【作者】 王燕；

【导师】 徐进；

【作者基本信息】 山东大学 ， 内科学（内分泌与代谢病）， 2021， 博士

【摘要】 研究背景:骨关节炎(osteoarthritis,OA)是一种与衰老相关的、主要病理表现为关节软骨破坏的退行性疾病。OA治疗方法非常有限,常用的治疗措施主要是缓解症状,目前尚没有可以干预OA进展的药物。因此明确OA的发病机制,进而针对性的早期干预尤为重要。不同类型的OA发病机制不同。继发性OA的主要原因有:创伤、肿瘤等因素。原发性OA机制尚不明确,一些研究认为年龄、性别、肥胖等因素可能参与了其发病,为早期干预带来困难。流行病学研究表明OA主要影响中老年人群,发病率表明女性在绝经前与男性相似,但是,随着女性进入绝经期,绝经后女性OA发病率明显高于男性。传统观点将绝经后女性OA发病升高的原因归结于雌激素(E2)水平的下降。然而,临床上的一些现象并不能仅仅用E2水平的降低解释,提示其他因素可能参与了OA的发生发展。研究发现:围绝经期女性卵泡刺激素(follicle-stimulating hormone,FSH)水平升高早于E2水平的下降;在E2水平还处于相对正常的水平,仅有FSH升高时,OA的患病率已经开始显著升高。且课题组的前期研究发现FSH与绝经后女性膝OA的严重程度相关,是OA的独立危险因素。然而,FSH对关节软骨或OA影响的研究甚少,亟待明确。本研究从这一现象出发,探讨FSH对软骨细胞的影响,寻找绝经后OA的发病原因。FSH是由垂体合成的糖蛋白激素,其受体FSHR(FSH receptor,FSHR)是一个七次跨膜的G蛋白偶联受体(GPCR)。一般来说,当配体与细胞膜上的受体结合后,可以通过不同的Gα蛋白(Gαs、Gαi、Gαq、Gα12)激活下游第二信使,作用于不同的反应元件发挥功能。以往认为FSH主要在性腺轴上发挥作用,通过结合在卵巢上的FSHR,调控卵泡的发育和雌激素分泌。根据课题组前期发表的研究,FSHR在人和小鼠原代软骨细胞上有表达。我们推测FSH通过与FSHR结合,影响特定的Gα蛋白,活化下游信号通路,对软骨细胞产生影响。越来越多研究认为OA不仅仅累及关节软骨,而是与整个关节有关的疾病。但是软骨的病理改变贯穿OA的整个环节。软骨中只有一种细胞——软骨细胞,它参与了细胞外基质(extracellular matrix,ECM)的合成。ECM主要成分是胶原,占软骨干重的60%。其中Ⅱ型胶原(type Ⅱ collagen,Coll-Ⅱ)占所有胶原的90-95%,是关节软骨主要的胶原成分。研究表明OA时软骨细胞出现分化异常。软骨细胞分化异常是OA的核心环节,探索软骨细胞行为改变对了解OA发病机制十分必要。软骨细胞在一些病理刺激下可以经历去分化。因软骨细胞去分化在OA发生发展中发挥了重要作用,逐渐引起了大家的关注。去分化的软骨细胞也被认为是幼稚的软骨细胞。研究发现去分化的软骨细胞表现出间充质干细胞的特点,如细胞形态由类圆形向长梭形改变;且软骨细胞分化标志物Coll-Ⅱ表达减低。因此,软骨细胞去分化可以导致软骨细胞表型的消失、ECM重塑,最终导致软骨机械性能下降和OA的发生。故本研究通过检测FSH对软骨细胞分化相关指标、分化主要标志物Coll-Ⅱ、细胞骨架影响,探究FSH对软骨细胞去分化的影响。研究表明ERK1/2 MAPK和p38 MAPK信号通路参与软骨细胞去分化。抑制p38 MAPK信号通路可以防止软骨细胞向肥大细胞分化;而抑制ERK1/2信号通路则可以增加Coll-Ⅱ的表达,促进软骨细胞分化。由此,ERK1/2和p38信号通路对软骨细胞分化的作用是相反的。本研究观察FSH对ERK1/2和p38信号通路的影响,并利用信号通路抑制剂,明确这两个通路在FSH影响的去分化中的作用。FSH抗体(FSH antibody,FSHAb)阻断小鼠模型是研究FSH效应缺失的经典模型。研究表明阻断FSH可以改善小鼠的骨质疏松、改善肥胖和高胆固醇血症的发生。在本研究中,我们利用FSH抗体阻断FSH,观察阻断FSH是否可以预防小鼠的膝关节软骨退变和软骨细胞去分化。研究表明表面区蛋白(superficial zone protein,SZP)或者蛋白多糖 4(proteoglycan 4,Prg4)在 OA 早期即可发现表达减低,是OA的早期标志物。早期生长反应蛋白1(early growth response protein 1,Egr1)在OA软骨中明显升高,且调控OA进展,被认为是OA的标志物。我们检测了这两个指标,寻找FSH调控绝经后OA的证据。本研究以绝经后女性OA发病率增高为切入点,通过体内和体外研究探究FSH对软骨细胞的影响和机制。体外研究利用原代软骨细胞和ATDC5软骨细胞系,发现FSH可以促进软骨细胞去分化;体内利用FSH抗体阻断小鼠模型,发现阻断FSH可以预防软骨退变、抑制软骨细胞去分化。对其机制进行探索,发现FSH通过结合FSHR,偶联Gαi蛋白,一方面活化ERK1/2,抑制p38信号通路,一方面抑制SOX9-CREB信号通路,最终导致Coll-Ⅱ表达减低,ECM合成减少。我们的研究揭示了 FSH对软骨细胞去分化的影响,建立了 FSH调控去分化的信号通路,为FSH影响绝经后OA提供支持依据和治疗靶点。研究目的:1.利用原代软骨细胞及软骨的细胞系——ATDC5细胞,明确FSH可以引起软骨细胞去分化。2.利用ERK1/2和p38通路抑制剂,G αi蛋白抑制剂和Fshr小干扰RNA阐明FSH调控软骨细胞去分化的信号通路。3.通过检测关节软骨FSHR的表达,对FSHR进行组织定位。通过检测FSHAb小鼠膝关节软骨的去分化指标(Coll-Ⅱ)、软骨细胞数目、软骨厚度、OA评分和OA标志物的表达,探讨阻断FSH在预防关节软骨退变中的作用。研究方法:1.细胞培养:小鼠原代软骨细胞和ATDC5软骨细胞系按照培养条件培养。细胞处理之前无血清培养基饥饿两小时去除血清中激素的影响。2.小鼠模型:9周龄雌性C57BL/6J小鼠分为两组,一组注射FSH中性抗体(FSHAb)组,一组注射IgG抗体(IgG对照组),分别腹腔注射FSH和IgG抗体,持续8周。3.CCK8实验:设置时间梯度(24h、48h、72h)和浓度梯度(0ng/mL、10ng/mL、100ng/mL)观察FSH对原代软骨细胞活力的影响。4.实时荧光PCR(qRT-PCR)实验:FSH(10ng/ml)刺激原代软骨细胞后,提取RNA进行实时荧光定量PCR(qRT-PCR)实验,检测Co12al,Acan,Sox9,Sox5,Mmp3,Mmp13,Col10a1,Ihh,Pth1h等分化相关的基因的表达。观察对细胞分化的影响。检测FSH对原代软骨细胞Prg4和Egr1的影响,确定FSH对OA标志物(Prg4,Egr1)的影响。5.蛋白免疫印迹(Western blot)实验检测FSH对细胞分化标志物等指标的影响。6.细胞免疫荧光实验:检测ATDC5细胞表面FSHR的表达。细胞免疫荧光检测FSH(30ng/ml)对 ATDC5 细胞 Egr1 和 Prg4 的影响。7.鬼笔环肽染色:FSH(0ng/mL、3ng/mL、10ng/mL、30ng/mL)刺激 ATDC5 细胞后行鬼笔环肽染色对软骨细胞骨架蛋白(F-actin)进行检测,确定FSH对细胞形态的影响。8.Fshr siRNA干扰实验:设计3对siRNA(#1,#2,#3),转染siRNA后,利用qRT-PCR检测fshr的表达,选择效率最高的一条(#2)用于后续试验。原代软骨细胞转染#2 fshr siRNA后。提取蛋白进行Western blot实验,观察对主要分化标志物Coll-Ⅱ的影响。明确FSHR在FSH介导的软骨细胞去分化中的作用。9.cAMP检测实验:FSH刺激原代软骨细胞后,检测cAMP的表达。百日咳毒素(PTX)可以干扰Gαi的表达,用来确定Gαi的作用;毛喉素(forskolin)可以刺激cAMP,用来作为阳性对照。选用Cayman的cAMP试剂盒进行检测。10.免疫组化:小鼠膝关节软骨组织切片行免疫组化实验检测FSHR的表达,确定FSHR在关节软骨的定位;检测Coll-Ⅱ、Coll-Ⅹ的表达,确定阻断FSH对软骨细胞分化的影响。Ki67染色确定阻断FSH后对细胞增殖的影响。Egr1染色检测阻断FSH对Egr1的影响。11.番红/固绿染色:IgG和FSHAb小鼠取膝关节,进行石蜡包埋、切片后行番红染色指示软骨组织;固绿染色指示骨组织,观察软骨组织的损伤和软骨细胞情况。12.Mankin评分和OARSI评分:利用Mankin评分和OARSI评分对两组小鼠膝关节组织进行评价,分析关节软骨的损伤和退变情况。13.MicroCT:观察膝关节周围骨质的变化。14.统计学分析:用Graphpad对数据进行分析,两组比较采用非参数检验后的Mann-Whitney检验,多组比较采用非参数检验后的Kruskal-Wallis检验。P值小于0.05被认为有统计学意义。研究结果:1.FSH不影响软骨细胞凋亡和增殖利用CCK-8试剂盒,发现FSH(0ng/mL、10ng/mL、100ng/mL)在不同时间点(24h、48h和72h)不影响软骨细胞活力,提示FSH对细胞没有毒性。2.FSH促进软骨细胞去分化qRT-PCR结果显示FSH影响软骨细胞分化相关的指标。主要表现为FSH下调Col2a1,Acan,Sox9,Sox5,Mmp3,Mmp13,Col10a1;下调促进分化的Ihh基因,上调抑制软骨细胞分化的Pth1h基因,提示FSH抑制软骨细胞分化。Western blot结果显示FSH剂量依赖性的抑制Coll-Ⅱ、COMP、aggrecan的合成,抑制Coll-Ⅱ的转录因子SOX9的合成;抑制CREB的磷酸化水平,提示影响CREB/SOX9/Coll-Ⅱ信号通路。鬼笔环肽染色显示FSH引起ATDC5细胞骨架重塑,由类椭圆形向长梭形改变,提示去分化样改变。3.ATDC5软骨细胞系有FSHR的表达课题组前期发表的研究观察到原代软骨细胞上有FSHR的表达,但是软骨细胞系上是否有受体表达尚无研究。细胞免疫荧光显示FSHR在ATDC5细胞表面有表达,ATDC5细胞系可以用于后续的实验研究。4.FSH通过FSHR调控软骨细胞去分化利用siRNA干扰fshr,观察到干扰Fshr可以逆转FSH下调的Coll-Ⅱ,提示FSHR参与了 FSH调控的软骨细胞去分化。5.Gai蛋白参与FSH调控的软骨细胞去分化利用cAMP试剂盒检测FSH是否影响第二信使cAMP的表达,结果显示FSH可以抑制cAMP的合成。用PTX抑制Gαi蛋白后,可以逆转FSH下调的cAMP和Coll-Ⅱ,表明Gαi蛋白参与FSH调控的软骨细胞去分化。6.ERK1/2 MAPK和p38 MAPK信号通路介导了 FSH调控的软骨细胞去分化Western blot实验显示FSH可以上调ERK1/2 MAPK磷酸化水平,下调p38 MAPK的磷酸化水平。用ERK1/2的抑制剂PD98059可以逆转FSH下调的Coll-Ⅱ;用p38抑制剂SB203580可以促进FSH下调Coll-Ⅱ。结果提示ERK1/2和p38参与FSH调控的软骨细胞去分化。7.FSHR定位于小鼠膝关节软骨的表面区组织切片免疫荧光显示FSHR主要定位于关节软骨的表面区(superficial zone,SZ),提示FSH可能主要影响表面区的软骨细胞。8.FSH抗体(FSHAb)阻断可以改善骨小梁微结构、防止软骨细胞去分化和关节软骨退变FSHAb阻断可以改善骨小梁相关参数,增加软骨Coll-Ⅱ的表达;改善OA的相关评分如Mankin Score和OARSI Score。番红染色提示阻断FSH后番红染色加深,软骨面损伤减少,软骨厚度增加,软骨细胞数目增加。免疫组化显示阻断FSH可以提高Ki67阳性的细胞数和强度。以上提示阻断FSH可以防止软骨细胞去分化和软骨退变。9.FSH影响OA标志物近期研究表明Prg4和Egr1是OA的标志物,我们观察到FSH可以上调Egr1,降低Prg4的表达。阻断FSH效应后可以降低Egr1的表达。以上提示FSH可能通过影响OA标志物参与OA的发生。结论:1.FSH抑制软骨ECM合成,参与调控软骨细胞去分化。2.FSH通过结合FSHR,偶联Gai蛋白,调控ERK1/2和p38信号通路以及Sox9-CREB-Coll-Ⅱ信号通路调控的软骨细胞去分化。3.FSH影响OA的两个标志物Egr1和Prg4的表达。4.阻断FSH可以改善骨微结构,增加软骨细胞数目、软骨厚度,防止软骨损伤和软骨细胞去分化。更多还原

【Abstract】 BackgroundOsteoarthritis(OA)is a degenerative joint disease associated with aging,and related to the destruction of articular cartilage.Once OA occurs,treatment methods are very limited,and the commonly used treatment measures are mainly to relieve the pain symptoms.At present,there is no drug that can interfere with the progression of OA.Therefore,it is particularly important to clarify the pathogenesis of OA,and then targeted early intervention.Some specific causes,such as trauma or neoplasms,can cause secondary OA.Primary OA may be related to age,gender,obesity,and other factors,but the mechanism is not clear,which brings difficulties for early intervention.Epidemiological studies show that OA mainly affects the elderly people;the incidence of premenopausal OA in women is similar to that in men.From the beginning of perimenopause,the incidence of OA in women is significantly higher than that in men.Traditionally,it is believed that the main cause of OA in postmenopausal women is the decrease of estrogen(E2)level.However,some clinical phenomena cannot be explained by the decrease of E2 level alone,suggesting that other factors may play roles.Studies found that in perimenopausal women,follicle-stimulating hormone(FSH)levels rise earlier than E2 levels fall.When E2 level was still at a relatively normal level,but FSH has been rises,the prevalence of OA began to increase significantly.In addition,our previous study have found that FSH is related to the severity of knee osteoarthritis in postmenopausal women and is an independent risk factor for OA.However,the effects of FSH on articular cartilage or osteoarthritis are poorly studied and need to be clarified.Based on this phenomenon,this study explores the effect of FSH on chondrocytes and seeks for the pathogenesis of postmenopausal OA.FSH is a glycoprotein hormone synthesized by pituitary,and its receptor FSHR is a seven-fold transmembrane G protein coupled receptor(GPCR).In general,when ligands bind to receptors on the cell membrane,they can activate the downstream second messenger through different Gα proteins(Gαs,Gαi,Gαq,and Gα 12),acting on different reaction elements to perform functions.Traditionally,it was believed that FSH mainly plays a role in the gonadal axis and regulates follicle development and estrogen secretion by binding to FSHR on the ovary.According to our previous study,FSHR is expressed in human and mouse chondrocytes.We hypothesized that FSH affects specific Ga proteins by binding to FSHR and activates downstream signaling pathways,thus influencing chondrocytes.OA is a disease that affects the entire joint.Articular cartilage damage is existed through throughout OA.The only cell type is chondrocyte.They can produce extracellular matrix(ECM)components.The main component of ECM is collagen,which accounts for 60%of the dry weight.The main collagen component of articular cartilage is type Ⅱ collagen(Coll-Ⅱ)，accounting for 90-95%of all collagens.Studies have shown that chondrocytes appear abnormal differentiation during OA.Chondrocytes can undergo dedifferentiation under some pathological conditions(such as inflammation and long-term culture in vitro).Since dedifferentiation of chondrocytes plays an important role in the occurrence and development of OA,it has gradually attracted public attention.The dedifferentiated chondrocytes are considered as naive chondrocytes.The dedifferentiated chondrocytes showed the characteristics of mesenchymal stem cells,such as the cell morphology changed from round to fibroblast phenotype.The dedifferentiation of chondrocytes was mainly characterized by decreased expression of Coll-Ⅱ.Therefore,chondrocyte dedifferentiation can lead to the disappearance of chondrocyte phenotype,ECM remodeling,mechanical properties damaging and the occurrence of OA.Therefore,this study explored the effect of FSH on chondrocyte dedifferentiation by detecting the effects of FSH on Coll-Ⅱ and cytoskeleton of chondrocytes.ERK1/2 MAPK and P38 MAPK signaling pathways have been shown involving in chondrocyte dedifferentiation and OA.Inhibition of p38 signaling pathway can prevent the hypertrophic differentiation of chondrocytes.Inhibition of ERK signaling pathway can increase the expression of Coll-Ⅱ,and promote chondrocyte differentiation.Thus,ERK1/2 and p38 signaling pathways play opposite roles in regulating chondrocyte differentiation.This study aims to investigate the effect of FSH on chondrocyte differentiation by observing the effects of FSH on ERX1/2 and p38 signaling pathways.The roles of ERK1/2 MAPK and p38 MAPK signaling pathways in dedifferentiation were further clarified by using ERK 1/2 and p38 signaling inhibitors.The FSH antibody blocking mouse model is a classic model for investigating the loss of FSH effects.It has been adopted for studying osteoporosis and obesity after menopause.Studies have shown that blocking FSH by FSH antibody improves osteoporosis;improve the occurrence of obesity and hypercholesterolemia and in mice.In this study,we used FSH antibody to block FSH to observe whether blocking FSH could prevent knee cartilage degeneration and chondrocyte dedifferentiation in mice.Studies have shown that the superficial zone protein(SZP)or proteoglycan 4(Prg4)is decreased in the early stage of OA and is an early marker of OA.Early growth response protein 1(Egrl)is significantly increased in OA cartilage and regulates OA progression,which is considered as a marker of OA.We examined these two indicators to find evidence that FSH regulates postmenopausal OA.Based on the phenomenon that OA is increased in postmenopausal women,this study explored the effect of FSH on chondrocytes through basic studies in vivo and in vitro.In vitro studies using primary chondrocytes and ATDC5 chondrocytes showed that FSH promoted the dedifferentiation of chondrocytes.In vivo,FSH antibody was used to block FSH in the mouse model,and it was found that blocking FSH can prevent cartilage degeneration and inhibit chondrocytes dedifferentiation.Exploring its mechanism,it was found that FSH,by binding FSHR and coupling Gαi protein.On the one hand FSH activates ERK1/2 and inhibits the p38 signaling pathway,on the other hand inhibits CREB-SOX9 signaling,eventually leads to the decrease of Coll-II expression and ECM remodeling.Our study reveals the effect of FSH on chondrocyte dedifferentiation,and established the signaling pathway of FSH regulating dedifferentiation,thus providing a supporting basis and therapeutic target for FSH affecting postmenopausal OA.Objectives:1.Using primary chondrocytes and ATDC5 chondrocyte lines to observe whether high FSH can cause chondrocyte dedifferentiation2.Using ERK1/2 and p38 pathway inhibitors,Gαi protein inhibitors and FSHR small interfering RNAs to clarify the signaling pathway that FSH regulates chondrocyte dedifferentiation3.Tissue localization of FSHR was performed by detecting the expression of FSHR in articular cartilage.To investigate the role of blocking FSH in preventing articular cartilage degeneration,Coll-II expression,number of chondrocytes,cartilage thickness,OA score and OA marker expression of knee cartilage in FSHAb mice were detected.Methods:1.Cell culture:mouse primary cells and ATDC5 chondrocyte lines were cultured according to the culture conditions.Cells were starved to remove the serum hormones.2.Mouse model:9-week-old female C57BL/6J mice were divided into two groups:one group is FSH antibody(FSHAb)group and the other is IgG control group.Mice were intraperitoneally injected FSHAb and IgG antibody,respectively,for 8 weeks.3.CCK8 experiment was performed on primary chondrocytes:time gradients(24h,48h,72h)and concentration gradients(Ong/mL,10ng/mL,100ng/mL)were set to observe the effect of FSH on chondrocyte viability.4.Real-time quantitative PCR(qRT-PCR)assay:after the primary chondrocytes were stimulated by FSH(10ng),differentiation related genes were detected:Col2a1,acan,sox9,sox5,Mmp3,Mmp13,Coll0a1,Ihh,Pthlh.The effect on cell differentiation was observed.The effect of FSH on Prg4 and Egr1 of primary chondrocytes was detected to determine the effect of FSH on OA markers(Prg4,Egr1).5.Chondrocyte differentiation markers were then detected by Western blot(WB)assay6.Cellular immunofluorescence assay:to detect the expression of FSHR.The effect of FSH(30ng/mL)on Egrl and Prg4 was detected by cellular immunofluorescence in ATDC5 cells.7.Rhodamine phalloidin staining.After stimulating primary chondrocytes with FSH(0ng/mL,3ng/mL,10ng/mL,30ng/mlL).The expression of F-actin in cartilage cytoskeleton of ATDC5 cells was detected by Rhodamine phalloidin staining to determine the effect of FSH on cell morphology.8.Fshr siRNA interference experiment:3 pairs of siRNA(#1,#2,#3)were designed.After siRNA was transfected,the expression of Fshr was detected by qRT-PCR,and the most efficient one(#2)was selected for the subsequent experiment.Primary chondrocytes were transfected with#2 Fshr siRNA.9.cAMP assay:The expression of cAMP was detected after FSH stimulation of primary chondrocytes.The pertussis toxin(PTX)can interfere with the expression of Gαi,and was used to determine the role of Gai.Forskolin can stimulate cAMP and is used as a positive control.Cayman’s cAMP kit was used for detection.10.Immunohistochemical(IHC)assay.The expression of FSHR was detected by immunohistochemical(IHC)assay to determine the location of FSHR in articular cartilage.The expression of Coll-II and Coll-X was detected to determine the effect of FSH blockade on chondrocyte differentiation.Ki67 staining was used to determine the effect of blocking FSH on cell proliferation.Egr1 staining was used to determine the effect of blocking FSH on Egr1.11.Safranin O/fast green staining.The assay was used to distinguish cartilage and bone tissue,and to observe the damage of cartilage tissue.12.Mankin score and OARSI score were performed on the knee tissue sections of mice to analyze the injury and degeneration of articular cartilage.13.MicroCT was used to detect bone structure of mice.14.Statistical analysis:data were analyzed by GraphPad.The Mann-Whitney test after nonparametric test was used for comparison between two groups,and the Kruskal-Wallis test after nonparametric test was used for comparison between multiple groups.A p value less than 0.05 was considered statistically significant.Results:1.FSH did not affect chondrocyte apoptosis and proliferation CCK-8 kit was used to detect FSH at 0,10,100 ng at 24h,48h and 72h.Results shows that FSH did not affect chondrocyte proliferation and apoptosis.2.FSH promotes chondrocyte dedifferentiationqRT-PCR results showed that FSH affected the indexes related to chondrocyte differentiation.FSH mainly down-regulates chondrocyte differentiation indexes,such as Col2a1,Acan,Sox9 and Sox5 genes related to cartilage matrix synthesis.Mmp3,Mmp13,Coll0a1 genes related to hypertrophy;Ihh was down-regulated and pthlh was up-regulated,indicating FSH inhibits chondrocyte differentiation.Western blot results showed that FSH inhibited the synthesis of Coll-Ⅱ and its transcription factor SOX9 in a dose-dependent manner.Besides,FSH inhibits phosphor-CREB,indicating that CREB/SOX9/Coll-II was affected.Rhodamine phalloidin staining shows that FSH effects cell morphology,chondrocytes changes from a round phenotype to fibroblast phenotype.3.FSHR expressed on ATDC5 chondrocyte cell lineWe have previously observed the expression of FSHR on primary chondrocytes,but whether FSHR expresses on chondrocyte lines has never been studied.Cell immunofluorescence showed that FSHR receptor was expressed on the cell surface after induction.4.FSH regulates chondrocyte dedifferentiation by binding FSHRInterference with Fshr by siRNAs was performed.After Fshr siRNA interfering,FSH downregulated Coll-Ⅱ was reversed.5.Gαi protein is involved in FSH regulated chondrocyte dedifferentiationThe cAMP kit was used to detect whether FSH affected the expression of the second messenger cAMP.The results showed that FSH inhibited the synthesis of cAMP,and the inhibition of Gαi by PTX reversed the FSH-down-regulated cAMP and Coll-Ⅱ,suggesting that Gαi was involved in the FSH-regulated dedifferentiation of chondrocytes.6.ERK1/2 and p38 signaling pathways are involved in chondrocyte dedifferentiationFSH can up-regulate ERK1/2 but down-regulate the phosphorylation of p38.PD98059,an ERK1/2 inhibitor,can reverse FSH downregulation of Coll-Ⅱ.FSH down-regulated Coll-Ⅱ was promoted with p38 inhibitors.These results suggest that ERK1/2 and p38 have opposite regulatory effects on Coll-Ⅱ and chondrocyte dedifferentiation.7.FSHR is located in the superficial zone of mouse knee cartilageImmunohistochemistry of tissue sections showed that FSHR is mainly located at the superficial zone(SZ)of articular cartilage.8.Mouse FSH antibody(FSHAb)blockade can improve trabecular parameters,prevent chondrocyte dedifferentiation and articular cartilage injuryFSH antibody blockade improves trabecular parameters,increases Coll-Ⅱexpression,and prevents chondrocyte dedifferentiation.FSHAb improves chondrocyte loss,cartilage articular surface damage,and OA related scores such as Mankin Score and OARSI Score.Ki67-positive cells was increased after blocking FSH,suggesting that blocking FSH might be beneficial to the improvement of cartilage degeneration.9.FSH affects OA markersRecent studies have shown that Prg4 and Egr1 are markers of OA.We show that FSH can up-regulate the expression of Egrl and Egr1.Blocking FSH can decrease the expression of Egr1.Above results suggest that FSH may contribute to OA by affecting OA markers.Conclusions:1.FSH decrease cartilage anabolism and promotes chondrocyte dedifferentiation.2.FSH is involved in the reduction of Coll-Ⅱ synthesis and promotion of chondrocyte dedifferentiation through binding of FSHR,which then couples Gαi protein.On one hand FSH activates of ERK1/2 signaling and inhibits p38 signaling.On the other hand inhibits Sox9-CREB-Coll-Ⅱ.3.Blockage of FSH can improve bone structure and improve a series of cartilage degeneration,such as chondrocyte dedifferentiation,chondrocyte injury and chondrocyte loss.4.Prg4 and Egr1 are important molecular mechanisms and targets of FSHAb in improving cartilage degeneration.更多还原