Preliminary Investigation Of The Effect And The Mechanism Of Follicle-stimulating Hormone On The Inflammatory Response Of Articular Chondrocytes

Objective:Osteoarthritis,the most serious joint disease and one of the leading causes of disability in the elderly,is currently of unknown etiology.The prevalence of osteoarthritis is significantly higher in perimenopausal women,when one of the most important clinical features is an increase in FSH levels.To investigate the relationship between FSH and osteoarthritis,in previous studies,we found that FSH receptors are expressed on human and mouse chondrocytes,but it is not clear how FSH affects chondrocytes and what kind of effect it has on chondrocytes.This experiment is intended to verify the effect of FSH on chondrocytes in vivo through animal experiments.In vitro experiments were performed by transcriptomic sequencing and bioinformatics analysis to explore the effect of FSH on chondrocyte transcript levels and to verify the differential genes by RNA level and protein level,and to explore the changes in some pathways.Methods:1.Osteoarthritis was induced by medial meniscus instability in mice,and mice were sacrificed at 4 weeks and 8 weeks after intra-articular injection of FSH in the knee joint,respectively,and the effect of high FSH on cartilage in vivo was observed by staining with safranin O-fast green on tissue sections of mice injected with FSH in the knee joint cavity.2.The primary chondrocytes were divided into four groups based on the use of siRNA was to interfere with FSHR and the addition of FSH stimulation,i.e.,Si NC group(transfected of siRNA-NC),Si NC+FSH group(transfection of siRNA-NC and addition of FSH),Si FSHR group(transfection of siRNA-FSHR),and Si FSHR+FSH group(transfection of siRNA-FSHR and addition of FSH),with three biological replicates for each group.RNA was extracted using the TRIZOL method and sequenced based on the Illumina sequencing platform,and the raw data from the sequencing was filtered to obtain high quality clean data.3.HISAT2 was used to construct an index of the reference genome,and paired end clean reads were compared with the reference genome.FPKM method was used to quantify the gene expression level.DESeq2 software(1.16.1)was used for differential expression analysis between the two comparison combinations.GO enrichment analysis and KEGG enrichment analysis of differentially expressed genes were realized by clusterProfiler R software.PPI analysis of differentially expressed genes is based on STRING databases that know and predict protein-protein interactions.4.ATDC5 cells were cultured and induced to differentiate into chondrocytes by adding ITS.RNA and protein were extracted by adding FSH stimulation,and some of the screened differential genes were verified by qRT-PCR,Western blot and immunofluorescence.Results:1.After surgery,the knee joints of mice showed obvious lesions and Mankin’s score was significantly increased,and the osteoarthritis model was successfully established.After injection of FSH into the knee joint cavity of mice,both the sham operation group and the operation group showed different degrees of joint damage.The Mankin’s scores of each group were higher than those of the saline injection group after FSH injection.2.Filtering the raw transcriptomic data,we can see that the clean reads were greater than 4.4*10^7,the base length was greater than 6.73G,the error rate was only 0.02%,Q20>98%,Q30>94.4%.By comparing clean reads after quality control to reference genes,the number of mappings on the reference genome was 96.22%-97.85%for all samples compared to the reference genome.The unique map used for subsequent quantitative analysis accounted for 91.48%-93.1%,multi map was less than 5.01%.3.A total of 1308 differential genes were screened between the NC and FSH groups,of which 664 were up-regulated,such as Coll2al,Collal,Col5al,Col3a1,Egrl,etc.,and 644 were down-regulated,such as Mgp,Hmgn2,Emb,Timp1,Cd24a,etc.For enrichment analysis,the more significant GO subsets were enriched for extracellular matrix organization,extracellular structure organization,cartilage development,etc.The differential genes were subjected to KEGG enrichment analysis.The enrichment of differential genes for KEGG resulted in the enrichment of 293 pathways in total.It can be seen that the more significantly enriched KEGG pathways include protein digestion and absorption,extracellular-matrix receptor interaction,oxidative phosphorylation and other pathways.What’s more,IL-6 was found to interact with most collagen through PPI networks4.Validated at the RNA level,EGR1,and Collal expression were elevated after FSH stimulation,consistent with the transcriptomic results.At the protein level,the expression of EGR1 and Co11a1 increased gradually with the increasing concentration of FSH stimulation in a dose-effect dependent relationship.In addition,the expression of the inflammatory factor IL-6 increased at the RNA and protein levels after FSH stimulation,and immunofluorescence experiments also demonstrated that the expression of IL-6 increased in the FSH group.5.After FSH stimulation,cAMP and PKA expression were decreased,total JNK was unchanged,p-JNK level was decreased and JNK phosphorylation was inhibitedConclusion:FSH can cause or exacerbate osteoarthritis by inhibiting the cAMP/PKA pathway and inhibiting JNK phosphorylation,causing an inflammatory response and chondrocyte dedifferentiation in the cartilage,leading to an imbalance in chondrocyte homeostasis,which explains the sudden increase in the prevalence of osteoarthritis in menopausal women.