| Objective: To study the expression of RhoA/ROCK signaling pathway in knee osteoarthritis(KOA)and to explore the correlation between RhoA/ROCK signal transduction pathway and KOA by detecting and comparing the expression of RhoA and ROCK in synovial and cartilage tissues of patients with primary knee osteoarthritis and patients undergoing lower limb amputation.Methods:Thirty patients with primary KOA in General Hospital of Ningxia Medical University from January 2020 to December 2022 were collected.The synovial and cartilage tissues removed during total knee arthroplasty(TKA)were taken as the observation group.In the same period,25 cases of synovial and cartilage tissue removed from lower limb amputation due to lower limb malignant tumor or lower limb damage were used as the control group.The appearance of the specimens was observed first,and the specimens were treated in the laboratory.The morphological changes of the two groups were observed under the microscope by HE staining.The expression of RhoA and ROCK protein in the two groups was detected by immunohistochemistry(IHC)and Western blot(WB).The expression of RhoA and ROCK m RNA was detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR).Results:1.General morphology observation:(1)Synovial tissue:In the observation group,extensive hyperplasia of synovial tissue,obvious congestion and edema,and yellow were observed.In contrast to the control group,the synovial tissue of the control group was white or light red,and the surface of the synovial membrane was smooth and flashy,thin and no edema.(2)Cartilage tissue: In the observation group,the surface of the femoral condyle cartilage was rough and uneven,the defect was obvious,and it was ulcerated.The anatomical structure of the femoral condyle and intercondylar was blurred,and some osteophytes were formed at multiple edges.The subchondral bone plate was visible through the worn cartilage,gray and dull,and cancellous bone sclerosis was visible in the subchondral bone.In the control group,the surface of the femoral condyle cartilage was smooth and flat,and there was no obvious loss of cartilage defects.The surface of the cartilage was clear and uniform,milky white,and the edge of the femoral condyle was clear without osteophyte formation.2.HE staining results:(1)Synovial tissue: The edge of synovial tissue was extremely irregular and the shape was different.The stained synovial cells and matrix were significantly reduced compared with the control group,showing light staining or loss of staining.In the control group,the edge of the synovial tissue of the joint was smooth and neat,and more stained synovial cells and deeply stained cell matrix could be seen.The edge of the synovial membrane was smooth,the synovial cells were arranged in an orderly manner,and the cells and matrix were evenly stained.(2)Cartilage tissue: Microscopically,the cartilage surface of the observation group was uneven,with more defects,uneven cartilage staining,incomplete tidal line,disordered arrangement of cartilage sacs and chondrocytes,clustered distribution,diffuse focal hyperplasia of subchondral bone,and disappearance of different tissue layers.In the control group,the cartilage surface was smooth and flat,the stratification of various tissues was obvious,the color of cartilage surface was uniform,the number of chondrocytes was large,the matrix was rich,and there was no focal hyperplasia.3.Observation of cartilage safranin O-fast green staining: In the observation group,there were cracks on the surface of cartilage,and the staining intensity of cartilage layer was not uniform.Safranin staining was deeply stained around clustered chondrocytes,and there was a loss of staining on the surface of heavily worn cartilage.The number of cartilage matrix and cells decreased and arranged in clusters.There was no obvious boundary between cartilage layer and subchondral bone,and green stained bone tissue was interspersed under cartilage.The cartilage surface of the control group was flat,the cartilage area was evenly colored,there was no deep staining or loss of staining,the number of nuclei was more than that of the observation group,the arrangement was scattered and disordered,the cartilage matrix was rich,and there was a clear boundary with the subchondral bone.4.The results of cartilage scanning electron microscopy showed that the cartilage in the observation group was severely damaged,the structure was unclear,the cartilage surface was uneven,and there were a large number of hyperplasia protrusions and cavity fissures;the surface of the control group was relatively smooth,with a large number of cartilage cell protrusions and folds,and the cartilage stroma was uniform.5.Immunohistochemical results : The positive expression of RhoA and ROCK protein in the synovial and cartilage tissues of the observation group was light yellow or brown particles distributed in the plasma membrane and cytoplasm,while only a small amount of positive expression was found in the control group.The integral optical density(IOD)of the two groups was measured by Image-pro plus 6.0.The average IOD of the observation group was significantly higher than that of the control group.6.Western blot results: Compared with the control group,the expression of RhoA and ROCK protein in the synovial and cartilage tissues of the observation group was significantly higher than that of the control group.7.RT-qPCR results: Compared with the control group,the expression levels of RhoA and ROCK m RNA in the synovial and cartilage tissues of the observation group were significantly higher than those in the control group.Conclusion: The expression of RhoA and ROCK protein molecules in RhoA/ROCK signal transduction pathway in synovial and cartilage tissues of KOA patients was significantly higher than that in patients undergoing lower limb amputation,and the expression of RhoA and ROCK m RNA was significantly higher than that in patients undergoing lower limb amputation,suggesting that RhoA/ROCK signal transduction pathway is correlated with knee osteoarthritis and may play a role in the occurrence and development of knee osteoarthritis. |
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