BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene ma...BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.展开更多
OBJECTIVE:To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis(KOA).METHODS:The rabbits were divided into blank control,model,acupoto...OBJECTIVE:To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis(KOA).METHODS:The rabbits were divided into blank control,model,acupotomy and electroacupuncture(EA)groups,with 12 rabbits in each.Modified Videman's method was used to prepare KOA model.The acupotomy and EA group were given indicated intervention for 3 weeks.The behavior of rabbits in each group was recorded.Subsequently,cartilage–subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography.Compression test was used to detect the mechanical properties of subchondral bone,Western blot and real-time polymerase chain reaction(RT-PCR)were applied to detect the expression of bone morphogenetic protein 2-Smad1(BMP2-Smad1)pathway in subchondral bone.RESULTS:Compared with the control group,rabbits in the KOA group showed lameness,knee pain,and cartilage degradation;the subchondral bone showed active resorption,the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly.Both acupotomy and EA intervention could increase the thickness of trabecular bone(Tb.Th),the bone volume fraction(BV/TV)and the thickness of subchondral bone plate,reduce the separation of trabecular bone(Tb.Sp),improve the maximum load and elastic modulus of subchondral bone,and effectively delay cartilage degeneration in KOA rabbits.This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway.The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group.CONCLUSIONS:Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.展开更多
目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂...目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂量组及骨瓜大剂量组,分别于模型组、骨瓜小剂量组、骨瓜大剂量组大鼠右膝关节腔内多点注射小牛软骨Ⅱ型胶原加福氏完全佐剂0.1 m L,制备类风湿性关节炎动物模型,造模成功后,骨瓜小剂量组、骨瓜大剂量组分别用骨瓜提取物注射液肌注治疗21 d,于治疗第1、7、14、21天,分别测定四组大鼠关节积液中HA及IL-1β含量;检测滑膜BMPs及MMP-1阳性表达,测量右足趾关节以下部位的容积并计算足肿胀度,记录各组动物消肿时间,评价骨瓜提取物注射液对大鼠类风湿性关节炎的治疗效果。结果治疗下14、21 d时,骨瓜小剂量组、骨瓜大剂量组MMP-1阳性表达及关节积液中IL-1β明显降低,BMPs及HA含量明显升高,消肿时间缩短,足肿胀度明显优于模型组,与模型组比较,差异有统计学意义(P<0.05),骨瓜小剂量组、骨瓜大剂量组间各指标差异无统计学意义(P>0.05)。结论骨瓜提取物注射液可促进类风湿关节炎模型大鼠关节积液HA及BMPs分泌与合成,降低软骨细胞MMP-1阳性表达,抑制炎性介质IL-1β对关节滑膜的侵蚀而起到保护关节的作用。展开更多
文摘BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.
基金Longitudinal Research Development Fund of Beijing University of Chinese Medicine:to Investigate the Effect of Acupotomy on Subchondral Bone Remodeling in Knee Osteoarthritis Based on BMP2-Smad1 Pathway(2020-ZXFZJJ-031)National Natural Science Foundation of China:to Investigate the Mechanism of Acupotomy"Regulating Tendon and Treating Bone"in the Treatment of KOA based on the Vascularization of Hypoxic Cartilage Mediated by HIF-1α/VEGF Pathway(82074523)。
文摘OBJECTIVE:To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis(KOA).METHODS:The rabbits were divided into blank control,model,acupotomy and electroacupuncture(EA)groups,with 12 rabbits in each.Modified Videman's method was used to prepare KOA model.The acupotomy and EA group were given indicated intervention for 3 weeks.The behavior of rabbits in each group was recorded.Subsequently,cartilage–subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography.Compression test was used to detect the mechanical properties of subchondral bone,Western blot and real-time polymerase chain reaction(RT-PCR)were applied to detect the expression of bone morphogenetic protein 2-Smad1(BMP2-Smad1)pathway in subchondral bone.RESULTS:Compared with the control group,rabbits in the KOA group showed lameness,knee pain,and cartilage degradation;the subchondral bone showed active resorption,the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly.Both acupotomy and EA intervention could increase the thickness of trabecular bone(Tb.Th),the bone volume fraction(BV/TV)and the thickness of subchondral bone plate,reduce the separation of trabecular bone(Tb.Sp),improve the maximum load and elastic modulus of subchondral bone,and effectively delay cartilage degeneration in KOA rabbits.This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway.The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group.CONCLUSIONS:Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.
文摘目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂量组及骨瓜大剂量组,分别于模型组、骨瓜小剂量组、骨瓜大剂量组大鼠右膝关节腔内多点注射小牛软骨Ⅱ型胶原加福氏完全佐剂0.1 m L,制备类风湿性关节炎动物模型,造模成功后,骨瓜小剂量组、骨瓜大剂量组分别用骨瓜提取物注射液肌注治疗21 d,于治疗第1、7、14、21天,分别测定四组大鼠关节积液中HA及IL-1β含量;检测滑膜BMPs及MMP-1阳性表达,测量右足趾关节以下部位的容积并计算足肿胀度,记录各组动物消肿时间,评价骨瓜提取物注射液对大鼠类风湿性关节炎的治疗效果。结果治疗下14、21 d时,骨瓜小剂量组、骨瓜大剂量组MMP-1阳性表达及关节积液中IL-1β明显降低,BMPs及HA含量明显升高,消肿时间缩短,足肿胀度明显优于模型组,与模型组比较,差异有统计学意义(P<0.05),骨瓜小剂量组、骨瓜大剂量组间各指标差异无统计学意义(P>0.05)。结论骨瓜提取物注射液可促进类风湿关节炎模型大鼠关节积液HA及BMPs分泌与合成,降低软骨细胞MMP-1阳性表达,抑制炎性介质IL-1β对关节滑膜的侵蚀而起到保护关节的作用。
