| Knee osteoarthritis is a common disease caused by multiple factors,which seriously affects the health and quality of life of elderly people.Chondrocyte degeneration is the basis of the pathogenesis of KOA,and the mechanism of mechanical factors in the degeneration of chondrocytes has been a hot topic.In order to clarify the mechanism of traditional Chinese medicine treatment of KOA,based on the study of differential expression proteomics of chondrocytes after mechanical stimulation intervention,this study would further study the changes of integrin and Hippo/YAP signaling pathways closely related to mechanical stimulation,and the changes of these signaling pathways after intervention by rational manipulation.Part One: Differential expression of proteomics in chondrocytes of knee osteoarthritis under cyclic compressive stressObjective: Proteomics technique was used to observe the differential expression of protein in KOA chondrocytes under cyclic stress.Methods: Two-week-old male SD rats were randomly divided into normal group,model group and pressure group with 4 rats in each group.The chondrocytes of normal group were cultured under normal pressure and environment.The chondrocytes in the model group were cultured in the medium with TNF-α reagent to simulate the OA inflammatory environment under the condition of normal pressure.The chondrocytes in the pressure group were stimulated by TNF-α to simulate the inflammatory environment of OA in the medium.The Flexcell-5000 cells were loaded with the tensile stress loading system of 172 KPA,and the cyclic compressive stress of 2Hz was given for 3 days continuously for 60 min/d.Differential expression of proteins in chondrocytes of each group was analyzed by liquid chromatography-mass spectrometry(LC-MS/MS).Results: A total of 7874 peptides and 1697 proteins were identified by mass spectrometry.Compared with the normal group,there were 81 up-regulated and 128 down-regulated differentially expressed proteins in the model group.Compared with normal group,there were 333 down-regulated differentially expressed proteins and 194 down-regulated differentially expressed proteins in pressure group.The results of functional enrichment analysis of differentially expressed protein GO showed that the cellular components of membrane-binding organelles associated with integrin β 1 and intracellular cellular components associated with the core protein of Hippo/YAP signaling pathway were significantly changed.The enrichment analysis of KEGG pathway showed that valine,leucine and isoleucine degradation pathway,proteasome pathway,fatty acid degradation pathway and adhesion link pathway were significantly changed in the model group compared with the normal group.There were significant changes in splice pathway,carbon metabolism pathway,citric acid cycle pathway,pyruvate metabolism pathway,RNA transport pathway and proteasome pathway in the stress group compared with the normal group.Further differential protein screening analysis indicated that ITG-β 1 protein,actin closely related to ITG-β 1 protein,and MOB1,Cry61,Smad2 protein,which was closely related to Hippo/YAP signaling pathway,had significant changes.Conclusion: Under the stimulation of inflammatory factors and after periodic pressure intervention,ITG-β 1 protein and its closely related actin,Hippo/YAP signal pathway related protein MOB1,Cry61,Smad2 and so on were significantly changed.These results suggest that these signaling pathways may play an important role in the regulation of chondrocyte metabolism.Part Two: Experimental study on the effect of cyclic compressive stress on integrin β 1-Hippo/YAP signaling pathway in chondrocytesObjective: To observe the effects of cyclic compressive stress on the expression of ITG-β 1 and Hippo/YAP signaling pathway related proteins in KOA chondrocytes.Methods: Two-week-old male SD rats were randomly divided into normal group,model group and pressure group with 4 rats in each group.The chondrocytes of normal group were cultured under normal pressure and environment.The chondrocytes in the model group were cultured in the medium with TNF-α reagent to simulate the OA inflammatory environment under the condition of normal pressure.The chondrocytes in the pressure group were stimulated by TNF-α to simulate the inflammatory environment of OA in the medium.The Flexcell-5000 cells were loaded with the tensile stress loading system of 172 KPA,and the cyclic compressive stress of 2Hz was given for 3 days continuously for 60 min/d.The TUNEL and ELISA techniques were used to observe the changes of apoptosis and expression of apoptosis-related proteins in chondrocytes of rats in each group.RT-PCR and Western Blot techniques were used to observe the expression of core genes and proteins in ITG-β 1 and Hippo/YAP signaling pathway MST1,YAP1,TEAD and Logistic linear regression analysis was used to observe the correlation between ITG-β 1 and MST1,YAP gene and protein expression.Results: The apoptosis of chondrocytes in the model group was significantly lower than that in the pressure group(P < 0.01).Compared with the normal group,the expression of apoptosis-related protein Bcl-2 in the model group was significantly lower and the expression of Bax was significantly higher(P<0.01).Compared with the model group,the expression of apoptosis-related protein Bcl-2 in pressure group was significantly higher than that in model group(P < 0.05),while the expression of Bax was decreased(P < 0.05).The expression of ITG-β1 m RNA in chondrocytes of model group was significantly lower than that of normal cartilage(P< 0.01).The expression of ITG-β 1 m RNA and protein in chondrocytes was up-regulated after cyclic stress(P < 0.01 and P < 0.05).RT-PCR study showed that the expression of downstream transcription coactivator YAP1 and TEAD m RNA in the Hippo/YAP signaling pathway of chondrocytes in the model group was significantly lower than that in normal cartilage(P < 0.01).Compared with the model group,In the pressure group the expression of YAP1 protein,m RNA and TEAD m RNA were significantly increased(P<0.01),but the expression of MST1 m RNA was not significantly increased(P>0.05).Logistic linear regression analysis showed that there was a significant correlation between ITG-β 1 and MST1 gene expression(r = 0.864,P < 0.01).There was a significant correlation between ITG-β 1 and YAP gene and protein expression(r = 0.779 and r = 0.851,P < 0.01).Conclusion: ITG-β 1 may be involved in regulating the changes of YAP protein and its target protein through Hippo/YAP signaling pathway and other signaling pathways respectively,thus regulating the metabolism of chondrocytes.Part Three: Effect of regulating tendon manipulation on the signal pathway of integrin-Hippo/YAP in knee joint of KOA rat modelObjective: To observe the effect of regulating tendon manipulation on the expression of ITG-β 1 and Hippo/YAP signal pathway related proteins in the articular cartilage of KOA rats.Methods: SPF grade SD rats were randomly divided into normal group,model group and manipulation group with 10 rats in each group.KOA model was established by medial collateral ligament resection combined with partial patellar ligament resection in rats.After 4 weeks of modeling,the rats in the manipulation group were treated with manipulation intervention for 7 minutes each time,once every other day,for 6 weeks.No intervention in normal group and model group.HE staining was used to observe the morphological changes of the articular cartilage,TUNEL technique was used to observe the apoptosis of chondrocytes,and RT-PCR and Western Blot techniques were used to observe the ITG-β 1 and Hippo/YAP signal pathway MST1,YAP1,.Changes in the expression of core constituent genes and proteins in TEAD.Logistic linear regression analysis was used to observe the correlation between ITG-β 1 and MST1,YAP gene and protein expression.Results: In the normal group,the cartilage of knee joint was smooth,the chondrocytes were spindle-shaped,horizontal arrangement,and the structure of tidal line was clear and complete.In the model group,there was moderate wear on the articular cartilage surface and the loss of chondrocytes was obvious in the model group.In manipulation group,the cartilage surface of knee joint was slightly worn,the morphology of chondrocytes was close to normal,and the phenomenon of chondrocyte clustering was less.The Mankin score of the model group was significantly higher than that of the normal group(P<0.01).After manual therapy,the Mankin score of the manipulation group was significantly lower than that of the model group(P<0.01),but it was still higher than that of the normal group(P<0.01).The results of flow cytometry showed that there was a significant difference between the model group and the normal group(P<0.01).The apoptosis rate of chondrocytes in the manipulation group was significantly different from that in the model group(P<0.05).The expression of ITG-β 1 m RNA in the cartilage of the model group was significantly lower than that of the normal group(P<0.01).The expression of ITG-β 1 m RNA and protein in the cartilage of the model group was obviously up-regulated after the intervention of reinforcement manipulation,compared with the model group(P<0.01).RT-PCR and Western blot studies showed that the expression of MST1,YAP1 and TEAD m RNA and YAP1 proteins in the Hippo/YAP signaling pathway of cartilage in the model group were significantly lower than those in the normal control group(P<0.01).Compared with the model group,the expression of YAP1 protein,m RNA and TEAD m RNA were significantly increased in the manipulation group(P<0.01),but the expression of MST1 m RNA was not significantly increased(P>0.05).Logistic linear regression analysis showed that there was a significant correlation between ITG-β 1 and MST1 gene expression(r = 0.748,P < 0.01).There was a significant correlation between ITG-β 1 and YAP gene and protein expression(r = 0.926 and r = 0.858,P < 0.01).Conclusion: The manipulation of regulating tendons could delay the degeneration of chondrocytes and that mechanical stimulation to chondrocytes was partly mediated by ITG-β 1 involved in the classical Hippo/YAP signaling pathway,and there were other unknown signaling pathways involved in the regulation of YAP protein by ITG-β1. |
PDF Full Text Request