Construction method, intervention target and application of a fetal osteoarthritis model caused by prednisone exposure during pregnancy

Abstract

The present invention discloses a method for constructing a fetal osteoarthritis model caused by prednisone exposure during pregnancy, an intervention target, and its application. circGtdc1 is a newly identified circRNA that is most significantly altered in dysplastic cartilage caused by prednisone exposure during pregnancy. RNase R, AcD, and Sanger sequencing confirmed its circularization. Prednisolone concentration-dependently reduced circGtdc1 expression in chondrocytes. Silencing circGtdc1 significantly inhibited chondrocyte proliferation and matrix synthesis gene expression, while overexpression of circGtdc1 had the opposite effect and could reverse the effects of prednisolone. Intra-articular injection of AAV‑circGtdc1 to overexpress circGtdc1 can reverse chondrodysplasia caused by prednisone exposure during pregnancy. The intervention target established by the present invention is novel, reliable, and simple, providing a possibility for the early prevention and treatment of fetal osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of gene function and application, and in particular to a method for constructing a fetal osteoarthritis model caused by prednisone exposure during pregnancy, an intervention target and an application thereof. Background Art

[0002] Osteoarthritis is the leading cause of joint pain and chronic disability in the elderly, and its prevalence increases with age. Currently, there are approximately 360 million osteoarthritis patients worldwide; among them, the incidence of osteoarthritis in people over 60 years old in China exceeds 50%, and the incidence in people over 70 years old even exceeds 80%, far exceeding the incidence of cardiovascular disease. As China enters an aging society, the increasing number of osteoarthritis patients has caused a huge health, economic and medical burden on the country. However, the pathogenesis of osteoarthritis is still not fully understood. In the 1990s, based on the results of a large-scale epidemiological survey, Barker found that the incidence of coronary heart disease, hypertension, hyperlipidemia and obesity in low-birth-weight children increased in adulthood, and based on this, he proposed the intrauterine origin theory of adult diseases [1]. As the research deepened, more and more epidemiological evidence showed that adult osteoarthritis also has an intrauterine origin. In 2011, Aigner and Richter proposed that age-related osteoarthritis may be related to fetal cartilage development [2]. Subsequently, Sayer and Jordan also found through epidemiological surveys that low-birth-weight children had a higher incidence of hand and lumbar osteoarthritis. Our laboratory's previous animal studies have demonstrated that exposure to various exogenous substances (caffeine, nicotine, and ethanol, among others) and dietary restriction during pregnancy can induce low cartilage quality in offspring and susceptibility to adult osteoarthritis. In summary, osteoarthritis has a fetal origin. Exploring the intrauterine origins and mechanisms of osteoarthritis provides an experimental basis for further treatment.

[0003] Chondrocytes and the extracellular matrix they secrete are the main components of articular cartilage, and their content reflects the quality of articular cartilage. Cartilage destruction, decreased chondrocyte number, and decreased extracellular matrix content are typical pathological characteristics of osteoarthritis. Under physiological conditions, the proliferation and apoptosis of chondrocytes, and the synthesis and degradation of extracellular matrix are in a state of balance. When this balance is broken, that is, the number of chondrocytes decreases or the content of extracellular matrix decreases, the cartilage quality is poor. Related studies have also shown that the quality of articular cartilage is closely related to the occurrence and development of osteoarthritis [3]. Changes in the biomechanics of articular cartilage lead to poor cartilage quality, thereby mediating the occurrence of osteoarthritis [4]. Our laboratory's series of studies also confirmed that adverse maternal environments (such as exposure to exogenous substances and food restriction) can lead to poor cartilage quality in offspring and susceptibility to osteoarthritis in adulthood. Since the articular cartilage of mammals and rodents is mainly formed in the embryonic period, the number of articular cartilage cells and their relative thickness gradually decrease with age after birth, and the cartilage lacks sufficient vascular control, and its self-repair ability after injury is extremely poor. Therefore, cartilage development during the intrauterine embryonic period plays an important and decisive role in the quality of cartilage in adulthood and may lead to the occurrence and development of osteoarthritis in adulthood.

[0004] Circular RNA (circRNA) is a type of RNA molecule that is connected end to end to form a closed loop without 5′ and 3′ end structures. This makes its structure very stable and not easily degraded by the nuclease exonuclease RNase R. Therefore, circRNA plays a vital role in the development and pathology of tissues and organs. For example, it has been confirmed that multiple circRNAs (such as circHIPK3, circ_ATRNL1, etc.) are involved in the chondrogenic differentiation of bone marrow mesenchymal stem cells and the proliferation of chondrocytes, thereby mediating the occurrence and development of osteoarthritis [5,6]; multiple circRNAs are involved in the development of skeletal muscle and intestine [7,8]. However, the effect of circRNA on cartilage development has not been reported. Intrauterine programming alteration refers to the process of permanent changes in tissue morphology and function caused by damage in the early development of the body. These changes in tissue and organ function or gene expression patterns usually last from the developmental period to adulthood, or even throughout life, leading to a series of effects on the body after adulthood [9]. Studies have shown that multiple non-coding RNAs program the effects of the intrauterine environment on the health of offspring. For example, miRNAs regulate the effects of maternal exercise during pregnancy on the liver glucose and lipid metabolism of adult offspring

[10] ; long noncoding RNA H19 mediates adrenal hypoplasia in offspring caused by caffeine exposure during pregnancy and its transgenerational genetic effects

[11] ; miRNA-92-3p, miRNA-200b-3p, etc. also mediate offspring chondrodysplasia caused by adverse maternal environment[12,13]. As one of the noncoding RNAs, circRNA programming of offspring cartilage development and susceptibility to osteoarthritis in adulthood has not been reported and needs further study.

[0005] References:

[0006] 1.Barker, DJ, The origins of the developmental origins theory. JIntern Med, 2007.261(5):p.412-7.

[0007] 2.Aigner,T.and W.Richter,OA in 2011:Age-related OA--a concept emerging from infancy? Nat Rev Rheumatol,2012.8(2):p.70-2.

[0008] 3.Dahlberg,L.,Cartilage quality,overweight and osteoarthritis:a casefor new behaviour?Ann Rheum Dis,2012.71(1):p.1-3.

[0009] 4.Pitsillides,A.A.and F.Beier,Cartilage biology in osteoarthritis--lessons from developmental biology.Nat Rev Rheumatol,2011.7(11):p.654-63.

[0010] 5.Li,S.,et al.,Mesenchymal stem cell-derived extracellular vesiclesprevent the development of osteoarthritis via the circHIPK3 / miR-124-3p / MYH9axis.J Nanobiotechnology,2021.19(1):p.194.

[0011] 6.Zheng,J.,et al.,Promotive role of CircATRNL1 on chondrogenicdifferentiation of BMSCs mediated by miR-338-3p.Arch Med Res,2021.52(5):p.514-522.

[0012] 7.Wu,P.,et al.,Identification of crucial circRNAs in skeletal muscleduring chicken embryonic development.BMC Genomics,2022.23(1):p.330.

[0013] 8.Li,S.Y.,et al.,RNA-Seq profiling of circular rnas duringdevelopment of hindgut in rat embryos with ethylenethiourea-induced anorectalmalformations.Front Genet,2021.12:p.605015.

[0014] 9.Meaney,M.J.,M.Szyf,and J.R.Seckl,Epigenetic mechanisms of perinatalprogramming of hypothalamic-pituitary-adrenal function and health.Trends MolMed,2007.13(7):p.269-77.

[0015] 10.Zhou,L.,et al.,Maternal exercise programs glucose and lipidmetabolism and modulates hepatic mirnas in adult male offspring.Front Nutr,2022.9:p.853197.

[0016] 11.He,Z.,et al.,H19 / let-7axis mediates caffeine exposure duringpregnancy induced adrenal dysfunction and its multi-generationinheritance.Sci Total Environ,2021.792:p.148440.

[0017] 12.Tie,K.,et al.,Low miR-92a-3p in oocytes mediates themultigenerational and transgenerational inheritance of poor cartilage qualityin rat induced by prenatal dexamethasone exposure.Biochem Pharmacol,2022.203:p.115196.

[0018] 13.Ni,Q.,et al.,miR-200b-3p / ERG / PTHrP axis mediates the inhibitory effect of ethanol on the differentiation of fetal cartilage into articularcartilage. Chem Biol Interact, 2022.368:p.110201. Summary of the Invention

[0019] To address the above-mentioned problems and deficiencies, the present invention aims to determine the relationship between altered circGtdc1 expression and fetal chondrodysplasia and osteoarthritis, identify circGtdc1 as an intervention target for fetal adult osteoarthritis, and provide the use of circGtdc1 as an intervention target in screening drugs for ameliorating fetal chondrodysplasia and adult osteoarthritis, as well as the use of circGtdc1 analogs in the preparation of drugs for ameliorating fetal adult osteoarthritis.

[0020] The above objectives are achieved through the following technical solutions:

[0021] In a first aspect, the present invention provides a method for constructing a fetal osteoarthritis model induced by prednisone exposure during pregnancy, characterized by comprising the following steps:

[0022] S1: Normal pregnant rats were given 0.125 and 0.25 mg / kg prednisone orally daily from gestational day 0 to 20;

[0023] S2: The litter size was adjusted to 14 pups per day after natural birth, with a sex ratio of 1:1. The mice were weaned at 4 weeks of age and had free access to food and water.

[0024] S4: running treatment 24–28 weeks after birth;

[0025] S5: Articular cartilage tissues were obtained from rats at 20 days of gestation, 12 weeks of age, and 28 weeks of age. Knee cartilage quality was assessed, and changes in cartilage matrix synthesis and degradation were detected.

[0026] In step S1, the rats are Wistar rats; the prednisone is a clinically used prednisone tablet;

[0027] The running conditions in step S3 are as follows: Day 1, 10 minutes, 10 m / min; Day 2, 15 minutes, 12 m / min; Day 3, 20 minutes, 15 m / min; Day 4, 30 minutes, 18 m / min; Day 5, 35 minutes, 20 m / min. On Day 5 and thereafter, the first 10 minutes consist of 12 m / min, followed by 55 minutes of running at 20 m / min each day.

[0028] The cartilage quality in step S4 includes the depth of safranin fast green staining; the ratio of cartilage in the proliferative zone and hypertrophic zone in the fetal mouse cartilage tissue; the smoothness of the cartilage surface, the reduction of cartilage thickness, whether the cartilage structure is disordered, whether the tide line is interrupted, and the osteoarthritis (OARSI) score; the detection of cartilage matrix synthesis indicators including the mRNA and protein expression of Sox9, Acan and Col2a1; the cartilage matrix synthesis indicators including the mRNA and protein expression of Mmp3, Mmp13 and Adamts5.

[0029] In a second aspect, the present invention provides an intervention target for fetal-induced adult osteoarthritis, characterized in that: the intervention target is circGtdc1 in rat cartilage; and overexpression of circGtdc1 in vivo can improve the poor cartilage quality caused by prednisone exposure during pregnancy.

[0030] In a third aspect, the present invention provides a use of the above-mentioned intervention target (circGtdc1) in screening drugs for fetal adult osteoarthritis, fetal adult chondrodysplasia, or fetal adult osteoporosis.

[0031] Preferably, the circGtdc1 is a newly identified circular RNA molecule; the expression of the circGtdc1 is reduced in the offspring cartilage, resulting in chondrodysplasia and an increased likelihood of osteoarthritis in adulthood.

[0032] The circGtdc1 is used as an intervention target, and the drug for improving fetal osteoarthritis includes a circGtdc1 gene mimetic.

[0033] The circGtdc1 gene mimic is a circRNA overexpression plasmid.

[0034] The circRNA overexpression plasmid is pLV-ciR-circGtdc1 or AAV-circGtdc1.

[0035] The technical principle and research process of the present invention are as follows:

[0036] The present invention establishes a fetal model of chondrodysplasia and osteoarthritis susceptibility by exposing the offspring to an exogenous substance (prednisone) during pregnancy. In this fetal model, expression of cartilage proliferation-related genes (Ki67) decreased at 20 days of gestation and 12 weeks after birth, safranin staining became lighter, and mRNA and protein expression of chondrocyte matrix synthesis genes (Col2a1 and Acan) decreased. After a second challenge (long-distance running) between 24 and 28 weeks after birth, the prednisone-exposed group exhibited a more pronounced osteoarthritis phenotype (e.g., lighter safranin fast green staining, more pronounced cartilage surface destruction, and a higher osteoarthritis RSI score) than the normal control group. Further RNA sequencing revealed that prednisone exposure during pregnancy led to an imbalance in circular RNA expression, with 37 circRNAs upregulated and 35 downregulated, of which 83% were exon-derived, 4% were intron-derived, and 5% were intergenic. The present invention selected 15 circRNAs with the most significant increases and decreases. Further tissue validation by PCR revealed that circGtdc1 had the most significant fold change. RT-qPCR further confirmed that circGtdc1 was most significantly decreased. RNaseR and AcD assays demonstrated that circGtdc1 was more stable than linear mGtdc1, and Sanger sequencing confirmed its circularization. These results suggest that prednisone exposure during pregnancy may lead to chondrodysplasia, and the mechanism by which this may be related to decreased circGtdc1 expression.

[0037] At the same time, the present invention demonstrated at the cellular level that prednisolone (but not prednisone) inhibited chondrocyte proliferation (EdU-positive cell number decreased) and the expression of matrix synthesis genes (Col2a1 and Acan) at both mRNA and protein levels in a concentration-dependent manner, while having no significant effect on the expression of cartilage matrix degradation-related genes (MMP13 and ADAMTS5) or chondrocyte apoptosis. In fetal chondrocytes, prednisolone reduced circGtdc1 expression in a concentration-dependent manner, a phenomenon reversed by the glucocorticoid receptor (GR) inhibitor RU486 (Sigma-Aldrich, Cat# M8046). These findings suggest that prednisolone inhibits circGtdc1 expression through the GR.

[0038] Furthermore, the present invention showed that the administration of circGtdc1 overexpression plasmid (pLC-ciR-Gtdc1) or siRNA (siRNA-circGtdc1) to fetal chondrocytes showed that pLC-ciR-Gtdc1 promoted chondrocyte proliferation and increased the mRNA and protein expression of matrix synthesis genes (Col2a1 and Acan), while siRNA-circGtdc1 had the opposite effect. Moreover, after pLC-ciR-Gtdc1 and prednisolone were co-treated with fetal chondrocytes, pLC-ciR-Gtdc1 could reverse the decrease in chondrocyte proliferation and the decrease in mRNA and protein expression of matrix synthesis genes (Col2a1 and Acan) caused by prednisolone. These results indicate that circGtdc1 mediates the inhibition of chondrocyte proliferation and the reduction in extracellular matrix synthesis caused by prednisolone.

[0039] Finally, the present invention utilized an animal model of chondrodysplasia in rat offspring induced by prednisone exposure during pregnancy. An adeno-associated virus (AAV)-encapsulated circGtdc1 overexpression plasmid (AAV-circGtdc1) was injected into the joints of rats 8-12 weeks after birth. The results showed that AAV-circGtdc1 reversed the decreased safranin fast green staining and increased osteoarthritis RSI scores induced by prednisone exposure during pregnancy, and increased the mRNA and protein expression of matrix synthesis genes (Col2a1 and Acan).

[0040] In conclusion, circGtdc1 can serve as an intervention target for fetal chondrodysplasia and susceptibility to osteoarthritis in adulthood caused by prednisone exposure during pregnancy, providing a new approach for the treatment of fetal osteoarthritis.

[0041] The present invention has the following advantages and effects compared to the prior art:

[0042] 1. This study discovered and identified a new circular RNA, circGtdc1.

[0043] 2. The present invention found that circGtdc1 mediates low cartilage quality in offspring and susceptibility to osteoarthritis in adulthood caused by prednisone exposure during pregnancy.

[0044] 3. Based on the application of circGtdc1 in fetal adult osteoarthritis caused by prednisone during pregnancy, it provides a target for the development of drugs to treat fetal osteoarthritis.

[0045] 4. The present invention found that intra-articular injection of the circGtdc1 mimetic AAV-circGtdc1 can be used to prepare therapeutic drugs for fetal osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .Exposure to prednisone during pregnancy leads to poor cartilage quality in offspring rat fetuses.

[0047] Figure 1 Middle: (A) Safranin fast green staining of fetal mouse knee joint (R, C, and H represent the circular, columnar, and mast cell layers of cartilage, respectively), scale bar = 500 μm, n = 5; (B) Quantitative statistics of mean optical density (MOD) of safranin fast green staining, n = 5; (CE) Area statistics of circular, columnar, and mast cell layers of cartilage, n = 5; (F) RT-qPCR detection of Col2a1, Acan, Mmp3, Mmp13, and Adamts5 mRNA expression in fetal cartilage tissue, n = 12; (GH) Immunohistochemistry detection of Acan and Col2a1 protein expression, scale bar = 300 μm, n = 5; (I) Quantitative statistics of mean optical density (MOD) of immunohistochemical staining, n = 5. The results are expressed as mean ± standard error. Compared with the respective normal control groups, * P<0.05, ** P<0.01, *** P<0.001.

[0048] Figure 2 .Exposure to prednisone during pregnancy results in low cartilage quality and susceptibility to osteoarthritis in adult offspring.

[0049] Figure 2 Middle: (A) Safranin fast green staining of articular cartilage in rats at 12 weeks after birth, scale bar = 1000 μm, n = 5; (B) Quantitative statistics of mean optical density (MOD) of Safranin fast green staining and RSI score of osteoarthritis, n = 5; (C) RT-qPCR detection of Col2a1, Acan, Mmp3, Mmp13 and Adamts5 in fetal cartilage tissue mRNA expression, n = 12; (D) Immunofluorescence and histochemical analysis of Col2, Acan and Mmp13 protein expression, fluorescence scale = 100 μm, histochemical scale = 50 μm, n = 5; (EG) Quantitative analysis of Col2, Acan and Mmp13 protein expression, n = 5 × 6; (HI) Safranin fast green staining and mean optical density (MOD) quantitative analysis of articular cartilage of rats at 28 weeks after birth, scale = 1000 μm, n = 5; (J) RSI score of osteoarthritis in articular cartilage of rats at 28 weeks after birth, n = 5; (L) Immunohistochemical detection of Col2 and Mmp13 protein expression, scale = 50 μm; (M) Quantitative analysis of mean optical density (MOD) of Col2 and Mmp13 protein expression, n = 5 × 6. The results are expressed as mean ± standard error. Compared with the respective normal control groups, * P<0.05, ** P<0.01, *** P<0.001.

[0050] Figure 3 .Pregnancy exposure to prednisone leads to decreased expression of circGtdc1.

[0051] Figure 3 Middle: (A) Volcano plot showing circular RNA sequencing expression; (B) Distribution of differentially expressed circular RNA genes; (C) RT-qPCR validation of the most significantly altered circular RNAs in sequencing results, n = 12; (D) RT-qPCR detection of circGtdc1, circRab31, and circMlip expression in cartilage tissue at 12 weeks after birth, n = 12; (E) Sanger sequencing validation of circGtdc1 circularization; (F) Northern blot detection of circGtdc1 and mGtdc1 expression; (G) circGtdc1 and mGtdc1 expression after RNase R treatment, n = 3; (H) circGtdc1 and mGtdc1 expression after actinomycin treatment, n = 3; (I) Nuclear and cytoplasmic distribution of circGtdc1, n = 3; (J) RT-qPCR detection of Acan and Col2a1 mRNA expression, n = 3; (K) EdU detection of chondrocyte proliferation and quantitative statistics, scale bar = 500 μm, n = 3. The results are expressed as mean ± standard error. Compared with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001.

[0052] Figure 4 .circGtdc1 mediates the decreased proliferation and extracellular matrix synthesis of fetal chondrocytes induced by prednisolone.

[0053] Figure 4Middle: (A) RT-qPCR detection of Acan and Col2a1 expression in fetal chondrocytes after prednisolone treatment, n=6; (BC) EdU detection of chondrocyte proliferation and quantitative statistics, scale bar=500μm, n=3; (D) Safranin detection of chondrocyte matrix content and quantitative statistics of average optical density, scale bar=500μm, n=6; (F) Immunofluorescence detection of Acan and Col2 protein expression in fetal chondrocytes after prednisolone treatment, scale bar=25μm, n=6; (G) EdU detection of chondrocyte proliferation and quantitative statistics, n=3; (HI) Safranin detection of chondrocyte matrix content and quantitative statistics of average optical density, scale bar=500μm, n=6; (J) RT-qPCR detection of Acan and Col2a1 expression in fetal chondrocytes after prednisolone treatment, n=3; (KL) Western Blot analysis and quantitative analysis of Acan and Col2 protein expression, n = 3; (M) RT-qPCR analysis of circGtdc1 expression in fetal chondrocytes after prednisolone treatment, n = 6. The results are expressed as mean ± standard error. Compared with the respective normal control groups, * P<0.05, ** P<0.01, *** P<0.001.

[0054] Figure 5 .circGtdc1 reverses fetal chondrodysplasia induced by prednisone exposure during pregnancy in vivo.

[0055] Figure 5 Middle: (A) Schematic diagram and in vivo imaging of intra-articular injection of adeno-associated virus-encapsulated circGtdc1 to intervene in offspring rats with chondrodysplasia induced by prednisone exposure during pregnancy; (B) Safranin fast green staining of articular cartilage, scale bar = 50 μm, n = 5; (C, D) RSI score of osteoarthritis and quantitative analysis of average optical density of Safranin fast green staining, n = 5 × 6; (E, F) RT-qPCR detection of Acan and Col2a1 mRNA expression in cartilage tissue, n = 8; (G1) Immunohistochemistry detection of Col2 and Acan protein expression and quantitative analysis of articular cartilage, scale bar = 50 μm, n = 5 × 6. The results are expressed as mean ± standard error. Compared with the respective normal control groups, * P<0.05, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0056] The following further describes the above content of the present invention in detail through specific implementation methods in the form of embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. The technologies implemented based on the above content of the present invention are all within the scope of the present invention.

[0057] All reagents involved in the experiments of the present invention can be purchased in domestic and foreign markets, or prepared by oneself according to the recipe in the instructions; experimental methods not otherwise specified are conventional methods known in the art.

[0058] [Example 1] Establishment of a fetal osteoarthritis susceptibility model

[0059] 1 Experimental methods

[0060] 1.1 Experimental Animals and Treatment

[0061] The present invention purchased pathogen-free (SPF) healthy Wistar rats from SPEIF (Beijing) Biotechnology Co., Ltd., animal license number: SCXK (Beijing) 2019-0010. All animal experimental protocols involved in this study were approved by the Experimental Animal Care and Use Committee (IACUC) of the Animal Experimental Center of Wuhan University (IACUCNO.WP20210060). After all rats were adaptively fed for 7 days, they were caged in a female:male ratio of 2:1 at 18:00 every night. Vaginal secretions were taken for microscopic examination the next morning, and sperm was found to be on gestational day 0 (GD0). Pregnant mice were given 0.125 and 0.25 mg / kg·d of prednisone by gavage on G0-20, respectively, and the normal control group was given an equal amount of sodium carboxymethyl cellulose by gavage. After some pregnant mice (n=12) were euthanized on GD20, the knee joints of the fetuses were obtained. From five litters, one right knee joint from each litter was fixed in 4% paraformaldehyde for histological analysis. The remaining knee joint tissue was immediately frozen at -80°C for subsequent analysis. From the remaining rats, one rat from each litter was selected for rearing and, after birth, the knee joints of some rats were harvested at postnatal week 12 (PW12) and PW28. The remaining rats were run-trained and their knee joints were harvested at PW24-28. Parts of the harvested knee joints were fixed in 4% paraformaldehyde, and the remaining parts were immediately frozen at -80°C for subsequent analysis.

[0062] 1.2 Safranin fast green staining and histological scoring

[0063] After decalcification and embedding, cartilage tissue was cut into 4-6 μm thick sections. Sections were placed on glass slides, allowed to dry, and fixed at 60°C for 15 minutes. Sections were then washed sequentially in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and distilled water. Sections were then immersed in Safranin O solution and allowed to stand at room temperature for 5 minutes. Sections were gently rinsed with water twice for 5 seconds each, and excess water was removed on filter paper. Sections were then immersed in 0.5% Fast Green solution for staining for 30-60 seconds. Sections were gently rinsed with water twice for 5 seconds each, and excess water was removed on filter paper. Finally, sections were covered with Xylene and blocked. Images were taken using a light microscope, with 6 fields of view per sample, and 5 samples per group (n = 5). Semiquantitative statistical analysis was performed using Image J, and the Osteoarthritis Research Society International (OARSI) score was calculated. The OARSI scoring system includes the following: Grade 1.0: intact chondrocytes; Grade 1.5: cell death, including apoptosis or necrosis; Grade 2.0: surface discontinuity, including fibrosis; Grade 2.5: surface wear and loss of a portion of the superficial cartilage zone; Grade 3.0: simple cracks or cracks invading the central zone; Grade 3.5: cracks extending and becoming bifurcated or complex cracks; Grade 4.0: loss of only the superficial cartilage zone (erosion); Grade 4.5: loss of the central zone; Grade 5.0 (stripping): presence of a bony surface composed of intact calcified cartilage or sclerotic bone; Grade 5.5: reparative fibrocartilage tissue or new bone formation; Grade 6.0: distortion of the joint geometry at the joint margin; and Grade 6.5: distortion of both the joint margin and the weight-bearing area. Osteoarthritis staging is based on the horizontal extent of affected cartilage surface. Stage 1: Less than 10% involvement; Stage 2: 10-25% involvement; Stage 3: 25-50% involvement; Stage 4: Greater than 50% involvement. The final osteoarthritis RSI score = Grade score × Stage score. The final score is the average of the two independent scores.

[0064] 1.3Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

[0065] The total RNA of cartilage tissue and cells was extracted by TRIzol. Briefly, after the sample was added to a 1.5 ml enzyme-free EP tube, 200 μl of TRIzol reagent was added. For cartilage tissue, 200 μl of chloroform was added after grinding, while 200 μl of chloroform was directly added to the cell sample. After inversion to mix, let it stand on ice for 10 minutes. After centrifugation at 12000g for 10 minutes, 400 μl was transferred to a new EP tube, an equal amount of isopropanol was added to mix, and then let it stand at room temperature for 10 minutes. After centrifugation at 12000g for 10 minutes, it was washed twice with 1 ml of 75% ethanol. After drying, enzyme-free water was added. The A260 / 280 ratio was then determined to be between 1.8 and 2.1 using the NanoDrop 2000 nucleic acid analyzer. It was then reversed to cDNA using a reverse transcription kit. Then, StepOnePlus was used TM Real-time PCR system was used to detect the expression of Acan, Col2a1, Mmp3, Mmp13, Adamts5, circGtdc1, and mGtdc1. Reaction conditions were as follows: 95°C / 30s, 95°C / 10s, and 62°C / 30s (40 cycles). Primer sequences are shown in Table 1:

[0066] Table 1. Primer sequences.

[0067]

[0068] 1.4 Immunohistochemical staining and tissue immunofluorescence staining

[0069] After the knee joint specimens were dewaxed to water, antigen retrieval was performed with sodium citrate buffer and incubated with BSA for 30 minutes. Then, primary antibodies (Acan, Col2a1 and Mmp13 antibodies at 1:200) were added at 4°C overnight. Biotin secondary antibodies were added on the second day of immunohistochemistry, while fluorescent secondary antibodies were added on the second day of immunofluorescence. DAB was used to detect peroxidase activity in immunohistochemistry. Changes in corresponding indicators were observed under optical microscope and fluorescence microscope respectively. The images after staining were obtained by The staining level was determined by calculating the mean optical density (MOD) or mean gray value of six fields of view for each sample.

[0070] 2 Experimental results

[0071] 2.1 Pregnancy-induced chondrodysplasia in rat fetuses

[0072] First, the present invention detects the effect of prednisone exposure during pregnancy on the quality of fetal cartilage. The results of safranin fast green showed that compared with the normal control group, the safranin fast green staining of the prednisone exposure group during pregnancy became lighter ( Figure 1 Middle A), the mean optical density decreased (P<0.001, Figure 1 Middle B), cartilage was reduced in the round and columnar regions (P<0.01, P<0.001, Figure 1 C, D), while the cartilage in the hypertrophic area increased (P<0.001, Figure 1 E), and it was obviously dose-dependent. RT-qPCR results showed that the expression of Col2a1, Acan, Mmp3, Mmp13 and Adamts5 mRNA in the prednisone exposure group during pregnancy decreased in a dose-dependent manner (P<0.05, P<0.01, Figure 1 Immunohistochemical staining further showed that the expression of Col2a1 and Acan proteins was significantly decreased in the prednisone exposure group during pregnancy (P<0.001, Figure 1 These results suggest that prednisone exposure during pregnancy induces impaired cartilage matrix synthesis, leading to chondrodysplasia.

[0073] 2.2 Prenatal exposure to prednisone leads to susceptibility to chondrodysplasia and osteoarthritis in adult rat offspring

[0074] Next, the present invention examined the effects of gestational prednisone exposure on the cartilage of postnatal rat offspring. The results showed that gestational prednisone exposure caused the cartilage of PW12 rats to stain lighter, the cartilage surface to be uneven, the thickness to be reduced, the structure to be disordered, and the tide line to be interrupted or even disappeared, and the osteoarthritis OARSI score to be reduced (P<0.01, P<0.001, Figure 2 RT-qPCR, immunofluorescence, and immunohistochemistry further demonstrated that prednisone exposure during pregnancy dose-dependently decreased the mRNA and protein expressions of Acan and Col2a1, while increasing the mRNA expressions of Mmp13 and Adamts5 and the protein expression of Mmp13 (P<0.05, P<0.01, P<0.001, Figure 2 The above changes could even continue until PW28 (P<0.05, P<0.01, P<0.001, Figure 2 After the second shock (long-distance running) at PW24-28, the animals exposed to prednisone during pregnancy showed more severe cartilage safranin staining reduction, cartilage surface destruction and higher OARSI scores than the unexposed animals (P<0.05, P<0.01, P<0.001, Figure 2 RT-qPCR and immunohistochemistry also showed that the expression of Cola1 and Acan mRNA and Cola1 protein in the prednisone exposure + running group was lower than that in the running group alone, while the expression of Mmp3, Mmp13 and Adamts5 mRNA and Mmp13 protein was higher (P<0.05, P<0.01, P<0.001, Figure 2 These results indicate that prednisone exposure during pregnancy leads to reduced cartilage quality and susceptibility to osteoarthritis in adult offspring rats.

[0075] The present invention gives Wistar rats 0.125, 0.25mg / kg prednisone by 0-20 days of pregnancy, and gives a second blow (long-distance running) process in 24-28 weeks after birth after natural production. It is found that exposure to prednisone during pregnancy can cause fetal cartilage quality to be low, and can be continued after birth and cause osteoarthritis to be susceptible. Modeling method of the present invention is simple, and in the model, safranin fast green staining, cartilage matrix content, cartilage matrix synthesis and degradation gene expression are stable, and obvious pathological changes of osteoarthritis occur after the second blow (long-distance running). Show that this modeling method is stable, reliable, and highly repeatable.

[0076] [Example 2] circGtdc1 mediates prednisolone-induced reduction in fetal chondrocyte proliferation and extracellular matrix synthesis

[0077] 1 Experimental methods

[0078] 1.1 Circular RNA Sequencing

[0079] Total RNA is extracted from the tissue, and linear RNA is removed using exonucleases such as RNase R to retain circular RNA. After preliminary quantification by Nanodrop, specific oligonucleotide primers are used to separate circular RNA from other RNA and then transcribe it into cDNA. Circular RNA is transcribed into cDNA using reverse transcriptase, and then the cDNA is amplified twice to obtain sufficient sample volume. Qubit 2.0 is used for preliminary quantification, and then the insert size of the library is detected using Agilent 2100. After the insert size meets expectations, the effective concentration of the library (the effective concentration of the library is higher than 3nM) is accurately quantified using the qRT-PCR method to ensure the quality of the library. The cDNA fragments are connected to the adapters required for the Illumina sequencing chip and PCR amplified to construct the cDNA library. High-throughput sequencing is performed on the Illumina PE150 platform to obtain sequence information of circular RNA. The sequencing data were quality controlled, low-quality reads were removed, rRNA and tRNA sequences were removed, and the data were aligned to the reference genome and annotated transcript database. Finally, differential expression analysis and functional annotation were performed.

[0080] 1.2 RNase R and actinomycin experiments

[0081] For RNase R experiments, total RNA was extracted from samples using TRIzol or other RNA extraction reagents. A portion of the RNA was digested with RNase R to remove linear RNA. To an RNase R reaction system, 10 μg of total RNA and an appropriate amount of RNase R (according to the manufacturer's recommendations) were added, and the reaction was performed (typically at 37°C for 1 hour). A portion of RNA was reserved as a linear RNA control and not treated with RNase R. After treatment, the expression of circGtdc1 and mGtdc1 in the samples was assessed by RT-qPCR. For the actinomycin experiment, tissue RNA was extracted after cells were treated with actinomycin for 0, 4, 8, 12, and 24 hours, and the expression of circGtdc1 and mGtdc1 in the samples was assessed by RT-qPCR.

[0082] 1.3 Cell culture

[0083] Primary cartilage chondrocytes were extracted from the cartilage tissue of rats on GD20. Briefly, fresh rat joints were excised, the skin and muscle were removed, and after washing with sterile PBS, the cartilage was cut into small pieces with a sterile scalpel and digested with 0.25% collagenase solution for 6-8 hours. The cell suspension was then centrifuged with culture medium (such as DMEM / F12) and the supernatant was removed. New culture medium was added and the operation was repeated to completely disperse the cells. The cell suspension was centrifuged and resuspended with culture medium containing 10% fetal bovine serum and double antibody. The suspension was transferred to a sterile culture dish and placed in a constant temperature incubator at 37°C and 5% CO2 for cell culture. After the amplified cells were treated with different concentrations of prednisone or prednisolone (0, 10, 50 and 250nM) for 48 hours, the relevant indicators were analyzed according to the experimental design.

[0084] 1.4 Real-time quantitative polymerase chain reaction (RT-qPCR) as described in Experimental Method 1.3 of Example 1

[0085] 1.5 Nuclear and cytoplasmic RNA isolation experiment

[0086] Nuclear and cytoplasmic RNA were isolated using the Norgen Biotek Cytoplasmic & Nuclear RNA Purification Kit (Cat. 2100). Following the initial protocol, nuclear and cytoplasmic fractions were prepared. Cytoplasmic and nuclear RNA were then bound to separate columns. After washing the columns, the nuclear and cytoplasmic RNA were eluted separately with RNA elution buffer. The extracted RNA was reverse transcribed and analyzed by RT-qPCR.

[0087] 1.6 RNA interference and overexpression experiments

[0088] Three siRNAs targeting circGtdc1 were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. Briefly, cells were seeded into 6-well plates and, when approximately 70% of cells were grown, 5 μl of siRNA and lip3000 were resuspended in 125 μl of serum-free medium per well. After 5 minutes, the two were mixed and incubated for 15 minutes. The plates were then added to the 6-well plates, and the volume was replenished to 2 ml with serum-free medium. For overexpression experiments, a circGtdc1 overexpression plasmid was synthesized by Ruibo. When cells reached approximately 70% of cells in the 6-well plates, 5 μl of siRNA, P3000, and lip3000 were resuspended in 125 μl of serum-free medium per well. After 5 minutes, the two were mixed and incubated for 15 minutes. The plates were then added to the 6-well plates, and the volume was replenished to 2 ml with serum-free medium. After 8 hours of treatment, the cells were cultured in serum-free medium and other treatments were performed. After 72 hours, changes in relevant indicators were measured according to the experimental design.

[0089] 1.7 EdU cell and immunofluorescence experiments

[0090] Meilun Biotech's EdU kit (MA0425) was used for cell experiments. After cell treatment, 20 μM EdU reagent was added to a 6-well plate. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. After three washes with PBS, click solution was added in the dark. The cells were then observed using a fluorescence microscope. For immunofluorescence, the cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. After blocking with BSA for 1 hour, primary antibodies against Acan (13880-1-AP, Proteintech) and Col2a1 (ab34712, Abclonal) were added at 1:200 dilution overnight. The next day, the cells were washed three times with PBS and incubated with secondary antibodies for 1 hour. After three washes with TBST, DAPI was added. Finally, the corresponding indicators were observed using a confocal microscope. Three samples were collected for each group, and six fields of view were randomly selected for each sample. Image J was used for EdU count and immunofluorescence gray value statistics.

[0091] 1.8 Western Blotting Experiment

[0092] After protein extraction, protein concentration was measured using the BCA assay. Proteins were mixed with SDS to impart a uniform negative charge and then placed on a polyacrylamide gel. Under the influence of an electric field, proteins separated into distinct bands based on size and charge. The separated proteins were transferred to a PVDF or NC membrane. Nonspecific binding sites were blocked by adding 5% skim milk powder or 3% BSA to the membrane, and the membrane was gently rocked for 1 hour at room temperature. Specific primary antibodies against Acan (13880-1-AP, Proteintech), Col2a1 (ab34712, Abclonal), and Gapdh (AC001, Abclonal) were added at a 1:200 ratio and allowed to bind to the target proteins, followed by overnight incubation. After washing with TBST to remove unbound primary antibody, an HRP-conjugated secondary antibody was added and allowed to bind to the primary antibody, followed by incubation for 1 hour. Unbound secondary antibody was removed by washing with TBST. Target proteins were visualized using chemiluminescence or staining (n = 3 per group). Image J was used for grayscale analysis.

[0093] 2 Experimental results

[0094] 2.1 Pregnancy-related prednisone exposure leads to decreased expression of circGtdc1

[0095] To further explore the mechanism by which gestational prednisone exposure leads to poor cartilage quality and susceptibility to osteoarthritis, the present invention performed circRNA sequencing on cartilage tissue. The results showed that gestational prednisone exposure led to the upregulation of 37 circRNAs and the downregulation of 35 circRNAs, of which 83% were exon-derived circRNAs, 4% were intron-derived, and 5% were intergenic circRNAs ( Figure 3 The present invention selected 15 circRNAs with the most significant increase and decrease, and further PCR verification on tissues showed that circGtdc1 had the most significant change fold, and this change could last until after birth (P<0.05, P<0.01, P<0.001, Figure 3 C, D). Sanger sequencing showed that circGtdc1 was connected end to end ( Figure 3 Northern blot showed that both cDNA and gDNA could amplify linear Gtdc1 RNA (mGtdc1), while only cDNA could amplify circGtdc1 ( Figure 3 RNase R and actinomycin experiments also showed that circGtdc1 had higher stability (P<0.01, P<0.001, Figure 3 Nuclear-cytoplasmic fractionation experiments further showed that circGtdc1 was mainly located in the nucleus ( Figure 3The present invention further interfered with circGtdc1 and found that the expression of Acan and Col2a1 mRNA in fetal chondrocytes was significantly reduced after siRNA interference (P<0.01, Figure 3 EdU results showed that the proliferation of fetal chondrocytes was also significantly reduced after siRNA interference (P<0.01, Figure 3 These results suggest that decreased circGtdc1 expression may mediate chondrodysplasia induced by prednisone exposure during pregnancy.

[0096] 2.2circGtdc1 mediates the reduction of fetal chondrocyte proliferation and extracellular matrix synthesis induced by prednisolone

[0097] After exposure to prednisone, there are two main forms of prednisone and prednisolone in the body. Therefore, the present invention treats cells with prednisone or prednisolone respectively. The results show that prednisone has no significant effect on Acan and Col2a1 mRNA and protein expression, chondrocyte proliferation and matrix content ( Figure 4 Prednisolone significantly reduced chondrocyte proliferation, decreased extracellular matrix content, and decreased Acan and Col2a1 mRNA and protein expressions (P<0.01, P<0.001, Figure 4 At the same time, the present invention detected the effect of prednisolone on the expression of circGtdc1 in chondrocytes. The results showed that prednisolone could reduce the expression of circGtdc1 in a concentration-dependent manner, while its receptor inhibitor RU486 could reverse the above changes (P<0.05, P<0.01, Figure 4 Furthermore, the present invention overexpressed circGtdc1 and combined it with prednisone to treat fetal chondrocytes, and confirmed that the circGtdc1 overexpression plasmid pLV-ciR-Gtdc1 could partially reverse the effects of prednisolone (P<0.05, P<0.01, P<0.001, Figure 4 In conclusion, prednisolone, but not prednisone, mediates the reduced cartilage quality induced by prednisone exposure during pregnancy, while circGtdc1 mediates the prednisolone-induced impairment of chondrocyte proliferation and matrix synthesis.

[0098] This study found that prednisolone exposure during pregnancy reduced expression of an unidentified circGtdc1 gene, which persisted after birth. Sanger sequencing identified a reverse cleavage site in circGtdc1, and RNase R and actinomycin assays demonstrated its circularization. Knockdown of circGtdc1 impaired chondrocyte proliferation and matrix synthesis, while overexpression of circGtdc1 in vitro reversed the prednisolone-induced impairments in chondrocyte proliferation and matrix synthesis.

[0099] [Example 3] circGtdc1 can serve as an intervention target for poor cartilage quality caused by prednisone exposure during pregnancy

[0100] 1 Experimental methods

[0101] 1.1 In vivo circRNA intervention and in vitro live imaging experiments

[0102] The adeno-associated virus-packaged circGtdc1 plasmid was provided by Heyuan Biopharmaceuticals. Prednisone-exposed animals were housed as described above until PW8, at which time empty vector and AAV-circGtdc1 were injected into the joint cavity. On PW12, the animals were euthanized and their knee joints were harvested. Eight specimens were frozen at -80°C for genetic analysis, and five specimens were fixed with 4% paraformaldehyde for histological analysis. The remaining specimens were examined for circGtdc1 expression using a small animal live imaging system.

[0103] 1.2 Safranin fast green staining and OARSI scoring are as in [Example 1] Experimental method 1.2

[0104] 1.3 Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as described in Experimental Method 1.3 of Example 1

[0105] 1.4 Immunohistochemical staining as in [Example 1] Experimental method 1.4

[0106] 2 Experimental results

[0107] circGtdc1 reverses fetal achondroplasia induced by prednisone exposure during pregnancy in vivo

[0108] Finally, the present invention uses adeno-associated virus to test whether overexpression of circGtdc1 improves the poor cartilage quality caused by prednisone exposure during pregnancy. First, in vivo imaging showed that circGtdc1 overexpression was successful ( Figure 5 Safranin fast green results showed that overexpression of circGtdc1 reversed the lighter cartilage staining, thinner cartilage thickness and increased osteoarthritis OARSI score in offspring rats induced by prednisone exposure during pregnancy (P<0.05, P<0.01, P<0.001, Figure 5 RT-qPCR and immunohistochemistry further demonstrated that overexpression of circGtdc1 reversed the decreased expression of Acan and Col2a1 mRNA and protein in cartilage of offspring rats induced by prednisone exposure during pregnancy (P<0.05, P<0.01, P<0.001, Figure 5These results indicate that overexpression of circGtdc1 in vivo can improve the poor cartilage quality induced by prednisone exposure during pregnancy.

[0109] In summary, this study demonstrates that circGtdc1 downregulation, through prednisolone-induced impairment of chondrocyte proliferation and matrix synthesis, contributes to the poor cartilage quality and susceptibility to osteoarthritis in offspring following prenatal prednisolone exposure. In vitro overexpression of circGtdc1 significantly reverses the poor cartilage quality induced by prenatal prednisolone exposure in offspring, suggesting that circGtdc1 may serve as an intervention target for fetal chondrodysplasia and adult osteoarthritis susceptibility.

Claims

1. Use of circGtdc1 in the preparation of a drug for ameliorating fetal adult osteoarthritis caused by prednisone exposure during pregnancy, characterized by: circGtdc1 is a newly identified circular RNA molecule; its expression is reduced in offspring cartilage, leading to chondrodysplasia and an increased likelihood of osteoarthritis in adulthood; The circGtdc1 is used as an intervention target, and the drug for improving fetal osteoarthritis is a circGtdc1 gene mimetic; The circGtdc1 gene mimic is a circRNA overexpression plasmid; The circRNA overexpression plasmid is pLV-ciR-circGtdc1 or AAV-circGtdc1.

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