Effect Of Follicle-stimulating Hormone On Cartilage Damage Of Knee Osteoarthritis In Postmenopausal Women And Its Mechanism

Background:As a chronic degenerative joint disease,osteoarthritis(OA)is the main cause of pain and disability in the elderly,which seriously affects the patients' life quality.The knee is the most common affected joint in OA.At present,there is no specific clinical treatment for knee osteoarthritis(KOA),which is usually symptomatic treatment and pain relief.However,analgesics like non-steroidal anti-inflammatory drugs can cause side effects including gastrointestinal hemorrhage.If the disease progresses,knee replacement surgery is needed,but for older patients,the surgery risk is greater.The onset of KOA has been found to be affected by many factors,such as gender and aging.The study found a significant increase in the prevalence of KOA in postmenopausal women,which was significantly higher than in men.The classic view assumes that the underlying mechanism of the increased prevalence of KOA is attributed to an insufficiency of estrogen.There are estrogen receptors in the cytoplasm and nuclei of human chondrocytes,and the combination of estrogen and receptor in women of reproductive age can promote the differentiation and proliferation of chondrocytes.Therefore,many postmenopausal women have degenerative changes in knee cartilage due to the decrease of estrogen level in the body.Estrogen provides a protective effect on the cartilage surface of the knee joint in ovariectomized rats by effective inhibition of type II collagen degradation.However,it has also been reported that the incidence of KOA increased significantly during perimenopause,when only follicle-stimulating hormone(FSH)was increased but estrogen was not significantly reduced,suggesting that FSH may directly affect the occurrence of KOA.FSH secreted by the pituitary and estrogen secreted by the ovary have a feedback-negative feedback regulation in the body.However,in addition to a decrease in estrogen,circulating FSH levels become elevated at menopause.Increased FSH is also considered as a sign of menopause.Studies have shown that postmenopausal female aged 50-60 years have the highest levels of FSH,which then gradually decline with age.FSH has been found to regulate bone metabolism independently of estrogen.FSH has been shown to accelerate osteoclast formation through Gi2a-coupled FSHR.Furthermore,FSH receptor(FSHR)stimulation activates AKT/NF-?B pathway to produce osteoclasts.FSH injection increased alveolar bone loss in ovariectomized rats in vivo experiments,independent of estrogen.Osteoporosis(OP)and OA are two closely related diseases.When OP occurs in the subchondral bone,joint instability or protective hyperplasia of unstable areas will occur to promote the development of OA.However,whether FSH is independent of estrogen and directly affects the cartilage of postmenopausal women with KOA has not been reported at home and abroad.Previous study has found that FSHR protein is located on the human chondrocyte membrane.Since the prevalence of KOA in postmenopausal women aged 50-60 years is increasing rapidly,it is interesting to note that postmenopausal women aged 50-60 years have the highest levels of FSH,so we speculate that the combination of FSH and FSHR on the cartilage cell membrane may cause adverse effects on the cartilage of postmenopausal women aged 50-60 years with KOA.Therefore,this study intends to explore from this perspective.In response to this hypothesis,we will proceed from clinical and cell experiments,respectively,in the hope of further studying the pathogenesis of KOA in postmenopausal women.PI3K/AKT signaling pathway has a wide range of functions in the body and is involved in many physiological and pathological processes.Among them,it is also closely related to the degeneration and destruction of OA articular cartilage.Therefore,it is considered a feasible strategy to alleviate the disease of OA by acting on the PI3K/AKT signaling pathway.Relevant studies have found that PI3K/AKT/mTOR pathway could affect articular chondrocyte autophagy and participate in the inflammatory response in OA rats.Allicin alleviates the progression of OA by inactivating the PI3K/AKT/NF-?B pathway.Studies have shown that the anti-inflammatory and chondroprotective effects of nobletin on IL-1?-induced human OA chondrocytes and in the surgical DMM mice OA models.Furthermore,nobletin dramatically suppressed the IL-1?-stimulated phosphorylation of PI3K/AKT and activation of NF-?B in human OA chondrocytes.The above studies demonstrated that PI3K/AKT pathway may be a key signaling pathway for the pathogenesis of KOA.In this clinical study,we analyzed the effects of different FSH levels on subjective symptoms and imaging changes in postmenopausal women aged 50-60 years with KOA.In addition,we investigated whether FSH contributes to the destruction of articular cartilage in postmenopausal female KOA patients via PI3K/AKT/NF-?B signaling pathway.The mouse embryonal carcinoma-derived cell line ATDC5 is a widely used cellular model of chondrogenesis.Under the appropriate stimulation,ATDC5 cells not only can be introduced into cartilage cells proliferate,aggregation and differentiation stage,but eventually differentiate into fertile chondrocytes,and ATDC5 cells can synthesize main components of extracellular matrix(ECM),such as aggrecan,type ? collagen.At present,the effect of PI3K/AKT pathway on ATDC5 chondrocyte metabolism has been studied.Such as laquinimod could ameliorate IL-1?-induced production of TNF-? and IL-6 in human chondrocytes,and prevent the degradation of type ? collagen by inhibiting MMP-3 and MMP-13.Meanwhile,the presence of laquinimod attenuates the reduction in aggrecan by mediating thrombospondin motifs-4(ADAMTS-4)and thrombospondin motifs-5(ADAMTS-5).Mechanistically,laquinimod ameliorates IL-1?-induced inflammation and degeneration of ECM by suppressing the activation of PI3K/AKT/NF-?B signaling pathway.In contrast,non-coding RNA LncHIFCAR promotes the aggravation of ATDC5 chondrocytes by activating the PI3K/AKT/mTOR pathway.These positive and negative results suggest that the PI3K/AKT pathway plays an important role in ATDC5 chondrocytes.The specific mechanism of the effect of FSH on cartilage injury is still unclear.The cell experiment in this study was partly based on the ATDC5 chondrocyte model.First,we focused on the effect of FSH on the ECM of ATDC5 cells,and then verified whether FSH enhanced the destruction of ATDC5 chondrocytes via PI3K/AKT/NF-?B signaling pathway.Objectives:1.To determine the effects of different FSH levels on the degree of clinical symptoms,biochemical indicators and imaging indicators in postmenopausal women aged 50-60 years with KOA.2.To observe whether FSH can promote articular cartilage destruction in postmenopausal women aged 50-60 years with KOA via the PI3K/AKT/NF-?B pathway.3.To observe whether FSH can promote the degradation of ECM of ATDC5 chondrocytes via the PI3K/AKT/NF-?B pathway in vitro,and to identify the molecular mechanism of FSH aggravating cartilage injury.Methods:1.215 postmenopausal female KOA patients over 50 years old were recruited in this study,which were divided into 50-60 age group(n=79),61-70 age group(n=88)and>70 age group(n=48).In the 50-60 age group,the patients were divided into high FSH group(>40mIU/ml;n=45)and low FSH group(<40mIU/ml;n=34).BMI,WOMAC scoring and knee magnetic resonance T2-mapping were performed.Serum FSH,estradiol(E2),fasting plasma glucose(FPG),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C)were examined.2.Serum TC,LDL-C,FSH,E2,WOMAC scoring and T2-mapping were compared between high FSH group and low FSH group.Then,Safranin O-fast green staining was performed.The p-PI3K/PI3K,p-AKT/AKT,p-NF?B/NF?B levels in articular cartilages were examined using western blot.3.ATDC5 chondrocytes were stimulated with different concentrations of FSH.The expressions of NF-?B p65,collgan2A and aggrecan were observed by immunofluorescence.Furthermore,collgan2A,p-PI3K/PI3K,p-AKT/AKT,P-NF?B/NF?B and p-I?Ba/I?B? levels were detected using western blot.Results:1.Serum FSH level was the highest in postmenopausal female patients with KOA aged 50-60 yearsIn this study,postmenopausal female patients with KOA were divided into three groups:50-60 age group,61-70 age group and>70 age group.The results showed that there were no significant differences in height,weight,BMI,E2,HDL-C and TG among the three groups(P>0.05).The levels of TC,LDL-C in patients 50-60 years were increased than those in the other two groups,and significantly increased than those in patients>70 years(P<0.05).There were no significant differences between other groups.E2 levels were low in all the three groups,with no significant difference.More importantly,we found that FSH in patients 50-60 years was significantly increased than that in the other two groups(P<0.05 or P<0.001).2.FSH exacerbates magnetic resonance cartilage damage and lipid metabolism disorder in postmenopausal women with KOAIn the 50-60 age group,all patients were classified into high FSH group and low FSH group.We found that there were no significant differences in BMI and E2 between the two groups(P>0.05).Furthermore,average T2 values and TC levels were both significantly higher in high FSH group than low FSH group(P<0.05).LDL-C and WOMAC scores were both higher in high FSH group than low FSH group,but there were no significant differences(P>0.05).3.FSH exacerbates pathological damages of articular cartilage tissues in postmenopausal women with KOAWe further observed the pathological changes of cartilages in postmenopausal female KOA patients with low FSH and high FSH by Safranin O-fast green staining.For low FSH,superficial cracks could be found on the surface of cartilage,with slightly disordered arrangement of chondrocytes,and clustered in the transitional layer.We also found that dehydrated,pyrettic and necrotic chondrocytes were at the junction of the transitional layer and the radiation layer,forming defect areas,and multiple voids and sockets appeared on the surface,with relatively complete tidal lines,fair distribution density of cells.Furthermore,the numbers of cells in the low FSH were more than those in the high FSH group.As for the pathological changes of cartilages in the high FSH,the surface of cartilage was rough and multiple cracks appeared,extending from the surface of cartilage to the radiation layer.Moreover,loose network connections could be seen in the cracks,with the phenomenon of chondrocyte clustering.The number of chondrocytes was significantly decreased,and the four-layer structure was disordered,which was not easy to distinguish.We further observed the pathological changes of cartilages of postmenopausal female KOA patients with high FSH under 100 x microscope,as follows:first,tidal markers were duplicated,drifted,and the gap expanded.Multiple tidal markers and significant thickening of the calculation layer were found.Second,the calculation layer thickened as the blood vessels.Third,breaks and lack of tide marks were observed.Fourth,the calcified layer and deep cartilage were defective and the subchondral bone was directly exposed.4.FSH could significantly activate PI3K/AKT/NF-?B pathway in postmenopausal women with KOAThe protein expression levels of p-PI3K,PI3K,p-AKT,AKT,p-NF?B and NF-?B were detected in articular cartilage tissues of postmenopausal female KOA patients with low FSH group and high FSH group using western blot.Compared with the low FSH group,the expression levels of p-PI3K/PI3K,p-AKT/AKT and p-NF?B/NF?B were significantly higher in the high FSH group than those in the low FSH group(P<0.05 or P<0.01).5.FSH could inhibit the expression of collgan2A and aggrecan and promote the nucleation of NF-?B p65 in ATDC5 chondrocytesIn this study,we examined the expression of collgan2A and aggrecan after ATDC5 chondrocytes were stimulated by different concentrations of FSH(Ong/?l;90ng/?l).90ng/?l FSH significantly inhibited the expression of collgan2A and aggrecan in ATDC5 chondrocytes.The nuclear translocation of NF-?B p65 was also measured using immunofluorescence staining.The results showed that 90ng/?l FSH stimulation in ATDC5 chondrocytes significantly promoted a dramatic increase in the translocation of p65 into the nucleus.6.FSH could activate PI3K/AKT/NF-?B pathway in ATDC5 chondrocytesThe protein expression levels of collgan2A,p-PI3K/PI3K,p-AKT/AKT,p-NF?B/NF?B,p-I?Ba/I?Ba were detected in ATDC5 chondrocytes stimulated with different concentrations of FSH(Ong/?l;9ng/?l;30ng/?l;90ng/?l)using western blot.We found that FSH significantly decreased the expression of collgan2A and total I?B? in ATDC5 chondrocytes,with a concentration dependent manner.Rather,FSH significantly increased the expression levels of p-PI3K/PI3K,p-AKT/AKT,p-NF?B/NF?B,p-I?Ba/I?Ba in ATDC5 chondrocytes,with a concentration dependent manner(P<0.05,P<0.01,P<0.001 or P<0.0001).Conclusions:1.FSH exacerbates subjective symptoms and imaging cartilage damage in postmenopausal women aged 50-60 years with KOA.FSH may aggravate cartilage damage in KOA patients by activating PI3K/AKT/NF-?B pathway.2.FSH significantly inhibits the expression of collgan2A and aggrecan in ATDC5 chondrocytes,promotes a dramatic increase in the translocation of NF-?B p65 into the nucleus,and aggravates the degradation of ECM components by activating PI3K/AKT/NF-?B pathway,suggesting that FSH is involved in cartilage damage.