytes.Conclusion:A decrease in glycolytic flux and an increase in oxidative phosphorylation have been confirmed during aging.Knockdown of MFN2 reverses the age-related metabolic changes in rat chondrocytes.Chapter III The pro-inflammatory effect of MFN2 in osteoarthritis both in vitro and in vivo Objective:To study the pro-inflammatory effect of MFN2 in osteoarthritis both in vitro and in vivo.Methods:We Infected chondrocytes with Lenti-OE,Lenti-KD and their control NC-OE/KD in vitro,Infected chondrocytes were then incubated with IL-1? for 24 h,expression of COX2,INOS,MMP9,MMP13,COL2 were detected by Western-blot.Further,infected chondrocytes were then incubated with IL-1? for 10 min,expression of P65,P-P65,IKB?,P-IKB? were detected by Western-blot.50 male Sprague-Dawley rats were used XII and randomly divided into 5 groups: Sham group received sham surgery as control,the other 40 rats received DMM surgery and were regarded as OA rats.Intra-articular injection of 20?L control-overexpression Mfn2 particles,lenti-overexpression Mfn2 particles,control-knockdown Mfn2 particles,lenti-knockdown Mfn2 particles was performed to each group respectively at 7 days pre-operation and 7,21,35 days postoperation.Rats were euthanized in 8 weeks post-operation.The knee samples were cut into 5?M sections.These sections were stained with SO staining,immunohistochemical staining.Results:In vitro data showed that matrix-degrading related protein expression,including COX2,INOS,MMP9 and MMP13,was increased in Lenti-OE group,while decreased in Lenti-KD group.Expression of cartilage-specific protein COL2 was decreased in Lenti-OE group,while increased in Lenti-KD group.Further,the NF-?B pathway,including P-P65/P65 and P-I?B?/I?B?,was tested and showed a significant increase in Lenti-OE group and a decrease in Lenti-KD group.SO staining showed Lenti-KD group had considerably less cartilage destruction than NC-KD,while Lenti-OE group had more serious cartilage destruction.Immunohistochemical showed that expression of COX2 and MMP13,which were used to evaluate OA progression,were significantly higher in Lenti-OE group than NC-OE group and significantly lower in Lenti-KD group than NC-KD group.Conclusion:MFN2 contributed to OA development both in vitro and in vivo.Chapter IV The regulatory mechanism of abnormal MFN2 expression Objective:To study the regulatory mechanism of abnormal MFN2 expression.Methods:Articular cartilage was obtained from knees of 3-week-old,5-month-old and 12-month-old SD rats.The mRNA levels of MFN2 in cartilage during aging were detected by RT-PCR.Then articular cartilage of OA model and sham control was also obtained.The protein levels of MFN2 and PARKIN in cartilage during aging and OA were detected by Western blot.siRNA was used to transfect rat chondrocytes in vitro to interfere the expression of PARKIN.The effect of siRNA interference on MFN2 expression was detected.Finally,endogenous co-immunoprecipitation was performed to detect the interaction between MFN2 and PARKIN,as well as ubiquitination of MFN2.Results:There was no significant difference in the mRNA levels of MFN2 in cartilage during aging.The protein levels of MFN2 were elevated during aging and OA,while,the protein levels of PARKIN were declined during aging and OA.Knockdown of PARKIN with siRNA showed a significant increase in MFN2 expression.Co-immunoprecipitation showed that there is a direct interaction between these two proteins under physiological conditions,and ubiquitination of MFN2 was significantly decreased in chondrocytes via knockdown of PARKIN.Conclusion:Elevated expression of MFN2 during aging and OA could be a result from the declined level of PARKIN. |
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