Ultrasonic Imaging Changes Of Infrapatellar Fat Pad In Knee OA And The Mechanism Of TGF-β1 It Participated Synthesis In Inhibiting Chondrocyte Pyroptosis By Exercise Therapy

Objectives:Osteoarthritis(OA)is a kind of chronic and progressive disease,mainly characterized by the degeneration of articular cartilage.The pathological changes of OA included joint space changes,remodeling of subchondral bone,osteophyte formation,inflammation of synovium and infrapatellar fat pad(IFP).Knee OA has a high incidence,high disability rate and high medical costs,causing huge economic and social burden to individuals and families.Therefore,the effective treatment of OA will further promote the realization of the goal of healthy China.High-resolution musculoskeletal ultrasound(MSUS)has been widely used clinically,which can clearly display the soft tissue around the joint and has high sensitivity for the detection of inflammation and injury in OA soft tissue.It has high reference value for evaluating the damage degree and prognosis of articular cartilage in knee OA.As the adipose tissue inside the joint capsule and outside the synovium,IFP can disperse the mechanical stress of the joint,absorb external shocks,stabilize the structure of the patella,promote the distribution of synovial fluid in the joint and slow down the friction in the joint.Inflammatory cytokines secreted by IFP can cause structural and functional changes in articular cartilage,but their biological effects on cartilage remain unclear.In this study,the rat treadmill training model and Flexcell stress loading system were used to observe the influence of exercise therapy on the joint IFP of OA rats,and to detect the changes of pro-inflammatory factors secreted by IFP after exercise and related indicators of cartilage microenvironment,so as to clarify the role of IFP in the exercise therapy of OA.GEO database was used to find the differential molecules in the articular cartilage of rats undergoing treadmill training,and the GO and KEGG pathways were used to analyze them.Finally,TGF-β1 was the key factor to maintain the stability of the joints.In this study,the imaging changes of IFP in different degrees of knee OA were detected by ultrasound,so as to clarify the correlation between IFP and knee OA and improve the ultrasonic diagnosis system of knee OA.On this basis,TGF-β1 from IFP source was further used as the entry point to explore the relationship between TGF-β1 secreted by IFP and knee OA exercise therapy,to clarify the important role of TGF-β1 from IFP source in knee OA exercise therapy;At the same time,chondrocyte culture was performed to explore the effect of TGF-β1 mediated NF-κB/NLRP3 signaling pathway on chondrocyte pyrodeath under mechanical stimulation through Flexcell stress loading system,which led to in-depth research on the mechanism of knee OA.Methods:1.Imaging changes of IFP in knee OA were explored by ultrasound examination.Patients meeting the inclusion criteria were selected for anterolateral radiographs of the affected knee joint and Kellgren-Lawrence(K-L)grading.Ultrasound examination was performed to measure the maximum IFP sagittal cross-sectional area and thickness,echo change,blood flow distribution and maximum vessel diameter,IFP fat lobular compressibility and other indicators,and to study the correlation between it and different degrees of knee OA.2.To explore the effect of TGF-β1 from IFP on articular cartilage of OA rats through treadmill training.(ⅰ)Through bioinformatics analysis of microarray,GO and KEGG pathway enrichment analysis,microarray subtype,gene set enrichment analysis and diagnostic ROC curve were performed to screen important Hub genes.(ⅱ)OA rat model was established,and different intensity of treadmill training was performed at the same time to detect the content of TGF-β1 in IFP;The content of TGF-β1 in articular cartilage was detected,and the correlation between the two was explored according to the OA microenvironment theory of knee joint.(ⅲ)The OA model of rats with different intensity of treadmill training was established,and the gross joint,X-ray and CT images of the rats were explored by intraperitoneal injection of TGF-β1 inhibitor(RepSox).Histopathologic sections and ELISA were used to detect related inflammatory factors,and the content of scorche-related proteins in the cartilage of OA rats was detected by western blot.3.To explore the mechanism of chondrocyte pyrogenesis mediated by TGF-β1 through Smad2/3 and NF-κB signaling pathways in patients with OA through flexcell stress loading system.(ⅰ)Collect cartilage samples of the affected side of knee OA patients for western-blot detection and verification of corresponding protein content.(ⅱ)Primary culture and identification of chondrocytes in OA patients,and the optimal stretching conditions of CTS cells and concentration of TGF-β1 were selected.(ⅲ)According to the optimal CTS conditions and TGF-β1 concentration,western-blot assay was performed to detect the expression of related proteins in chondrocytes,reactive oxygen species detection,immunofluorescence of p65 and Smad2/3 into the nucleus of chondrocytes,cell morphology observation by transmission electron microscopy and SYTOX green staining.Results:1.Part One:According to the classification of non-knee OA,mild knee OA and severe knee OA,the maximum sagittal cross-sectional area and maximum thickness of IFP,the measurement of IFP blood flow distribution and maximum vessel diameter,and the compressibility of IFP fat lobule were examined by ultrasound,and it was found that each index was correlated with the degree of OA injury(P<0.05).There was no significant correlation between the internal echo of IFP and OA(P>0.05).2.Part Two:(i)Prediction of TGF-β1 signaling pathway by biogenesis plays a key role in the process of OA.The content of TGF-β1 from IFP decreased in OA rats with knee OA by western-blot analysis,but the content of TGF-β1 from IFP increased after treadmill training.The results were consistent with the change of TGF-β1 content in articular cartilage.(ⅱ)OA rats were selected for different intensity treadmill training,and TGF-β1 inhibitor(RepSox)was injected into the abdomen to evaluate knee OA.The knee joint of rats was roughly visible to the naked eye,and the knee cartilage of rats in the normal control group was smooth and complete,and no injury was observed.However,the OA model showed that the surface of cartilage degeneration was uneven,with fissures and synovial hyperplasia.Pathological sections showed:normal(CG group)surface cartilage integrity,no obvious degeneration,uniform distribution of chondrocytes,no obvious chondroblast proliferation,complete toluidine blue staining.In OA model(OAG group),the cartilage surface was damaged,uneven and cracked,and toluidine blue stain was found.Pathological correlation scores(OARSI and Mankin)showed that there was significant statistical difference between OAG and CG(P<0.05).Appropriate exercise intensity was selected and TGF-β1 receptor inhibitor RepSox was injected intraperitoneally.Pathological score showed that there was significant difference between the RepSox injection group and the appropriate intensity running group(P<0.05).The correlation detection method showed that the expression trend of related proteins was consistent with the pathological results.(ⅲ)X-ray imaging,CT imaging,gross observation,H&E staining and toluidine blue staining,IL-1β and TNF-α in serum of OA rats in each group,collagen Ⅱ,NLRP3,caspase-1,pro-caspase-1,TGF-β1,Collagen Ⅱ,NLRP3,Caspase-1,TGF-β1 were detected by western-blot analysis.The results of IL-1βshowed that TGF-β1 inhibitor(RepSox)group reduced the effects of moderate intensity treadmill training on cartilage injury.3.Part Three:(ⅰ)Western-blot analysis was performed on cartilage of injured and normal sites of OA patients.The results showed that TGF-β1 in articular cartilage decreased significantly with the increase of OA,and there was a significant negative correlation between the two.(ⅱ)The chondrocytes of OA patients in primary culture were treated with CTS of the same frequency,duration and different mechanical strengths.western-blot analysis revealed that 8%of CTS significantly increased the content of collagen Ⅱ in chondrocytes.(ⅲ)Western-blot results showed that NLRP3,pro-caspase,caspase 1,IL-1β and GSDMD-N in chondrocytes of OA were significantly decreased under CTS(8%,0.5Hz,12h)stimulation.The contents of TGF-RI,Smad2,Smad3 and IκB were significantly increased.That trend reversed with RepSox.(ⅳ)Western-blot analysis of IκB-α,IL-1βand NLRP3 in cytoplasm and NF-κB p65 in nucleus inhibited the Smad pathway with SIS3,a 4 ng/mL TGF-β1 and 10 μM Smad3 inhibitor.(ⅴ)Transmission electron microscopy images of chondrocytes showed that CTS(8%,0.5Hz,12h)could improve chondrocyte swelling,cell membrane protrusion,and cell atrophy in patients with OA.Conclusion:Ultrasonography has high clinical application value in the detection of IFP in patients with knee OA,and can be used as an auxiliary diagnosis or imaging prediction index.Mechanical loading under appropriate conditions can co-activate Smad2/3 by stimulating the secretion of TGF-β1 and inhibit NF-κB signaling pathway to reduce chondrocyte pyrodeath and exert the anti-OA effect.