Construction method and application of animal model of patellar osteoarthritis based on two-step method

Abstract

The application discloses a construction method of a parent-derived osteoarthritis animal model based on a two-step method and application thereof. The parent-derived osteoarthritis animal model is a male rat model of pre-pregnancy caffeine exposure (60 mg / kg.d, 2 months) of a father in a daily life environment of a rodent (such as a Wistar rat) in a fertile age, which is then mated with a normal female rat to obtain offspring, and after normal feeding after birth, 4 weeks of medium-intensity running is given, and typical characteristics similar to human osteoarthritis appear. The animal model construction method provided by the application is simple and easy to implement, has high success rate, is highly repeatable, and reduces the influence of human operation factors, creates a favorable disease animal model basis for research on the pathogenesis and prevention and treatment of parent-derived osteoarthritis, and has important practical significance and application value.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a two-step method for constructing an animal model of paternal osteoarthritis and its application. Background Technology

[0002] Osteoarthritis is a chronic degenerative joint disease that commonly affects the elderly, characterized primarily by degeneration of articular cartilage, osteophyte formation, subchondral bone sclerosis, and synovitis. Osteoarthritis is also the leading cause of joint pain and chronic disability in the elderly, with its prevalence increasing with age, reaching as high as 50% in those over 65. Iranian scholar Safiri and colleagues, through analysis of the osteoarthritis burden in multiple countries and regions worldwide from 1990 to 2017 in the 2017 Global Burden of Disease Study, found that the annual incidence of osteoarthritis in 2017 increased by 8.2% compared to 1990. Furthermore, a commentary published in *The Lancet* in 2020 indicated that the incidence of osteoarthritis accounts for 7% of the global population, affecting over 500 million people. Osteoarthritis not only severely impacts the quality of life of individual patients but also imposes a heavy economic burden on society. In developed countries, the healthcare costs associated with osteoarthritis can reach 1% to 2.5% of GDP. In addition, osteoarthritis is listed by the U.S. Food and Drug Administration as a "serious disease with unmet medical needs." [1] Therefore, the prevention and treatment of osteoarthritis is a major challenge facing the global public health field. There is an urgent need to understand the pathophysiological mechanisms driving osteoarthritis in order to achieve early prevention and diagnosis, and thus select better individualized treatment methods.

[0003] In recent years, the "developmental origins of health and disease (DOHaD)" theory has received widespread attention, with increasing evidence supporting a developmental origin for osteoarthritis. Studies have found that adverse paternal environments can induce adverse pregnancy outcomes in offspring (such as intrauterine growth retardation) and cause susceptibility to multiple diseases in adulthood, as well as multi-generational genetic effects. For example, paternal alcohol consumption can cause growth retardation in offspring and lead to spatial learning disabilities; paternal nicotine exposure can lead to changes in metabolic function and increase the risk of liver fibrosis; poor paternal nutrition and dietary habits can lead to impaired glucose tolerance in offspring and increase the risk of obesity and cardiovascular disease, with effects lasting for more than three generations. Furthermore, adverse paternal environmental exposure can also lead to susceptibility to osteoarthritis in offspring. Studies have found that osteoarthritis has a paternal origin; male mice fed a high-fat diet showed increased susceptibility to trauma-induced osteoarthritis in their F1 and F2 generation female mice, exhibiting changes in bone microstructure, i.e., the occurrence of paternal osteoarthritis.[2] All of the above suggests that an unfavorable environment during spermatogenesis is a significant risk factor for susceptibility to paternal osteoarthritis. Therefore, exploring the role of adverse environmental factors during the father's reproductive years in the development of osteoarthritis, and rationally avoiding exposure to adverse environments during spermatogenesis, has important practical guiding significance for eugenics and improving population quality.

[0004] In research on the occurrence and development of osteoarthritis, animal models exhibiting similar manifestations to human osteoarthritis are of paramount importance. Current experimental animal models primarily include surgical models resembling traumatic arthritis (e.g., medial meniscectomy); chemical models resembling cartilage destruction (e.g., intra-articular injection of papain); and degenerative arthritis models (e.g., external fixation with plaster casts on the lower limbs). These models mainly target the physical, chemical, or biological destruction of intra-articular structures in adult animals, leading to degenerative changes in articular cartilage. However, models for understanding the developmental origins of osteoarthritis and the occurrence and development of paternal osteoarthritis remain a blind spot and a significant challenge. Furthermore, while these modeling methods can simulate the disease phenotype of osteoarthritis, the phenotype is only localized within the joint cavity, failing to explore the relationship between osteoarthritis and systemic metabolism. [3] Furthermore, these animal models are created after birth, rather than through paternal factors affecting spermatogenesis and thus only reflect damage to intra-articular tissues caused by the "second blow" after birth, neglecting the developmental origin of osteoarthritis. This results in significant differences in the pathogenesis of paternal osteoarthritis. Therefore, current research urgently needs to establish animal models that simultaneously induce a high incidence of paternal osteoarthritis, highly similar to human osteoarthritis, to further explore its pathogenesis, early warning, and prevention, thereby benefiting humanity.

[0005] Articular cartilage is primarily formed during the embryonic period, and the proliferation and differentiation of articular chondrocytes in early life plays a decisive role in the metabolic homeostasis of the joints in later life. With age, the joints lack local blood supply, leading to a gradual decline in the number of chondrocytes and the quality of articular cartilage. Damaged articular cartilage gradually becomes unable to repair itself, manifesting as degenerative changes in the joints. Therefore, the occurrence of osteoarthritis in adulthood is closely related to the occurrence and differentiation of chondrocytes during intrauterine development. Hypertrophic differentiation of chondrocytes is the most common abnormal differentiation; hypertrophic differentiation of chondrocytes during intrauterine development can lead to increased degradation of the extracellular matrix of articular chondrocytes after birth, thereby inducing the development of osteoarthritis. [4]The mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in the proliferation, differentiation, transformation, and phenotypic maintenance of articular cartilage. In adult osteoarthritis, activation of the MAPK signaling pathway leads to increased extracellular matrix degradation in chondrocytes, while in utero and childhood, it mediates chondrocyte hypertrophy and differentiation. Previous studies have suggested that MAPKs are non-catalytically active in their basic forms. To become active, multiple phosphorylation events are required in their activation loop. Therefore, the phosphorylation level of the MAPK signaling pathway plays a vital role in articular cartilage development and the occurrence and development of osteoarthritis.

[0006] Caffeine is a xanthine alkaloid widely found in various soft drinks and is a commonly used psychoactive drug worldwide. It is estimated that approximately 80% of the global population consumes caffeinated foods daily. In recent years, global coffee consumption has been increasing, and men of reproductive age are frequently exposed to caffeine. Survey data shows that the average caffeine intake of men of reproductive age is 240 mg / day, approximately 1.5 times that of women. [5] This invention established a paternal pre-pregnant caffeine exposure (PPCE) model achievable in daily living environments, with an exposure period of 2 months (covering the entire spermatogenesis cycle of male mice). Offspring were then bred with normal female mice to obtain pups. This invention observed that PPCE resulted in smaller fetal size, significantly reduced body weight and length, and a significantly increased incidence of intrauterine growth retardation, indicating that PPCE can establish a stable model of low birth weight in offspring. This suggests that PPCE is closely related to the occurrence of paternal osteoarthritis.

[0007] Main references:

[0008] 1. Latourte A, Kloppenburg M, Richette P: Emerging pharmaceutical therapeutics for osteoarthritis. Nat Rev Rheumatol 2020,16(12):673-688.

[0009] 2.Harasymowicz NS,Choi YR,Wu CL,Iannucci L,Tang RH,Guilak F:

[0010] Intergenerational Transmission of Diet-Induced Obesity,MetabolicImbalance,andOsteoarthritis in Mice. Arthritis Rheumatol 2020,72(4):632-644.

[0011] 3.van der Kraan PM:Factors that influence outcome in experimentalosteoarthritis.

[0012] Osteoarthr Cartilage 2017,25(3):369-375.

[0013] 4.Kozhemyakina E, Lassar AB, Zelzer E: A pathway to bone: signaling molecules and transcription factors involved in chondrocyte development and maturation.

[0014] Development 2015,142(5):817-831.

[0015] 5. Yeshurun ​​S, Rogers J, Short AK, Renoir T, Pang TY, Hannan AJ: Elevatedpaternal glucocorticoid exposure modifies memory retention in femaleoffspring.

[0016] Psychoneuroendocrino 2017,83:9-18. Summary of the Invention

[0017] The technical problem to be solved by the present invention is to provide a method for constructing an animal model of paternal osteoarthritis that is highly successful, effective, reliable, reproducible, simple and easy to implement.

[0018] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0019] In a first aspect, the present invention provides a method for constructing an animal model of paternal osteoarthritis based on a two-step method, characterized by comprising the following steps:

[0020] S1: Select healthy rodents of reproductive age and administer 60 mg / kg of caffeine via intragastric gavage daily for 8 weeks prior to conception (covering one complete spermatogenesis cycle). Male rodents are allowed free access to food and are mated with normal female rats to obtain pregnant rodents.

[0021] S2: The pregnant mice give birth naturally and produce offspring. The birth date is taken as day 0 after birth. One day after birth, select a litter with 12 to 14 pups and adjust each litter to have 6 male and 6 female pups for nursing.

[0022] S3: The baby mice were weaned 4 weeks after birth and the males and females were separated into different cages. Some continued to be fed a normal diet until 28 weeks after birth.

[0023] S4: Some offspring were raised to 28 weeks of age, and then given 4 weeks of moderate running (at a constant speed of 18 m / min for 30 minutes, 3 times a week). Osteoarthritis-related indicators were detected at 32 weeks to comprehensively determine the occurrence of osteoarthritis; finally, a paternal osteoarthritis animal model was obtained.

[0024] As a preferred embodiment, in step S1, the rodents are SPF-grade Wistar, SD, or other rats, or Kunming, C57, or other mice, as well as guinea pigs, hamsters, etc.

[0025] Furthermore, in step S3, the normal diet is formulated to be the same as the formula for mouse and rat feed specified in the National Standard of the People's Republic of China GB14924.3-2001.

[0026] Furthermore, in step S4, the relevant indicators for osteoarthritis detection are: morphological changes in articular cartilage and osteoarthritis score.

[0027] Secondly, the present invention provides the application of a paternal osteoarthritis animal model in screening for adverse paternal age-related environmental disturbances that can cause chondrogenic toxicity in offspring mice, characterized in that: the paternal osteoarthritis animal model is obtained by any of the above construction methods.

[0028] Thirdly, the present invention provides the application of an animal model of paternal osteoarthritis in screening early warning and intervention targets for paternal osteoarthritis, characterized in that: the animal model of paternal osteoarthritis is obtained by any of the above construction methods.

[0029] Fourthly, the present invention provides the application of paternal osteoarthritis animal models in screening drugs for the prevention and treatment of osteoarthritis, characterized in that: the paternal osteoarthritis animal model is obtained by any of the above construction methods.

[0030] The aforementioned animal model of paternal osteoarthritis can also be used to study the pathogenesis of osteoarthritis.

[0031] The technical principles and research process of this invention are as follows:

[0032] This invention establishes a paternal osteoarthritis animal model by subjecting PPCE (first-impact pediatric end-stage) offspring to moderate-intensity running or anterior cruciate ligament rupture (second-impact pediatric end-stage) and detecting osteoarthritis-related morphological indicators. This two-step method simulates the disease phenotype of osteoarthritis in offspring resulting from adverse environmental exposures during the reproductive years of the father, and is of great significance for elucidating the pathogenesis of paternal osteoarthritis and identifying early warning and prevention mechanisms.

[0033] The advantages and beneficial effects of this invention are as follows:

[0034] 1. This invention has a novel subject matter. Caffeine, as a chronic stressor, is more likely to be exposed daily. Establishing an animal model of pre-pregnancy paternal caffeine exposure is more consistent with daily life and is more suitable and novel.

[0035] 2. This invention offers a simple modeling method. During the reproductive period, caffeine is administered, and offspring are mated with normal females. After birth, the offspring are normally raised until 28 weeks of age, at which point a second stress test is administered. The occurrence and progression of osteoarthritis can be observed using indicators such as changes in articular cartilage morphology and osteoarthritis scores. This model exhibits good stability and reproducibility, providing a reliable method for constructing animal models of paternal osteoarthritis.

[0036] 3. This invention facilitates the investigation of pathogenesis. The animal model of paternal osteoarthritis constructed based on this invention can be used to explore the pathogenesis and development of paternal osteoarthritis. Compared with traditional animal models of osteoarthritis, this animal model places greater emphasis on the initial impact of adverse environmental exposure during the reproductive years of the father, allowing for earlier observation of the pathogenesis of osteoarthritis.

[0037] 4. This invention facilitates the identification of early warning targets. The paternal osteoarthritis animal model constructed based on this invention can be used to explore early warning targets for paternal osteoarthritis. Compared with traditional osteoarthritis animal models, this animal model places greater emphasis on changes in cartilage quality during development, which lead to the occurrence of osteoarthritis in adult individuals, thus enabling the identification of early warning targets for osteoarthritis during development.

[0038] 5. This invention facilitates the identification of intervention targets. The animal model of paternal osteoarthritis constructed based on this invention can be used to explore early intervention targets for paternal osteoarthritis. Compared with traditional animal models of osteoarthritis, this animal model places greater emphasis on the continuous changes in cartilage function from in utero to postnatal changes, seeking early intervention targets for osteoarthritis during development. Attached Figure Description

[0039] Figure 1This is a schematic diagram illustrating the construction of the animal experimental model of the present invention.

[0040] In the diagram: A: First blow: paternal preconception caffeine exposure and offspring acquisition; B: Second blow: moderate running model; C: Osteoarthritis occurrence.

[0041] Figure 2 This invention describes the changes in cartilage morphology in offspring rats after paternal pre-pregnancy caffeine exposure, both intrauterine and postnatal.

[0042] In the figure: A, C, E: Safranin staining of articular cartilage in utero, at 6 weeks after birth, and at 12 weeks after birth; B, D, F: Integrated optical density statistics of Safranin staining of articular cartilage in utero, at 6 weeks after birth, and at 12 weeks after birth. Compared with the control group, * P<0.05, ** P<0.01.

[0043] Figure 3 This invention describes the phenomenon of articular cartilage hypertrophy and differentiation in offspring rats after paternal pre-pregnancy caffeine exposure.

[0044] In the figure: A: Runx2 and Col10 mRNA expression in mast cells of articular cartilage during intrauterine period; B, C: COL10 protein expression in mast cells of articular cartilage during intrauterine period; D: Runx2 and Col10 mRNA expression in mast cells of articular cartilage at 6 weeks after birth; E, F: COL10 protein expression in mast cells of articular cartilage at 6 weeks after birth; G: Runx2 and Col10 mRNA expression in mast cells of articular cartilage at 12 weeks after birth; H, I: COL10 protein expression in mast cells of articular cartilage at 12 weeks after birth. Compared with the control group, * P<0.05, ** P<0.01.

[0045] Figure 4 This invention describes the changes in cartilage morphology in female offspring rats after moderate-intensity running following pre-pregnancy caffeine exposure in the paternal parent. In the figure: A: Safranin staining of articular cartilage; B: Osteoarthritis pathological score; C: Integrated optical density statistics of Safranin staining of articular cartilage. Compared with the control group, * P<0.05, ** P<0.01.

[0046] Figure 5 This invention modifies the degradation of extracellular matrix in chondrocytes of offspring rats after moderate-intensity running following pre-pregnancy caffeine exposure in the father.

[0047] In the figure: A, B: mRNA expression of articular cartilage extracellular matrix degradation genes Mmp13 and Adamts5; C, D: protein expression of articular cartilage extracellular matrix degradation gene MMP13. Compared with the control group, * P<0.05, **P < 0.01.

[0048] Figure 6 . Effects of paternal pre-pregnancy caffeine exposure on the phosphorylation of MAPK signaling pathway in the joints of offspring rats of the present invention.

[0049] In the figure: A, B: Detection of the phosphorylation level of MAPK signaling pathway in articular cartilage during intrauterine period by Western Blot; C, D: Detection of the phosphorylation level of MAPK signaling pathway in articular cartilage at 6 weeks after birth by immunohistochemistry; E, F: Detection of the phosphorylation level of MAPK signaling pathway in articular cartilage at 12 weeks after birth by immunohistochemistry. Compared with the control group, * P < 0.05, ** P < 0.01.

[0050] Figure 7 . Changes in cartilage morphology after overexpression of Dusp14 in the joint cavity of offspring rats of the present invention with paternal pre-pregnancy caffeine exposure.

[0051] In the figure: A: Safranin staining of articular cartilage; B: Statistical analysis of the integrated optical density of safranin staining of articular cartilage. Compared with the control group, * P < 0.05, ** P < 0.01.

[0052] Figure 8 . Changes in chondrocyte hypertrophy differentiation after overexpression of Dusp14 in the joint cavity of offspring rats of the present invention with paternal pre-pregnancy caffeine exposure.

[0053] In the figure: A, B: Expression of Runx2 and Col10 mRNA in hypertrophic cells of articular cartilage; C, D: Protein expression of COL10 mRNA in hypertrophic cells of articular cartilage. Compared with the control group, * P < 0.05, ** P < 0.01. Detailed implementation manners

[0054] The following further elaborates on the technical content of the present invention in detail in combination with specific embodiments and drawings.

[0055]

Example 1

[0056] 1 Method for constructing the animal model

[0057] 1.1 Experimental animals

[0058] SPF-grade healthy Wistar rats were purchased from Hubei Provincial Center for Disease Control and Prevention, and the animal license number is: SCXK(E)2012 - 2014. This study was approved by the Ethics Committee of the Medical Department of Wuhan University and was strictly carried out in accordance with the relevant treatment guidelines of the International Laboratory Animal Protection Certification and Evaluation Agency.

[0059] Laboratory animals were raised in a barrier environment at a temperature of 22-25 °C, a humidity of 50%, and a 12-hour day-night cycle.

[0060] 1.2 Experimental methods

[0061] 1.2.1 Thirty male Wistar rats (body weight 260-300 g) and sixty female Wistar rats (body weight 200-240 g) were used. They had free access to water and food. After 7 days of adaptive feeding, the experimental rats were randomly divided into two groups: a control group and a caffeine group, with 10 rats in each group. The caffeine group was intragastrically perfused with 60 mg / kg.d of caffeine daily, and the control group was given the same volume of normal saline. The dosing volume was 1 mL / 100 g, and the dosing time was 8 weeks.

[0062] 1.2.2 After 8 weeks of dosing, the rats were caged together at a male:female ratio of 1:2. The next morning, vaginal smears were taken to determine pregnant rats, which were recorded as gestational day 0 (GD0). The pregnant rats in each group had free and normal access to food. The feed was purchased from Wuhan Wanqian Jiaxing Biotechnology Co., Ltd., license number: SCXK(E)2011-0011. The feed formula was the same as the mouse and rat formula feed specified in the "National Standard of the People's Republic of China GB14924.3-2001".

[0063] 1.2.3 Six pregnant rats were randomly selected from each group and cesarean section was performed under 3% isoflurane anesthesia to obtain offspring fetal rats (GD20) for corresponding examinations. The remaining female rats gave birth naturally to obtain the F1 generation. The day of birth was taken as postnatal day 0. At postnatal day 1, litters with 12-14 pups in each group were selected, and the number of male and female pups in each litter was adjusted to 6 for lactation feeding to ensure balanced nutrition for the pups. The pups were weaned at postnatal week 4 (PW4) and separated by sex into cages. All the above-mentioned offspring rats were anesthetized and sacrificed at PW6 and PW12 for subsequent tests.

[0064] 2 Detection indexes and methods

[0065] 2.1 For RT-qPCR detection, total RNA was isolated from cartilage tissue using TRIzol reagent. The isolated RNA was aliquoted and stored at -80℃. 1 μg of purified RNA was reverse transcribed using a cDNA synthesis kit, followed by cDNA amplification. The reaction was performed for 40 cycles. Relative amplicon expression was calculated using the 2-ΔΔCt method. The expression of Runx2, Col10, and GAPDH in the RNA was determined. Rat primer sequences are shown in Table 1. All cDNA sequences were obtained from the NCBI Entrez nucleotide database, and primers were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA). The NCBI BLAST database was used to query each designed primer sequence for homology comparison to determine the final primer sequences used. Primer information is shown in Table 1.

[0066] Table 1.

[0067]

[0068] 2.2 For Safranin O and Fast Green staining, dewax rat knee joint paraffin sections were dewaxed to water: the sections were sequentially immersed in xylene I-xylene II-anhydrous ethanol I-anhydrous ethanol II-75% ethanol, followed by washing with tap water. The sections were immersed in Fast Green staining solution for 5-10 min, washed with water, and then immersed in Safranin staining solution for 15-30 s, followed by rapid dehydration with three batches of anhydrous ethanol. The sections were then cleared with clean xylene for 5 min and mounted with neutral resin.

[0069] 2.3 For immunohistochemical staining, rat femurs were fixed in 4% paraformaldehyde solution for 3 days and then processed using paraffin embedding. Knee joints were sectioned in 5 μm sagittal sections for morphological staining analysis. After dewaxing, fluid replenishment, and antigen retrieval, paraffin sections were treated with EDTA antigen retrieval buffer (pH 8.0). Primary antibody (BSA) and primary antibody (MMP13) were used for blocking, and detection was performed using a DAB staining kit (GeneTech Company, Ltd., Shanghai, China).

[0070] 3 Experimental Results

[0071] PPCE can increase susceptibility to osteoarthritis in offspring rats, specifically manifested as follows: Safranin O and Fast Green results showed that, compared with the CON group, the PPCE group exhibited irregular arrangement of articular chondrocytes, hypertrophy of cells in both quiescent and proliferative areas, and significantly reduced and uneven Safranin staining density. Figure 2(A) The quantification of the intensity optical density (IOD) confirmed the reduced staining of PPCE fetal rat cartilage. Figure 2 In the PPCE group, some articular chondrocytes at PW6 and PW12 still showed significant hypertrophic differentiation, with a significantly reduced extracellular matrix (ECM) staining density and markedly lighter staining of the cartilage surface. Quantitative IOD analysis confirmed this weakened staining. Figure 2 (CF).

[0072] Further examination of Col10, a marker gene for cell hypertrophy and differentiation in articular cartilage, revealed that in the PPCE group, the expression of COL10 mRNA and protein in the progeny cartilage of GD20, PW6, and PW12 was upregulated, and the expression of Runx2 mRNA was also upregulated. Figure 3 Medium AI).

[0073] 4. Research Conclusions

[0074] This embodiment demonstrates that PPCE can lead to hypertrophic differentiation and poor cartilage quality in offspring before and after birth; PPCE can induce a stable, effective, and reliable animal model of paternal osteoarthritis susceptibility.

[0075] [Example 2] Construction of a two-step animal model of paternal osteoarthritis

[0076] 1. Animal Model Construction Methods

[0077] 1.1 Laboratory Animals

[0078] The experimental animals were the same as in Example 1.

[0079] 1.2 Experimental Methods

[0080] The methods for constructing the animal model in the early stages are the same as those in Examples 1.2.1-1.2.2.

[0081] 1.2.3 The rats were fed a normal diet until PW28. Twelve rats were randomly selected from each group. Six of them continued to be fed a normal diet until PW32, while the other six rats underwent a 4-week period of moderate-intensity running (at a constant speed of 18 m / min for 30 minutes, 3 times a week) until PW32. All the offspring rats were then anesthetized and sacrificed for subsequent testing.

[0082] 2. Detection Indicators and Methods

[0083] 2.1 The RT-qPCR detection method is as described in Example 1. Primer information is shown in Table 2.

[0084] Table 2.

[0085]

[0086] 2.2 The Safranin O and Fast Green staining detection method and the primers used are as described in Example 1.

[0087] 2.3 The immunohistochemical staining detection method and primers used are as described in Example 1.

[0088] 3 Experimental Results

[0089] PPCE can increase the susceptibility of offspring rats to osteoarthritis, specifically manifested as follows: Analysis of articular cartilage development in the CON and PPCE groups using safranin staining revealed that the CON group had a smooth cartilage surface with clearly distinguishable four-layer structure; the cells were numerous, neatly arranged, and the matrix staining was uniform; the PPCE group had a smooth cartilage surface and normal deep structure, but the cartilage matrix staining was lighter and the resting area of ​​the articular cartilage was thinner; the CON+Running group had a smooth cartilage surface, normal deep structure, uniform matrix staining, normal cartilage matrix structure, mild chondrocyte hypertrophy, and a clear tide line; the PPCE+Running group showed obvious degenerative changes on the cartilage surface, relatively uniform matrix staining, mildly disordered cartilage matrix structure, reduced chondrocyte number, and a still clear tide line. Figure 4 (A)

[0090] Further investigation by Mankin's, involving modifications to the knee joint, revealed that compared to the CON group, the PPCE group showed a significant increase in osteoarthritis scores, but no obvious signs of osteoarthritis. All groups showed an increase in osteoarthritis scores after chronic running, with the PPCE+Running group showing a significantly higher increase than the CON+Running group, meeting the diagnostic criteria for osteoarthritis. Figure 4 (B, C)

[0091] Further examination of the extracellular matrix degradation marker gene MMP13 in articular cartilage revealed that MMP13 mRNA and protein expression were upregulated in the PW32 progeny cartilage of the PPCE group, as was Adamts5 mRNA expression. After moderate running, the expression of MMP13 mRNA and protein in the PW32 progeny cartilage of the PPCE group was significantly upregulated, and Adamts5 mRNA expression was significantly upregulated. Figure 5 (Chinese AD).

[0092] 4. Research Conclusions

[0093] This embodiment found that the PPCE animal model exhibited typical osteoarthritis manifestations after a "second blow" of subthreshold stimulation. Paternal osteoarthritis occurs through a "two-blow" process: adverse preconception environmental exposure (the first blow) and postnatal subthreshold stimulation (the second blow) jointly cause paternal osteoarthritis.

[0094] [Example 3] The pathogenesis of paternal-derived adult osteoarthritis susceptible animal model established using the method of Example 1 was discovered.

[0095] 1. Animal Model Construction Methods

[0096] 1.1 Laboratory Animals

[0097] The experimental animals were the same as in Example 1.

[0098] 1.2 Experimental Methods

[0099] The experimental animals were the same as in Example 1.

[0100] 2. Detection Indicators and Methods

[0101] 2.1 Western blotting was used to detect phosphorylation levels of the MAPK signaling pathway in offspring intrauterine (GD20) cartilage, including p-ERK, p-JNK, p-P38, and GAPDH.

[0102] Protein extraction was performed according to the instructions of the EpiQuik Total Protein Extraction Kit. GAPDH was used as an internal control. 50 μg of total protein was separated by SDS-PAGE, then electrotransferred to a PVDF membrane. The membrane was blocked with 1×TBST containing 5% skim milk powder at room temperature with gentle shaking for 1 h. p-ERK, p-JNK, p-P38, and GAPDH monoclonal antibodies were added, and the membrane was incubated overnight at 4°C. The membrane was washed four times with 1×TBST, and then incubated with secondary antibody at room temperature for 1 h. After washing four times with 1×TBST, the membrane was reacted in Super Signal chemiluminescence reagent for 2 min, and then exposed to X-ray film in a dark room. Development and fixing were performed using standard methods. The gray values ​​of the protein bands were analyzed using ImageJ software. Using GAPDH as an internal control, the gray values ​​of the p-ERK, p-JNK, and p-P38 protein bands were normalized.

[0103] 2.2 Immunohistochemistry was used to detect phosphorylation levels of the MAPK signaling pathway in progeny PW6 and 12 articular cartilage, including p-ERK, p-JNK, p-P38, and GAPDH. The immunohistochemical detection methods and primers used were as described in Example 1.

[0104] 3 Experimental Results

[0105] Using the method described in Example 1, a paternal pre-pregnancy caffeine exposure-induced osteoarthritis susceptibility animal model was established. The phosphorylation levels of the MAPK signaling pathway in offspring were detected in utero and at 6 and 12 weeks postnatally. Western blot analysis showed increased phosphorylation levels of the MAPK signaling pathway in the articular cartilage of offspring during the utero period. Figure 6 (A, B); Immunohistochemical results showed similar changes in the expression of PW6 and PW12 proteins. Figure 6In the PPCE group, the phosphorylation levels of ERK, JNK, and P38 proteins in articular cartilage were significantly increased. These results suggest that PPCE leads to continuous activation of the MAPK signaling pathway in the articular cartilage of offspring rats during utero and after birth, mediating prenatal and postnatal chondrocyte hypertrophy and differentiation, and poor articular cartilage quality.

[0106] 4. Research Conclusions

[0107] This embodiment demonstrates that the MAPK signaling pathway in the cartilage of PPCE offspring is activated before and after birth. The PPCE animal model can be stably used to explore the pathogenesis of susceptibility to paternal adult osteoarthritis.

[0108] [Example 4] Identifying intervention targets for osteoarthritis based on an established paternally susceptible adult animal model.

[0109] 1. Animal Model Construction Methods

[0110] 1.1 Laboratory Animals

[0111] The experimental animals were the same as in Example 1.

[0112] 1.2 Experimental Methods

[0113] The methods for constructing the animal model in the early stages are the same as those in Examples 1.2.1-1.2.2.

[0114] 1.2.3 Mother mice gave birth naturally, with the day of birth designated as day 0 after birth. On day 1 after birth, litters of 12-14 pups were selected from each group, and the number of male and female pups in each litter was adjusted to 6 for nursing to ensure balanced nutrition. Pups were weaned at PW4 and separated into male and female cages, and fed a normal diet until PW6. PW6 rats were anesthetized with 3% isoflurane and injected intra-articularly with 50 μl of the target adeno-associated virus. After recovery from anesthesia, they were fed normally until PW12. All offspring were euthanized under anesthesia for subsequent testing.

[0115] 2. Detection Indicators and Methods

[0116] 2.1 The RT-qPCR detection method is as described in Example 1, and the primer information is as described in Example 1.

[0117] 2.2 The Safranin O and Fast Green staining detection method and the primers used are as described in Example 1.

[0118] 2.3 The immunohistochemical staining detection method and primers used are as described in Example 1.

[0119] 3 Experimental Results

[0120] Using the method in Example 1, a paternal pre-pregnancy caffeine exposure-induced osteoarthritis susceptibility animal model was established. At PW6, local intervention with the target gene (Dusp14) in the joint cavity via adeno-associated virus for 6 weeks partially reversed the osteoarthritis susceptibility induced by PPCE in offspring. Specifically, Safranin O and Fast Green assays showed that in the PPCE group, after 6 weeks of Dusp14 overexpression (PW12), the number of hypertrophic chondrocytes was significantly reduced, and the ECM staining density was significantly increased. Figure 7 In the middle A), quantitative IOD analysis confirmed that its staining was weakened ( Figure 7 (B)

[0121] Further examination was conducted on Col10, a marker gene for cell hypertrophy and differentiation in articular cartilage. Compared with the PPCE control group, the expression of COL10 mRNA and protein in PW12 progeny cartilage was upregulated in the PPCE Dusp14 overexpression group, and the expression of Runx2 mRNA was also upregulated. Figure 8 (Chinese AD).

[0122] 4. Research Conclusions

[0123] This embodiment demonstrates that targeted therapy can correct the poor cartilage quality in PPCE offspring and inhibit the development of susceptibility to adult osteoarthritis in the long term. In other words, the PPCE animal model can be effectively used for the identification of intervention targets for paternal adult osteoarthritis.

[0124] In summary, the PPCE animal model constructed in this invention produces offspring exhibiting chondrocyte hypertrophy and differentiation, and low articular cartilage quality, leading to osteoarthritis in adult rats after a "secondary shock." This demonstrates that this method is effective in establishing animal models of paternal osteoarthritis and can be used to study the mechanisms of susceptibility to paternal osteoarthritis. Based on this, it can further guide the screening of chondrogenic toxicity caused by pre-pregnancy environmental disturbances or drugs, and its application in identifying early warning and intervention targets for paternal osteoarthritis.

Claims

1. A method for constructing an animal model of paternal osteoarthritis based on a two-step approach, characterized in that: Includes the following steps: S1: Select healthy rodents of reproductive age, covering a complete spermatogenesis cycle in the 8 weeks before conception, administer 60 mg / kg of caffeine via gastric gavage daily, allow male rodents to eat freely, and obtain pregnant rodents by mating with normal female rodents. S2: The pregnant mouse gives birth naturally and obtains pups. The birth date is taken as day 0 after birth. One day after birth, select a litter with 12-14 pups and adjust each litter to 6 male and 6 female pups for nursing. S3: The baby mice were weaned 4 weeks after birth and the males and females were separated into different cages. Some continued to be fed a normal diet until 28 weeks of age. S4: Some offspring were raised until 28 weeks after birth, and then given 4 weeks of moderate-intensity running. Osteoarthritis-related indicators were detected at 32 weeks to comprehensively determine the occurrence of osteoarthritis; ultimately, an animal model of paternal osteoarthritis was obtained. In step S1, the rodent is an SPF-grade animal, selected from any one of Wistar rat, SD rat, Kunming mouse, C57 mouse, guinea pig or hamster; In step S3, the normal diet is formulated in the same way as the formula feed for mice and rats specified in the National Standard of the People's Republic of China GB14924.3-2001. In step S4, the relevant indicators for osteoarthritis detection are: morphological changes in articular cartilage, osteoarthritis score, and detection of genes related to articular cartilage matrix degradation.

2. The application of an animal model of paternal osteoarthritis in screening for adverse paternal age-related environmental disturbances that can cause chondrogenic toxicity in pups, characterized in that: The paternal osteoarthritis animal model is obtained by the construction method as described in claim 1.

3. The application of an animal model of paternal osteoarthritis in screening intervention targets for paternal osteoarthritis, characterized in that: The paternal osteoarthritis animal model is obtained by the construction method as described in claim 1.

4. The application of a paternal osteoarthritis animal model in screening drugs for the prevention and treatment of osteoarthritis, characterized in that: The paternal osteoarthritis animal model is obtained by the construction method as described in claim 1.

5. The application of an animal model of paternal osteoarthritis in screening early warning targets for the occurrence of paternal osteoarthritis, characterized in that: The paternal osteoarthritis animal model is obtained by the construction method as described in claim 1.

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