Expression Of PENK In Synovial Tissues And Cells Of Osteoarthritis And Its Protective Effect On Osteoarthritis

Objective:Osteoarthritis(OA)is currently the most common chronic joint disease in the elderly,and the number of people suffering from knee osteoarthritis has reached nearly400 million worldwide.In China,the prevalence of osteoarthritis has exceeded 50% in people over 60 years old,and the prevalence of people over 75 years old has reached80%.OA most commonly affects joints,including the knees,hands,hips,and spine,and is the leading musculoskeletal cause of mobility disability in older adults.A number of risk factors associated with OA including genetic predisposition,aging,obesity,and joint malalignment have been proposed.The main clinical manifestations of OA are chronic pain,joint instability,stiffness,joint deformity,and narrowing of joint space on imaging.Traditional treatments for osteoarthritis include reduction of pain,relief of stiffness,maintenance of physical activity,and improvement of living status.Specific treatments include weight loss,acupuncture,injections of sodium hyaluronate,glucosamine and chondroitin sulfate,and joint replacement surgery.At present,the pathogenesis of OA is still not well understood.Although it was previously thought that OA only led to disease progression due to the "wear and tear" of cartilage,it is now believed to be due to a complex interaction between local and systemic factors and multiple effects of inflammatory mediators released in cartilage,bone and synovial membrane.Among them,synovial inflammation in OA has been paid more and more attention by researchers in recent years,however,only joint replacement surgery is an effective intervention for the treatment of OA.Pproenkephalin(PENK)is a classically defined opioid protein-coding gene.It was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine system.Among the inflammatory pathways associated with osteoarthritis,PENK is involved in the regulation of NF-κB signaling pathway and PI3K/AKT/m TOR signaling pathway,and PENK is also present in the apoptotic pathway of fibroblast synoviocytes.This thesis mainly focused on the mechanism of PENK in osteoarthritis,and normal people were used as control group.At the same time,the expression of PENK in osteoarthritis patients and its effect on osteoarthritis synovial cells were studied.This may provide new potential molecular targets for the prevention and treatment of OA.Methods: 1.The literature related to osteoarthritis and gene information websites were searched,and PENK was identified as the target gene.2.Synovial tissue samples were collected,and immunohistochemistry,Western Blot and q PCR were used to detect the differences in the protein and transcription levels of PENK between human osteoarthritis synovial tissue and normal synovial tissue.3.Four concentration gradients of IL-1β(0,5,10,15 ng/ml)were set to induce human normal fibroblast-like synoviocytes(HFLs)inflammation model for 24 hours,and the optimal concentration of IL-1β(10ng/ml)was finally determined.The protein expression level of IL-6 was detected by Western Blot.The cell morphology was observed by inverted microscope,and the cell activity was detected by CCK-8.4.The PENK overexpression vector was constructed by molecular biological methods and infected with normal fibroblast-like synoviocytes to establish a PENK-overexpressing fibroblast-like synoviocyte line.The expression of PENK was identified by Western Blot and the successful establishment of the fibroblast-like synoviocytes with PENK overexpression was verified by CCK-8 assay.The relationship between PENK and OA was verified.5.The PENK knockdown lentiviral vector was constructed by molecular biology technology and infected with normal fibroblast-like synoviocytes to establish a PENK knockdown fibroblast-like synoviocyte line.Western Blot was used to identify PENK and CCK-8 was used to verify the successful establishment of PENK knockdown FLS cell line.The relationship between PENK and OA was verified.Results: 1.Immunohistochemistry,Western Blot and q PCR showed that the protein and transcription levels of PENK in OA synovial tissues were significantly lower than those in normal human synovial tissues.2.The expression level of IL-6 was detected by Western Blot,and the expression level of IL-6 showed an upward trend at the concentration of 10ng/ml,and decreased slightly at the concentration of 15 ng/ml(P<0.05),and the optimal concentration of IL-1β(10ng/ml)was determined.Western Blot and q PCR showed that the protein and transcription levels of PENK in osteoarthritis synoviocytes were significantly lower than those in normal synoviocytes.CCK-8 assay showed that the proliferation rate of synoviocytes in OA model was significantly higher than that in normal synoviocytes.3.Western Blot showed that the protein expression levels of OA inflammatory mediators(IL-6,IL-8,TNF-α)in the fibroblast-like synoviocytes with PENK overexpression were significantly lower than those in the OA inflammation model group(P<0.05).CCK-8 test showed that the proliferation ability of fibroblasts in PENK overexpression group was lower than that in control group.4.Western Blot showed that the protein expression level of OA inflammatory mediators(IL-6,IL-8,TNF-α)in the PENK knockdown HFLs line was significantly higher than that in the OA inflammation model group(P<0.05).CCK-8 assay showed that the proliferation ability of fibroblasts in PENK knockdown group was higher than that in control group.Conclusion: 1.There are some differences in the expression of PENK between human osteoarthritis fibroblast-like synoviocytes and human normal fibroblast-like synoviocytes.The expression level of PENK in human osteoarthritis fibroblast-like synoviocytes is significantly lower than that in normal synoviocytes.2.Overexpression of PENK in human normal fibroblast like synoviocytes and human osteoarthritis fibroblast like synoviocytes reduced the protein level of OA-related inflammatory mediators and inhibited the proliferation rate of human osteoarthritis fibroblast like synoviocytes.The results showed that PENK had a corresponding inhibitory effect on the proliferation of human osteoarthritis synoviocytes.3.Knockdown of PENK in human fibroblast-like synoviocytes and human osteoarthritis fibroblast-like synoviocytes increased the protein levels of OA-related inflammatory mediators and increased the activity of human osteoarthritis fibroblast-like synoviocytes,indicating that PENK deficiency may be one of the causes of the development of human osteoarthritis.