The Study On The Role Of Delaying Articular Cartilage Degeneration In Osteoarthritis By Knocking Down HIF-2α

Objective:HIF-2a targeted in vivo in articular cartilage reduces chondrocyte apoptosis and delays the progression of osteoarthritis(OA),while exploring the correlation between HIF-2α,HDAC4,and OA.Methods:(1)Knee joint cartilage tissue samples were collected from patients who underwent total knee replacement surgery and stored in the Biobank of the Department of Orthopedics Laboratory,Second Hospital of Shanxi Medical University.The samples were classified as relatively normal(Outerbridge grade Ⅰ)or osteoarthritic(Outerbridge grade Ⅳ).The overall condition of the cartilage tissue was observed through safranin-o staining and imaging examinations.The expression of indicators such as HIF-2α and HDAC4 was detected at the protein and mRNA levels using techniques such as immunohistochemistry staining,Western blot,and real-time qPCR.(2)An OA model was established in rats by anterior cruciate ligament transection(ACLT)surgery,and the expression levels of HIF-2a and HDAC4 as well as chondrocyte apoptosis were observed at different time points during OA progression.The experimental groups were divided into the bone arthritis group(Control),normal control group(Normal),and blank control group.(3)Adeno-associated virus containing HIF-2α siRNA was intra-articularly injected into the knee joint cavity of rats 2 weeks after ACLT surgery to observe the transfection of adeno-associated virus in the articular cartilage.The animal experiments were divided into the treatment group(AAV9-HIF-2α),negative control group(AAV9-HIF-2α-NC),and blank control group(Sham).(4)Gait analysis was used to evaluate the effect of intra-articular injection of HIF-2a siRNA adeno-associated virus on the progression of osteoarthritis.(5)Imaging examinations(X-ray,MicroCT,MRI,FMT)were used to evaluate the effect of intra-articular injection of HIF-2α siRNA adeno-associated virus on the progression of osteoarthritis.(6)The expression of indicators such as HIF-2α,HDAC4,ATF4,and CHOP was detected using immunohistochemistry and real-time qPCR.Results:(1)Compared to relatively normal human cartilage tissue,Safranin-o staining of human OA cartilage tissue samples showed loss of cartilage proteoglycans,lighter staining,disordered cell arrangement,and incomplete surface layer of articular cartilage.Results from immunohistochemistry,Western blot,and real-time qPCR showed that the expression levels of HIF-2α in OA cartilage tissue were significantly higher than those in normal cartilage tissue,while the expression levels of HDAC4 were decreased,and the expression levels of ATF4,CHOP,caspase-3,and caspase-9 were significantly increased,indicating severe chondrocyte apoptosis.(2)A successful OA model was established in rats,and as OA progressed,Safranin-o staining showed loss of proteoglycans in the Control group cartilage tissue;bone spurs were significantly increased in the Control group rats,the bone trabeculae were sparse,and MRI showed bone marrow edema.Immunohistochemistry and real-time qPCR results showed that at 12 weeks after ACLT surgery,the expression levels of HIF-2α in the OA cartilage tissue were significantly higher than those in normal cartilage tissue,while the expression levels of HDAC4 were decreased,and the expression levels of ATF4,CHOP,caspase-3,and caspase-9 were significantly increased,indicating severe chondrocyte apoptosis.(3)The adenovirus was successfully transfected into the rat articular cartilage,and the transfection was complete after 8 weeks of joint injection.(4)Gait analysis results showed that compared to the AAV9-HIF-2α-NC group,the AAV9-HIF-2α group had a longer duration of maximum contact area,stride length,and single-leg standing time,and obvious relief of pain behavior.(5)Imaging results showed that the AAV9-HIF-2α group had reduced bone spurs,no significant narrowing of joint space,and no significant high T2 signal on MRI.The level of matrix metalloproteinases(MMPs)was lower in the negative control group.(6)Immunohistochemistry and real-time qPCR results showed that the expression level of HIF-2α in the AAV9-HIF-2α group was significantly decreased,while the expression level of HDAC4 was increased,and the expression levels of ATF4,CHOP,caspase-3,and caspase-9 were significantly decreased,indicating reduced chondrocyte apoptosis.Conclusion:Intra-articular injection of adeno-associated virus targeting HIF-2α in chondrocytes can effectively inhibit chondrocyte apoptosis and alleviate the progression of osteoarthritis by affecting the expression of HDAC4 in chondrocytes,providing a new strategy for the treatment of osteoarthritis.