Effects Of FSH On Cartilage Matrix Through The PKA/CREB/SOX9 Signaling Pathway

Background:Osteoarthritis(OA)is a chronic,degenerative joint disease characterized by articular cartilage destruction and bone hyperplasia.The main clinical manifestations of OA are joint pain and limited mobility,which seriously affects the quality of life of patients.There are many reasons for OA,and gender and age are the most important factors.Epidemiological investigation shows that the prevalence of OA in the population aged 55 years in China reached 50%,and it is increases with age.With the increasing aging in China,OA has become a hot issue in society.In addition,the incidence of OA is higher in women than in men,and the incidence and severity of OA has increased significantly in postmenopausal women,but the mechanism is still unclear.During menopause,due to the decline of ovarian function,women will experience fluctuations in various hormone levels,which are mainly manifested by a decrease in sex hormone synthesis and secretion,and an increase in gonadotropin.Decreased estrogen levels have always been considered the cause of postmenopausal OA,but studies have found that in perimenopausal women with relatively normal estrogen levels,only when follicle stimulating hormone(FSH)increases,the incidence of OA has begun to increase,so it is proposed that FSH may be directly involved in the regulation of cartilage metabolism.Articular cartilage is mainly made up of chondrocytes and extracellular matrix(ECM).Chondrocytes are the only cells found in healthy articular cartilage,and they are responsible for the production and maintenance of the cartilage matrix.Normally,aggrecan and type Ⅱ collagen,which are the main matrix components of cartilage,are in a dynamic balance due to anabolic and catabolic processes.Once this homeostatic balance is disrupted,cartilage will inevitably degrade.Therefore,the imbalance in the synthesis and decomposition of ECM is considered to be a key factor of articular cartilage degeneration.In this study,we took the high risk of OA in perimenopausal women as the entry point,focused on the degradation of ECM,and studied the effect of FSH on cartilage.Objectives:To study the effect of FSH on cartilage ECM metabolism and to explore its mechanism of action.Methods:1.Eight-week-old C57BL/6 mice were selected,then microsurgery was performed to transect the medial meniscotibial ligament of the right knee joint,which contributed to the DMM model.In the sham-operated group,only the skin of the knee joint was resected.Mice were sacrificed 8 weeks after surgery,and the knee joints of the mice were taken for histopathological observation to evaluate the degree of cartilage damage.2.Eight weeks after DMM surgery,the mice were randomly divided into two groups:the follicle-stimulating hormone(FSHR)knockdown group and the control group,with 10 mice in each group.The knockdown group was injected with adeno-associated virus(AAV)-mediated FSHR-shRNA intra-articularly,and the control group was injected with an equal amount of unloaded control adenovirus(AAV-NC).The animals were sacrificed 3 weeks after the injection,and the knee joints of the mice were collected.Histopathological staining of the articular cartilage was performed to evaluate the knockdown efficiency,the degree of articular cartilage damage and the degradation of articular cartilage matrix.3.ATDC5 cells were cultured in vitro and induced to differentiate into mature chondrocytes for 2 weeks.Then FSH stimulation was given.Cell viability was measured by the Cell Counting Kit-8 assay(CCK-8),cartilage matrix-specific proteins Collagen Ⅱ,Aggrecan,and PKA pathway-related proteins were detected by Western blot analysis and immunofluorescence staining.Results:1.A mouse osteoarthritis model was successfully constructed.In the DMM operation group,the joint space narrowed,the cartilage layer became thinner,the cartilage surface integrity was damaged,the cartilage cell structure was disorderly arranged,the cartilage matrix was largely lost,the subchondral bone sclerosis and osteophyte formation was obvious.In the sham operation group,the cartilage surface was smooth and the chondrocytes were evenly distributed.2.Histopathological staining results showed that compared with the control group,the expression of FSHR protein on the surface of articular cartilage and the growth plate area in the FSHR knock-down group was significantly reduced,and the degree of cartilage disruption and cartilage matrix degradation were improved.3.FSHR was detected on the membranes of ATDC5 cells.The CCK8 analysis results show that there was no obvious effect of FSH on cell viability,Western blotting results showed that FSH reduced the protein expression of Collagen Ⅱ and Aggrecan,and was verified in the results of immunofluorescence,besides,Western blotting results confirmed the association between cartilage degradation induced by FSH and the PKA/CREB/SOX9 pathway.Summary and conclusions:1.Blocking FSH signaling in joint tissues effectively delayed the development of posttraumatic OA in mice.2.FSH was able to induce the degradation of Collagen Ⅱ and Aggrecan,the main ECM-related proteins,in ATDC5 chondrocytes.Mechanistically,its catabolic effects were due to the suppression of the PKA-CREB-SOX9 signaling pathway.3.Blocking FSH and its related signaling pathways is expected to be a new strategy for the treatment of OA.