Female osteoarthritis intervention target based on INSL3 / RXFP2 signal channel and application thereof

Abstract

The invention discloses a parent osteoarthritis intervention target based on an INSL3 / RXFP2 signal channel and application of the parent osteoarthritis intervention target. The pregestation caffeine exposure of the male body has obvious influence on the cartilage development of the male offspring, and the susceptibility of osteoarthritis is increased. Through screening and verification, INSL3 secreted by testicular interstitial cells plays a key role in the parent osteoarthritis, and cAMP / PKA / CREB signal channels are reduced by locally knocking out cartilage RXFP2 or inhibiting combination of INSL3 and RXFP2, so that synthesis and development of cartilage matrixes are inhibited. And the exogenous recombinant INSL3 protein is injected into the knee joint cavity, so that the cartilage dysplasia caused by pregestation caffeine exposure of the parent can be obviously improved. The invention discloses a key effect of an INSL3 / RXFP2 signal channel in regulation and control of cartilage matrix synthesis, and provides a new biological basis and an early prevention and treatment strategy for targeted therapy of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an intervention target for paternal osteoarthritis based on the INSL3 / RXFP2 signaling pathway and its application. Background Technology

[0002] Osteoarthritis is a chronic degenerative disease that primarily affects the joints of the hands, hips, and knees[1]. Its core pathogenesis is the continuous wear and degeneration of articular cartilage, which has become the leading cause of joint pain and chronic disability in the elderly[2]. Nevertheless, its pathogenesis has not been fully elucidated. Existing evidence shows that, in addition to individual factors (such as age, sex, obesity, etc.) and joint-level factors such as joint injury and abnormal load, developmental factors also play a crucial role in the occurrence and progression of osteoarthritis[3]. For example, Hussain's retrospective study found that low birth weight fetuses have a 2.04 times higher risk of developing hip osteoarthritis than normal birth weight fetuses[4]. Furthermore, previous studies in our laboratory have confirmed that maternal exposure to substances such as caffeine, nicotine, and ethanol during pregnancy can reduce the quality of offspring cartilage and increase susceptibility to osteoarthritis[5, 6]. In recent years, the "Paternal Origin of Health and Disease" (POHaD) theory has prompted researchers to pay widespread attention to the impact of paternal environmental exposure on offspring health[7-9]. However, research on paternal osteoarthritis remains scarce and requires further in-depth investigation.

[0003] Caffeine (1,3,7-trimethylxanthine) is a common central nervous system stimulant, widely found in coffee, tea, soft drinks and compound drugs. Caffeine can effectively refresh the mind and relieve fatigue by stimulating the HPA axis. In recent years, global caffeine consumption has continued to rise, especially among people of reproductive age, where men's caffeine intake is significantly higher than women's

[10] . According to data from the U.S. National Health and Nutrition Examination Survey, the average daily caffeine intake of men of reproductive age is 240 mg, which is about 1.5 times that of women, and more than 15% of men have an average daily intake of 300 mg

[10] . Therefore, caffeine has become an important chronic stressor that is prevalent in modern society. Based on the average daily caffeine intake of adult men, this study is the first to explore the effect of paternal pre-pregnancy caffeine exposure (PPCE) on offspring cartilage development, revealing the key role of INSL3 secreted by testicular interstitial cells in regulating cartilage matrix synthesis. Studies have found that the INSL3 / RXFP2 signaling pathway can serve as a potential target for intervention in paternal osteoarthritis, and provide theoretical and experimental evidence for the occurrence and development of paternal osteoarthritis.

[0004] Main references: 1.Hootman JM, Helmick CG, Barbour KE, Theis KA, Boring MA: Updatedprojected prevalence of self-reported doctor-diagnosed arthritis andarthritis-attributable activity limitation among US adults, 2015-2040.Arthritis Rheumatol 2016, 68(7):1582-1587. 2.Allen KD, Thoma LM, Golightly YM: Epidemiology of osteoarthritis.Osteoarthritis Cartilage 2022, 30(2):184-195. 3.Palazzo C, Nguyen C, Lefevre-Colau MM, Rannou F, Poiraudeau S: Riskfactors and burden of osteoarthritis. Ann Phys Rehabil Med 2016, 59(3):134-138. 4.Hussain SM, Wang Y, Wluka AE, Shaw JE, Magliano DJ, Graves S,Cicuttini FM: Association of low birth weight and preterm birth with theincidence of knee and hip arthroplasty for osteoarthritis. Arthritis Care Res(Hoboken) 2015, 67(4):502-508. 5.Li J, Xiao H, Luo H, Tan Y, Ni Q, He C, Magdalou J, Chen L, Wang H:GR / HDAC2 / TGFβR1 pathway contributes to prenatal caffeine induced-osteoarthritis susceptibility in male adult offspring rats. Food Chem Toxicol2020, 140:111279. 6.Luo H, Li J, Cao H, Tan Y, Magdalou J, Chen L, Wang H: Prenatalcaffeine exposure induces a poor quality of articular cartilage in male adultoffspring rats via cholesterol accumulation in cartilage. Sci Rep 2015, 5:17746. 7.Chan JC, Nugent BM, Bale TL: Parental Advisory: Maternal andpaternal stress can impact offspring neurodevelopment. Biol Psychiat 2018, 83(10):886-894. 8.Chang RC, Wang HQ, Bedi Y, Golding MC: Preconception paternalalcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming. Epigenet Chromatin 2019, 12. 9.Jawaid A, Jehle KL, Mansuy IM: Impact of parental exposure onoffspring health in humans. Trends Genet 2021, 37(4):373-388. 10.Grosso G, Godos J, Galvano F, Giovannucci EL: Coffee, caffeine, and health outcomes: An umbrella review. Annu Rev Nutr 2017, 37:131-156. Summary of the Invention To address the shortcomings of existing technologies, this invention studies and discovers that insulin-like growth factor 3 (INSL3) can serve as a novel biomarker and therapeutic target, exploring its crucial role in the occurrence and development of osteoarthritis through its interaction with its receptor RXFP2. Based on the research findings of the INSL3 / RXFP2 signaling pathway, this invention proposes an intervention target for paternal osteoarthritis based on the INSL3 / RXFP2 signaling pathway and its application.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an intervention target for paternal osteoarthritis based on the INSL3 / RXFP2 signaling pathway.

[0006] This invention, through systematic animal experiments, molecular biology, and histopathological studies, has clarified the core role of the INSL3 / RXFP2 signaling pathway in paternal osteoarthritis and constructed a three-in-one technical system of "target-formulation-strategy," the specific scheme of which is as follows: 1. Discovery and validation of core intervention targets Target screening: Transcriptome sequencing, RT-qPCR, immunofluorescence and ELISA were used to confirm that PPCE can significantly downregulate serum INSL3 levels and mRNA and protein expression of RXFP2 in cartilage tissue of male offspring, while inhibiting the activation of downstream cAMP / PKA / CREB signaling pathways, resulting in reduced expression of key genes in cartilage matrix synthesis such as Col2a1, Aggrecan and transcription factor SOX9.

[0007] Target function validation: Cartilage-specific RXFP2 knockout mice were constructed (RXFP2 gene exons were removed using CRISPR-Cas9 technology and bred with Col2-Cre mice). The knockout mice showed the same phenotype as the PPCE offspring: reduced cartilage safranin staining intensity, increased Mankin score, reduced COL2A1 and SOX9 expression, and a significant decrease in the PKACα+ / p-CREB+ positive cell rate, confirming that the INSL3 / RXFP2 signaling pathway is the core target for regulating cartilage development and susceptibility to osteoarthritis.

[0008] 2. RXFP2 knockout regulation method Local RXFP2 knockout in cartilage was achieved using CRISPR-Cas9 gene editing technology: a guide RNA specifically targeting the RXFP2 gene exon was designed, a conditional knockout vector was constructed, and mouse zygotes were transfected to obtain RXFP2flox / flox mice; these mice were then crossed with Col2-Cre mice that specifically express Cre recombinase to obtain heterozygous mice with locally knocked-out RXFP2 in cartilage (RXFP2[flox / -,Col2-Cre]). This method can specifically silence the expression of RXFP2 in cartilage tissue, inhibiting cartilage matrix synthesis and development by blocking INSL3 binding and downregulating the cAMP / PKA / CREB signaling pathway, providing a tool for mechanism research and target validation.

[0009] Secondly, the present invention provides a biological agent for treating paternal osteoarthritis, wherein the biological agent is an exogenous recombinant INSL3 protein, which can regulate chondrocyte function through the INSL3 / RXFP2 signaling pathway, thereby preventing or delaying the occurrence and progression of paternal osteoarthritis.

[0010] Biological agent preparation: INSL3 protein is recombinantly expressed using genetic engineering technology, and highly active exogenous recombinant INSL3 protein is obtained after purification with a purity of over 95%, which can specifically bind to RXFP2 on the surface of chondrocytes.

[0011] Administration method and dosage: Intra-articular injection of the knee joint was used to administer the drug, which allows the drug to act directly on the diseased cartilage tissue and avoid systemic side effects. The optimal administration regimen was determined to be: intervention to be initiated at 8 weeks after birth, with 500 ng of exogenous recombinant INSL3 protein injected each time, once every 2 weeks, for 4 consecutive weeks.

[0012] Mechanism of action: Exogenous INSL3 can specifically bind to RXFP2 in chondrocytes, activate the cAMP / PKA / CREB signaling pathway, reverse the downregulation of cartilage matrix gene and protein expression caused by PPCE, and improve the morphology and function of cartilage tissue.

[0013] Thirdly, this invention provides the application of the aforementioned biological agent in the early intervention of paternal osteoarthritis. Injection of exogenous recombinant INSL3 protein into the knee joint cavity effectively activates the RXFP2 / cAMP / PKA / CREB signaling pathway, thereby improving symptoms of paternal osteoarthritis caused by PPCE. It is suitable for the prevention and early treatment of osteoarthritis in male offspring following preconception caffeine exposure in the father.

[0014] The specific implementation plan for the above early intervention strategy is as follows: Population selection: For male offspring whose fathers have a long history of caffeine exposure before pregnancy, individuals at high risk of osteoarthritis can be screened by detecting serum INSL3 levels and cartilage RXFP2 expression.

[0015] Intervention timing and procedure: Intervention was initiated at 8 weeks post-birth (PW8) by injecting exogenous recombinant INSL3 protein into the knee joint cavity for 4 consecutive weeks. After intervention, cartilage tissue morphology (SO & FG staining), expression of matrix synthesis-related molecules, and Mankin score were regularly monitored to assess the intervention effect.

[0016] Efficacy verification: Animal experiments confirmed that this strategy can significantly increase the mRNA levels of Col2a1 and Aggrecan in the cartilage of PPCE progeny, upregulate the expression of COL2A1 and SOX9 proteins, increase the intensity of safranin staining, reduce the Mankin score, and significantly improve the osteoarthritis susceptibility phenotype.

[0017] The technical principle of this invention is as follows: 1. The role of INSL3 in paternal osteoarthritis: Studies have found that INSL3 plays a crucial role in cartilage development in male offspring with PPCE (Paternal-Cartilage-Induced Osteoarthritis). Under physiological conditions, local knockout of RXFP2 in cartilage can silence the binding of RXFP2 to INSL3, thereby downregulating the cAMP / PKA / CREB signaling pathway and inhibiting the synthesis and development of cartilage matrix. Therefore, the INSL3 / RXFP2 signaling pathway plays a central role in the occurrence and progression of paternal osteoarthritis, making it a potential therapeutic target.

[0018] 2. Treatment strategy targeting INSL3: This invention proposes an innovative treatment strategy based on the INSL3 / RXFP2 signaling pathway. Intra-articular injection of exogenous recombinant INSL3 protein effectively activates the RXFP2 / cAMP / PKA / CREB pathway and significantly alleviates symptoms of paternal osteoarthritis induced by PPCE.

[0019] 3. Clinical Application Prospects: This invention provides a new approach for the early intervention of paternal osteoarthritis. Specific activation of the INSL3 / RXFP2 signaling pathway can effectively prevent or delay the onset of paternal osteoarthritis. This treatment strategy offers a novel direction for precision treatment and individualized intervention of osteoarthritis, and has broad clinical application potential.

[0020] The advantages and beneficial effects of this invention are as follows: 1. Innovation and academic value: This invention reveals for the first time the key role of the INSL3 / RXFP2 signaling pathway in the pathogenesis of paternal osteoarthritis, filling a research gap in this field, providing new theoretical basis for the precision treatment and individualized intervention of osteoarthritis, and identifying potential biomarkers.

[0021] 2. Precise Targeted Intervention: This invention proposes local intervention through intra-articular injection of recombinant INSL3 protein in the knee joint cavity, which can specifically activate the RXFP2 / cAMP / PKA / CREB pathway and significantly alleviate osteoarthritis symptoms caused by PPCE. This intervention method is highly targeted and effectively reduces the risk of adverse reactions.

[0022] 3. Early Intervention Potential: This invention provides a novel strategy for early intervention in paternal osteoarthritis. By specifically regulating the INSL3 / RXFP2 signaling pathway, it can effectively prevent or delay the onset of osteoarthritis, providing strong technical support for early clinical intervention.

[0023] 4. Broad application potential: The treatment method of this invention is not only applicable to the intervention of paternal osteoarthritis, but also provides theoretical support for other types of osteoarthritis (such as maternal osteoarthritis and adult osteoarthritis), and has significant cross-disciplinary application potential, which helps to promote the development of osteoarthritis pathology and its treatment strategies.

[0024] 5. Simple to operate and highly scalable: The local injection treatment proposed in this invention is simple to operate, requires no complex equipment or cumbersome steps, and has strong operability and promotion prospects. Compared with traditional treatment methods, it has broad clinical application potential. Attached Figure Description

[0025] Figure 1 PPCE can lead to chondrodysplasia and increased susceptibility to osteoarthritis in male offspring. Figure 1 (A) Representative images of SO&FG staining in the cartilage of male offspring of GD20, PW6, PW12, and PW28; (B) Representative images of COL2A1 immunohistochemical staining in the cartilage of male offspring of GD20, PW6, PW12, and PW28; (C) Safranin staining intensity of SO&FG staining in the cartilage of male offspring of GD20; (D, G, J, M) Protein expression levels of COL2A1 in the cartilage of male offspring of GD20, PW6, PW12, and PW28 through immunohistochemical staining; (E, H, K, N) Cartilage matrix genes of male offspring of GD20, PW6, PW12, and PW28. Col2a1 and AggrecanmRNA level expression; (F, I, L) Mankin scores of SO&FG staining of cartilage in male offspring of PW6, PW12 and PW28; (O, P) Representative images of SO&FG staining of cartilage in male offspring before and after ACLT treatment and their Mankin scores.

[0026] Figure 2 PPCE can lead to decreased expression of the INSL3 / RXFP2 signaling pathway in the cartilage of male offspring; Figure 2 (A, C) Representative images of SOX9 immunohistochemical staining in GD20 and PW12 cartilage and their protein expression; (B) GD20 and PW12 cartilage Sox9 mRNA expression in PW12 cartilage; (D, E) Volcano plot and heatmap of differentially expressed genes in PW12 cartilage; (F) Venn diagram of differentially expressed genes in PW12 hippocampus, cartilage, testis, and adrenal gland; (G) Relative expression levels of RXFP2 in different tissues from the Human Protein Atlas public dataset; (H) PW12 in hippocampus, cartilage, testis, and adrenal gland. Rxfp2 mRNA expression of (I) GD20 and PW12 cartilage; Rxfp2 mRNA expression; representative images of GD20 and PW12 cartilage RXFP2 immunofluorescence staining and their protein expression; serum INSL3 levels of GD20 and PW12 (L).

[0027] Figure 3 PPCE can lead to decreased expression of the cAMP signaling pathway in the cartilage of male offspring; Figure 3 (A) Schematic diagram of the relationship between RXFP2 and downstream cAMP signaling pathway in KEGG public data; (B, C) KEGG and GO enrichment analysis of differentially expressed genes in PW12 cartilage; (D, E) Representative images and positive cell rates of GD20 and PW12 cartilage PKACα and p-CREB immunofluorescence staining.

[0028] Figure 4 : Knockout of RXFP2 in cartilage inhibits cartilage matrix synthesis and development by downregulating the cAMP signaling pathway; Figure 4 Chinese: (A, C, D, F, G, I) Representative images of cartilage RXFP2, SOX9, and COL2A1 stained with immunofluorescence or immunohistochemistry and their protein expression in GD20 and PW12 knockout mice; (B, E, H) Cartilage in GD20 and PW12 knockout mice Rxfp2, Sox9, Col2a1mRNA expression; (J) Representative images of SO&FG staining in cartilage of GD20 and PW12 knockout mice; (K, L) Representative images and positive cell rates of PKACα and p-CREB immunofluorescence staining in cartilage of GD20 and PW12 knockout mice; (M) Safranin staining intensity in cartilage of GD20 knockout mice; (N) Mankin score of cartilage in PW12 knockout mice.

[0029] Figure 5 Exogenous supplementation of INSL3 improves cartilage matrix synthesis and development in male offspring of PPCE; Figure 5 (A, C, D, F) Representative images of SOX9 and COL2A1 immunohistochemical staining in cartilage after exogenous INSL3 supplementation and their protein expression; (B, E) Cartilage after exogenous INSL3 supplementation Sox9, Col2a1 (F) mRNA expression in cartilage COL2A1 after exogenous INSL3 supplementation; (G, H) representative images of SO&FG staining of cartilage after exogenous INSL3 supplementation and the degree of safranin staining.

[0030] Figure 6 Exogenous supplementation of INSL3 upregulates RXFP2 / cAMP pathway expression; Figure 6 (A, C) Representative images of RXFP2 immunofluorescence staining in cartilage after exogenous INSL3 supplementation and its protein expression (400×, statistically based on average optical density, n=5); (B) Cartilage after exogenous INSL3 supplementation Rxfp2 mRNA expression (n=8); (D, E) Representative images of cartilage PKACα and p-CREB immunofluorescence staining after exogenous INSL3 supplementation and positive cell rates (200× and 800×, counted by mean optical density, n=5). Detailed Implementation

[0031] The technical content of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings: Example 1 1. Laboratory animals Male and female SPF-grade Wistar rats (6 - 8 weeks old, male body weight ranging from 250 - 280 g, female body weight ranging from 200 - 230 g) were purchased from Hubei Center for Disease Control and Prevention, with the experimental animal license number SCXK(E)2020 - 0018. Male and female SPF-grade C57bl6 mice were purchased from Cyagen Biosciences Inc. (Suzhou, China), with the experimental animal license number SYXK(S)2022 - 0061. All animal experiments were conducted at the Animal Experiment Center of Wuhan University (located in Wuhan, China) which is AAALAC International accredited, and were approved by the Animal Experiment Ethics Committee of the School of Medicine, Wuhan University before the experiment. Before the start of animal treatment, all rats and mice were adaptively housed for one week under standard environmental conditions (room temperature: 18 - 22 °C; humidity: 40% - 60%; 12 h light cycle).

[0032] 2. Experimental Methods In this study, 8-week-old male Wistar rats were divided into two groups: a control group (CON group, gavaged with 0.5 mL / kg normal saline daily) and a PPCE experimental group (gavaged with 60 mg / kg caffeine daily, and the caffeine solution was prepared with normal saline), with 12 rats in each group. The treatment lasted for 8 weeks to cover the entire spermatogenesis cycle, and the dose of caffeine was selected based on the daily intake of the human population. Throughout the experiment, all rats were housed under standard conditions, and their body weights were recorded once a week. After 8 weeks of administration, when the male Wistar rats were 16 weeks old, they were paired with normal female rats at a ratio of 1:2 at 6:00 p.m. If sperm were observed under a microscope in the vaginal smear the next morning, it was recorded as gestational day 0 (GD0). Then, the pregnant female rats were housed separately and this process was repeated until there were a sufficient number of pregnant rats. At GD20, pregnant rats (n = 10) were anesthetized and euthanized with 2% isoflurane, and the situation was recorded in detail (including live fetuses, dead fetuses, absorbed fetuses, etc.) to evaluate the pregnancy outcome. The blood samples of each litter of fetal rats were pooled for subsequent analysis of blood series indices (separated by male and female). The cartilage tissues of male fetal rats were immediately frozen with liquid nitrogen and stored at -80 °C for subsequent experiments. In addition, the cartilage tissues of 5 male fetal rats randomly selected from different litters were fixed overnight in 4% paraformaldehyde solution, then dehydrated and paraffin-embedded for morphological analysis.

[0033] Other pregnant rats that gave birth naturally (n=10), with litters ranging from 8 to 14 pups, were considered eligible. The sex and number of fetuses in different litters within the same group were adjusted to ensure each litter contained 10 fetuses with a 1:1 sex ratio. After weaning, 4-week-old pups were housed separately according to sex, with 4 pups per cage, provided with standard experimental feed. Male offspring were euthanized at 6, 12, and 28 weeks of age under anesthesia with 2% isoflurane, and blood was collected from the carotid artery for serological testing. Their cartilage tissue was flash-frozen in liquid nitrogen and immediately stored at -80°C for long-term preservation or fixed with 4% paraformaldehyde (samples were randomly selected from 5 offspring rats in each group) for subsequent experimental studies.

[0034] Intra-articular injection (IAI) is a common surgical procedure that aims to inject drugs directly into the joint cavity, often used to treat various joint diseases. In this study, we investigated the effect of local injection of recombinant INSL3 protein (500 ng each time, once every 2 weeks) on cartilage quality improvement via knee joint aspiration. Based on the experimental objectives, male PW8 offspring rats were divided into four groups: CON + saline group (Saline group), PPCE + Saline group, CON + INSL3 group, and PPCE + INSL3 group.

[0035] 3. Detection indicators and methods 3.1 Cartilage decalcification Before dehydration and embedding cartilage specimens, decalcification is necessary. The procedure is as follows: First, fix the sample with 4% paraformaldehyde and rinse with PBS to remove excess formaldehyde. Next, immerse the sample in decalcification solution and agitate it periodically to accelerate the decalcification process. Change the decalcification solution every 4 days. Check the degree of cartilage softening by gently pricking it. Once softened, rinse with PBS and continue with subsequent processing.

[0036] 3.2 Cartilage dehydration, embedding, and sectioning Cartilage tissue was dehydrated sequentially with an increasing concentration of alcohol until complete dehydration, then transferred to a clearing agent for clearing. Subsequently, the tissue was immersed in molten paraffin for infiltration, with the paraffin being replaced multiple times to ensure full penetration, ultimately forming a paraffin block. The paraffin block was then cut into 4-5 micrometer thin sections using a microtome, dried, and stored for subsequent staining experiments.

[0037] 3.3 Cartilage sections stained with Safranin & Fast Green (SO & FG) After dewaxing, the paraffin sections were stained with Fast Green and Safranin, respectively. Following staining, the sections were dehydrated and cleared, and finally mounted with neutral resin. Microscopic observation was performed, and the staining intensity was analyzed using ImageJ software or a Mankin score was calculated.

[0038] 3.4 Mankin score for cartilage histology The Mankin score is used to assess pathological changes in cartilage, mainly including four aspects: structure, cell morphology, matrix staining, and calcification. By observing SO & FG staining results, the surface structure of cartilage, cell morphology, loss of matrix proteoglycans, and degree of calcification are assessed and quantitatively scored.

[0039] 3.5 Immunohistochemical (IHC) staining of cartilage sections Paraffin sections were first dewaxed, then subjected to antigen retrieval and blocking of non-specific sites, followed by incubation with primary and secondary antibodies. Finally, DAB staining was used to develop the stain, and after counterstaining the cell nuclei, the sections were dehydrated, cleared, and mounted. Microscopic observation and semi-quantitative analysis of IHC labeling intensity and the number of positive cells were performed using ImageJ.

[0040] 3.6 Immunofluorescence (IF) staining of cartilage sections After dewaxing, paraffin sections underwent antigen retrieval and non-specific site blocking, followed by incubation with primary and secondary antibodies and DAPI counterstaining. Finally, sections were mounted with anti-fluorescence quenching mounting medium and observed under a microscope. ImageJ was used to analyze fluorescence intensity, distribution, and the number of positive cells.

[0041] 3.7 IF double staining of cartilage sections Double staining was performed using the TSA method. After dewaxing, paraffin sections underwent antigen retrieval and blocking, followed by primary antibody, HRP-labeled secondary antibody, and TSA staining. A second round of primary and secondary antibody incubation and DAPI counterstaining followed, and the sections were mounted. The double staining results were then analyzed using microscopy and image analysis software.

[0042] 3.8 Real-time quantitative PCR (RT-qPCR) detection of cartilage tissue RNA was first extracted from cartilage tissue. After separation using TRIzol reagent and chloroform, the RNA was precipitated with isopropanol and washed with 75% ethanol. Finally, the RNA was dissolved and stored. Subsequently, cDNA was synthesized using an RNA reverse transcription kit and further detected by real-time quantitative PCR. The expression of relevant genes was analyzed using designed primers (primer sequences are shown in Table 1).

[0043] Table 1. Primer sequences for rat real-time quantitative PCR

[0044] Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RXFP2, relaxin family peptide receptor 2; COL2A1, type II collagen α1 chain; Aggrecan, aggregate protein; SOX9, SRY box transcription factor 9; MMP13, matrix metalloproteinase 13; ADAMTS5, ADAM metalloproteinase with platelet adhesion protein type 1 domain 5.

[0045] 3.9 Statistical Analysis Data was analyzed and processed using GraphPad Prism software (6.5). All numerical results are calculated as mean ± standard error. Student... The St test was used to analyze significant differences between two groups, and one-way ANOVA was used to compare multiple groups. P <0.05 indicates that the difference is statistically significant.

[0046] 4. Experimental Results 4.1 PPCE can lead to chondrodysplasia and susceptibility to osteoarthritis (OA) in male offspring. This study investigated the effects of long-term caffeine exposure in adult males on cartilage development in offspring by administering 60 mg / kg of caffeine orally to male Wistar rats daily for 8 consecutive weeks. In SO&FG staining of cartilage from male offspring GD20 and PW6 / 12 / 28 in the PPCE group, compared to the control group, this invention found that PPCE reduced the safranin staining intensity of the intrauterine fetal cartilage matrix in male offspring. P <0.01, Figure 1 In the middle A and C), the chondrocyte population showed disordered cell arrangement and elevated Mankin score after birth. P <0.01, Figure 1 (A, F, I, L). RT-qPCR results further confirmed that the mRNA expression levels of the cartilage matrix genes Col2a1 and Aggrecan were reduced in male offspring of the PPCE group before and after birth. P <0.05, P <0.01, Figure 1 E, H, K, N). IHC results also showed that the protein expression level of COL2A1 in the prenatal and postnatal cartilage of male offspring in the PPCE group was decreased. P <0.01, Figure 1 (B, D, G, J, M). Furthermore, OA surgery was performed on male PW28 offspring using ACLT. Results showed that, compared to the sham-operated group, ACLT treatment exacerbated SO&FG pathological staining damage in the cartilage of male offspring in both the CON and PPCE groups, and further increased the Mankin score (…). P <0.01, Figure 1 In the O and P groups, the degree of damage in the PPCE group was significantly greater than that in the CON group, manifested as the disappearance of chondrocytes, coarse and irregular collagen fibers, and even microcracks or deep fractures. P <0.01, Figure 1 (O, P). These results indicate that PPCE can lead to prenatal and postnatal chondrodysplasia and susceptibility to osteoarthritis (OA) in male offspring.

[0047] 4.2 PPCE can lead to decreased expression of the INSL3 / RXFP2 signaling pathway in the cartilage of male offspring. Subsequently, this invention further explored the mechanisms of chondrodysplasia and susceptibility to osteoarthritis (OA) in male offspring of PPCE. Compared with the control group, this invention found that the mRNA and protein levels of SOX9, a key transcription factor in cartilage matrix synthesis, were reduced in male offspring of PPCE before and after birth. P <0.01, P <0.05, Figure 2 The transcriptome sequencing results of PW12 male progeny cartilage showed that 423 downregulated genes and 135 upregulated genes were present in the PPCE male progeny cartilage. Figure 2 (D). Among them, RXFP2 expression was significantly reduced in the cartilage of male offspring of PPCE ( Figure 2 (E). RXFP2 is known to be a G protein-coupled receptor that specifically binds to INSL3, a regulatory factor secreted by testicular interstitial cells, triggering downstream signal transduction. Public data from the Human Protein Atlas show that RXFP2 is expressed in multiple tissues, including the adrenal gland, testis, and basal ganglia. Figure 2 (G), verified by RT-qPCR and IF experiments, this invention found that the mRNA and protein expression levels of RXFP2 in the prenatal and postnatal cartilage of PPCE offspring were significantly reduced (G). P <0.01, Figure 2 (IK). Combined analysis of multi-organ transcriptome sequencing of PPCE progeny and experimental results confirmed that Rxfp2 mRNA levels were significantly downregulated in multiple organs, including the testes, hippocampus, and adrenal glands, of the male PPCE progeny PW12. P <0.01, Figure 2 (F, H). Meanwhile, ELISA experiments showed that serum INSL3 levels in male offspring of PPCE remained consistently low before and after birth. P <0.01, Figure 2 (L).

[0048] 4.3 PPCE can lead to decreased expression of the cAMP signaling pathway in the cartilage of male offspring. The KEGG database indicates that activation of RXFP2 triggers the classic G protein-cAMP-PKA signaling pathway. Figure 3 (A). In this process, the G protein increases cAMP levels, which in turn promotes the translocation of phosphorylated cAMP response element-binding protein (p-CREB) into the nucleus and binding to the cAMP response element in the promoter by activating the protein kinase A catalytic subunit alpha (PKACα), thereby regulating the transcription of target genes. Figure 3 (A). KEGG and GO enrichment analysis of cartilage transcriptome sequencing indicated significant alterations in the cAMP signaling pathway in the cartilage of male PPCE offspring. Figure 3 (B, C). Through IF double labeling experiments, this invention confirms that PPCE can significantly reduce the positive rate of PKACα+ / p-CREB+ cells in the pre- and post-natal cartilage of male offspring (B, C). P <0.01, Figure 3 (D, E). These results indicate that PPCE can lead to decreased serum INSL3 levels, cartilage RXFP2 / cAMP pathway expression, and matrix synthesis function in male offspring before and after birth. The INSL3 / RXFP2 signaling pathway may mediate chondrodysplasia and susceptibility to osteoarthritis in male offspring with PPCE.

[0049] 4.4 Cartilage knockout of RXFP2 inhibits cartilage matrix synthesis and development by downregulating the cAMP signaling pathway. To further observe the effects of INSL3 / RXFP2 physiological signals on prenatal and postnatal cartilage development at the overall level, this invention constructed RXFP2 cartilage local knockout mice. In short, RXFP2 conditional knockout mice were obtained by excising gene exons using CRISPR / Cas9 technology. These mice were then bred with Col2-Cre mice (cartilage-specific expression) and genotypes were determined using tail DNA. The results showed that, compared with wild-type and conditional knockout mice (Rxfp2[flox / -]), heterozygous knockout mice (RXFP2[flox / -, Col2-Cre]) exhibited significantly reduced mRNA and protein expression levels of RXFP2 in GD20 and PW12 cartilage. P <0.01, Figure 4 (AC). This invention further discovered that local knockout of RXFP2 cartilage induced a cartilage development inhibition phenomenon similar to that in male offspring of PPCE, manifested as a significant reduction in the mRNA and protein expression levels of SOX9 and COL2A1 ( P <0.01, Figure 4 The degree of safranin staining decreased and the Mankin score increased (in the middle DI). P <0.01, Figure 4J, M, N). Furthermore, local knockout of RXFP2 cartilage also suppressed the rate of downstream PKACα+ / p-CREB+ positive cells ( P <0.01, Figure 4 (K, L). These results indicate that local knockout of RXFP2 in cartilage inhibits cartilage matrix synthesis by downregulating the cAMP signaling pathway, thereby hindering normal cartilage development.

[0050] 4.5 Exogenous INSL3 supplementation improves cartilage matrix synthesis and development in male offspring of PPCE. This invention conducted an experiment on exogenous supplementation of recombinant INSL3 protein via intra-articular injection at the systemic level (500 ng each time, once every 2 weeks, for a total of 4 weeks). The results showed that exogenous INSL3 supplementation significantly improved the reduced cartilage quality in male PPCE offspring. Specifically, compared with the saline group, the INSL3 intervention group significantly upregulated the mRNA and protein expression of SOX9 and COL2A1 in the cartilage of male PPCE offspring. P <0.01, Figure 5 In the middle AF), and effectively reduced the Mankin score of cartilage SO&FG staining ( P <0.01, Figure 5 (G, H).

[0051] 4.6 Exogenous supplementation of INSL3 upregulates RXFP2 / cAMP signaling pathway expression Further investigation revealed that exogenous INSL3 supplementation promoted the mRNA and protein expression of RXFP2 in the cartilage of male PPCE offspring. P <0.01, Figure 6 (AC). Through IF double-labeling experiments, this invention confirms that exogenous supplementation of INSL3 also reversed the inhibitory effect of PPCE on the PKACα / p-CREB signaling pathway in male offspring cartilage (AC). P <0.01, Figure 6 (D, E). These results indicate that exogenous INSL3 supplementation improves cartilage matrix synthesis and development in male PPCE offspring by upregulating RXFP2 / cAMP pathway expression.

Claims

1. An intervention target, characterized in that: The intervention target is a paternal osteoarthritis intervention target based on the INSL3 / RXFP2 signaling pathway.

2. The intervention target according to claim 1, characterized in that: The intervention targets the INSL3 / RXFP2 signaling pathway; CRISPR-Cas9 gene editing technology is used to locally knock out RXFP2 in cartilage, silencing the binding of RXFP2 to INSL3, thereby downregulating the cAMP / PKA / CREB signaling pathway and inhibiting cartilage matrix synthesis and development.

3. The intervention target according to claim 2, characterized in that: INSL3 is a regulatory factor specifically secreted by testicular interstitial cells.

4. The use of an intervention target as described in any one of claims 1 to 3 in the development of diagnostic reagents, therapeutic agents or intervention techniques for paternal osteoarthritis.

5. A biological agent for treating paternal osteoarthritis, characterized in that: The biological agent is an exogenous recombinant INSL3 protein.

6. The biological agent according to claim 5, characterized in that: INSL3 protein regulates chondrocyte function through the INSL3 / RXFP2 signaling pathway, preventing or delaying the occurrence and progression of paternal osteoarthritis.

7. The biological agent according to claim 6, characterized in that: The biological agent is administered via intra-articular injection into the knee joint.

8. The biological agent according to claim 7, characterized in that: The biological agent improves chondrodysplasia caused by paternal preconception caffeine exposure by regulating the RXFP2 / cAMP / PKA / CREB pathway.

9. The application of the biological agent as described in claim 8 in the early intervention for the prevention and treatment of paternal osteoarthritis, characterized in that: Intra-articular injection of exogenous recombinant INSL3 protein into the knee joint activates the RXFP2 / cAMP / PKA / CREB signaling pathway, improving symptoms of osteoarthritis in male offspring caused by paternal preconception caffeine exposure.

10. The application according to claim 9, characterized in that: It is suitable for the prevention and early treatment of osteoarthritis in male offspring after paternal preconception caffeine exposure.

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