CN116814539A - Construction method of fetal osteoarthritis model caused by prednisone exposure during pregnancy, intervention target and application of intervention target - Google Patents
Construction method of fetal osteoarthritis model caused by prednisone exposure during pregnancy, intervention target and application of intervention target Download PDFInfo
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- CN116814539A CN116814539A CN202310766443.0A CN202310766443A CN116814539A CN 116814539 A CN116814539 A CN 116814539A CN 202310766443 A CN202310766443 A CN 202310766443A CN 116814539 A CN116814539 A CN 116814539A
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Abstract
The invention discloses a construction method, an intervention target and application of a fetal osteoarthritis model caused by prednisone exposure during pregnancy. circGtdc1 is a newly identified circRNA, which changes most significantly in dysplastic cartilage caused by prednisone exposure during pregnancy. RNase R, acD and Sanger sequencing confirmed their cyclization. Prednisolone concentration-dependent decreases circGtdc1 expression in chondrocytes. Silencing circGtdc1 can obviously inhibit chondrocyte proliferation and matrix synthesis gene expression, while overexpression of circGtdc1 has opposite effects and can reverse the effect of prednisolone. The injection of AAV-circGtdc1 over-expression circGtdc1 into the joint cavity can reverse the cartilage dysplasia caused by prednisone exposure during pregnancy. The intervention target established by the invention is novel, reliable and simple, and provides possibility for early prevention and treatment of fetal osteoarthritis.
Description
Technical Field
The invention relates to the technical field of gene functions and applications, in particular to a construction method, an intervention target and applications of a fetal osteoarthritis model caused by prednisone exposure during pregnancy.
Background
Osteoarthritis (osteoarthritis) is the leading cause of joint pain and chronic disability in the elderly, with its prevalence increasing with age. Currently, about 3.6 hundred million people are worldwide suffering from osteoarthritis; wherein, the incidence rate of osteoarthritis in people over 60 years old in China is over 50 percent, and the incidence rate of osteoarthritis in people over 70 years old is even over 80 percent, which is far over the incidence rate of cardiovascular diseases. As china enters an aging society, an increasing number of osteoarthritis patients create huge health economy and medical burden to the country. However, the pathogenesis of osteoarthritis is not completely understood. In the 90 s of the last century, barker found an increase in the incidence of coronary heart disease, hypertension, hyperlipidemia, obesity, etc. in low birth weight infants after adulthood based on the results of extensive epidemiological investigation, and proposed the theory of intrauterine origin of adult disease based thereon [1]. As research proceeds, more and more epidemiological evidence suggests that adult osteoarthritis also has an intrauterine origin. In 2011, aigner and Richter proposed that age-related osteoarthritis might be associated with fetal cartilage development [2]. Subsequently, sayer and Jordan also found, through epidemiological investigation, that low birth weight infants developed a higher proportion of hand and lumbar osteoarthritis. The early stage of the room is based on a series of animal experiments, and the early stage of the room proves that exposure and ingestion limitation of various exogenous matters (caffeine, nicotine, ethanol and the like) during pregnancy can induce low quality of offspring cartilage and susceptibility of adult osteoarthritis. In conclusion, osteoarthritis has fetal origin. The intrauterine origin process and the occurrence mechanism of the osteoarthritis are explored, and an experimental basis is provided for further treatment of the osteoarthritis.
Chondrocytes and their secreted extracellular matrix are the main components of articular cartilage, and their content reflects the quality of articular cartilage. Cartilage destruction, reduced chondrocyte numbers, and reduced extracellular matrix content are typical pathological features of osteoarthritis. Under physiological conditions, proliferation and apoptosis of chondrocytes, and synthesis and degradation of extracellular matrix are in equilibrium. When this balance is broken, i.e., the number of chondrocytes is reduced or the extracellular matrix content is reduced, the quality of cartilage is lowered. Related studies have also shown that the quality of articular cartilage is closely related to the occurrence and progression of osteoarthritis [3]. The biomechanical changes in articular cartilage lead to low cartilage quality, mediating the onset of osteoarthritis [4]. The present laboratory series of studies also demonstrate that poor conditions during pregnancy (such as exogenous exposure and ingestion limitation) can lead to poor quality of offspring cartilage and susceptibility to osteoarthritis in adulthood. Since mammalian and rodent articular cartilage is mainly formed in embryonic stages, the cell number and relative thickness of articular cartilage gradually decrease with age after birth, and cartilage lacks sufficient vascularization, and the ability to self-repair after injury is extremely poor. Thus, cartilage development during the period of intrauterine embryos has an important determinant of cartilage quality after adulthood and may lead to the development and progression of osteoarthritis after adulthood.
Circular RNAs (circrnas) are a class of closed-loop RNA molecules that are joined end-to-end to form closed-loop RNA molecules that do not have 5 'and 3' end structures, which make their structure very stable and not easily degraded by exonuclease RNase R. Thus, circRNA plays a central role in the development and pathology of tissues and organs. For example, it has been demonstrated that a plurality of circrnas (e.g., circHIPK3, circ_atrnl1, etc.) are involved in chondrogenic differentiation and chondrocyte proliferation of bone marrow mesenchymal stem cells, thereby mediating the occurrence, progression, of osteoarthritis [5,6]; multiple circrnas are involved in skeletal muscle and intestinal development [7,8]. However, the effect of circRNA on cartilage development has not been reported. Intrauterine programming changes (intrauterine programming alteration) refer to the process of permanent changes in tissue morphology and function resulting from early lesions in the development of the body, and changes in the function of these tissues and organs or in the pattern of gene expression are usually maintained from the development stage to adulthood, even throughout life, leading to a series of effects in the body after adulthood [9]. Studies have shown that a variety of non-coding RNAs program the effects of intrauterine environment on offspring health. For example, miRNAs regulate the effects of maternal pregnancy exercise on liver glycolipid metabolism in adult offspring [10]; long-chain non-coding RNA H19 mediates progeny adrenal dysplasia and its trans-generation genetic effects due to pregnancy caffeine exposure [11]; miRNA-92-3p, miRNA-200b-3p and the like also mediate the sub-cartilage dysplasia caused by the pregnancy bad environment [12,13]. As one of non-coding RNA, the development of cartilage in the programming progeny of circRNA and susceptibility to osteoarthritis in the adult stage have not been reported yet and are yet to be studied further.
Reference is made to:
1.Barker,D.J.,The origins of the developmental origins theory.J Intern Med,2007.261(5):p.412-7.
2.Aigner,T.and W.Richter,OA in 2011:Age-related OA--a concept emerging from infancyNat Rev Rheumatol,2012.8(2):p.70-2.
3.Dahlberg,L.,Cartilage quality,overweight and osteoarthritis:a case for new behaviourAnn Rheum Dis,2012.71(1):p.1-3.
4.Pitsillides,A.A.and F.Beier,Cartilage biology in osteoarthritis--lessons from developmental biology.Nat Rev Rheumatol,2011.7(11):p.654-63.
5.Li,S.,et al.,Mesenchymal stem cell-derived extracellular vesicles prevent the development of osteoarthritis via the circHIPK3/miR-124-3p/MYH9 axis.J Nanobiotechnology,2021.19(1):p.194.
6.Zheng,J.,et al.,Promotive role of CircATRNL1 on chondrogenic differentiation of BMSCs mediated by miR-338-3p.Arch Med Res,2021.52(5):p.514-522.
7.Wu,P.,et al.,Identification of crucial circRNAs in skeletal muscle during chicken embryonic development.BMC Genomics,2022.23(1):p.330.
8.Li,S.Y.,et al.,RNA-Seq profiling of circular rnas during development of hindgut in rat embryos with ethylenethiourea-induced anorectal malformations.Front Genet,2021.12:p.605015.
9.Meaney,M.J.,M.Szyf,and J.R.Seckl,Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health.Trends Mol Med,2007.13(7):p.269-77.
10.Zhou,L.,et al.,Maternal exercise programs glucose and lipid metabolism and modulates hepatic mirnas in adult male offspring.Front Nutr,2022.9:p.853197.
11.He,Z.,et al.,H19/let-7axis mediates caffeine exposure during pregnancy induced adrenal dysfunction and its multi-generation inheritance.Sci Total Environ,2021.792:p.148440.
12.Tie,K.,et al.,Low miR-92a-3p in oocytes mediates the multigenerational and transgenerational inheritance of poor cartilage quality in rat induced by prenatal dexamethasone exposure.Biochem Pharmacol,2022.203:p.115196.
13.Ni,Q.,et al.,miR-200b-3p/ERG/PTHrP axis mediates the inhibitory effect of ethanol on the differentiation of fetal cartilage into articular cartilage.Chem Biol Interact,2022.368:p.110201.
disclosure of Invention
In order to solve the problems and the shortcomings, the invention aims to determine the interrelation between the change of the expression of the circGtdc1 and the fetal cartilage dysplasia and osteoarthritis, determine the circGtdc1 as an intervention target of the fetal adult osteoarthritis, provide the application of the circGtdc1 as the intervention target in screening medicines for improving the fetal cartilage dysplasia and the adult osteoarthritis, and the application of the circGtdc1 analogue in preparing medicines for improving the fetal adult osteoarthritis.
The above purpose is achieved by the following technical scheme:
in a first aspect, the present invention provides a method for constructing a model of fetal osteoarthritis caused by prednisone exposure during pregnancy, which is characterized in that: the method comprises the following steps:
s1: normal pregnant rats were given a gastric lavage of prednisone of 0.125, 0.25mg/kg daily for 0-20 days of gestation;
s2: regulating each nest to 14 mice in the first day after the pregnant mice are naturally produced, wherein the sex ratio is 1:1; weaning 4 weeks after birth, and freely obtaining edible water;
s4: running treatment 24-28 weeks after birth;
s5: the method comprises the steps of gestating for 20 days, acquiring articular cartilage tissue of the rat at 12 weeks and 28 weeks after birth; evaluating the quality of knee cartilage, and detecting the change of related indexes of cartilage matrix synthesis and degradation;
the rats in the step S1 are Wistar rats; prednisone is a clinically used prednisone tablet;
the running conditions in the step S3 are as follows: day 1, 10min, speed 10m/min; day 2, 15min, speed 12m/min; day 3, 20min, speed 15m/min; day 4, 30min, speed 18m/min; day 5, 35min, speed 20m/min. On and after day 5, the first 10min includes 12m/min, followed by running at a speed of 20m/min for 55 minutes per day.
The quality of the cartilage in the step S4 comprises the solid green dyeing degree of safranin; cartilage proportion in proliferation area and hypertrophy area of the cartilage tissue of the fetal mouse; cartilage surface smoothness, reduced cartilage thickness, whether cartilage structure is disordered, whether the line is interrupted for hours, and osteoarthritis OARSI scores; detecting mRNA and protein expression of cartilage matrix synthesis indexes including Sox9, acan and Col2a 1; cartilage matrix synthesis indicators include mRNA and protein expression of mp3, mp13, and adamps 5.
In a second aspect, the present invention provides an intervention target for fetal adult osteoarthritis, characterized in that: the intervention target is circGtdc1 in rat cartilage; overexpression of circGtdc1 in vivo can improve cartilage quality deterioration caused by prednisone exposure during pregnancy.
In a third aspect, the invention provides an application of the intervention target (circGtdc 1) in screening medicines for fetal adult osteoarthritis or fetal adult chondrodysplasia or fetal adult osteoporosis.
Preferably, the circGtdc1 is a newly identified circular RNA molecule; the expression of circGtdc1 is reduced in the progeny cartilage, resulting in an increased likelihood of developing its cartilage dysplasia and osteoarthritis in adulthood.
The circGtdc1 is used as an intervention target, and the medicine for improving the fetal osteoarthritis comprises a circGtdc1 gene mimic.
The circGtdc1 gene mimetic is a circRNA over-expression plasmid.
The circRNA overexpression plasmid is pLV-ciR-circGtdc1 or AAV-circGtdc1.
The technical principle and the research process of the invention are as follows:
the invention constructs a model for susceptibility of fetal cartilage dysplasia and osteoarthritis through pregnancy exogenous (prednisone) exposure. In this fetal model, it was found that the progeny had reduced expression of the gene associated with cartilage proliferation (Ki 67) 20 days of gestation and 12 weeks after birth, the safranine staining was shallowed, the mRNA and protein expression of the chondrocyte matrix synthesis genes (Col 2a1 and Acan) was reduced, and that the prednisone-exposed group had a more pronounced osteoarthritis phenotype (e.g. safranine-fast green showed a shallower staining, cartilage surface damage more pronounced, and osteoarthritis RSI score higher) after 24 to 28 weeks post-natal second hit (long run). Further, by RNA sequencing, prednisone exposure during pregnancy resulted in unbalanced expression of circular RNA, up-regulation of 37 circRNAs, down-regulation of 35 circRNAs, of which 83% was of exon-derived circRNA,4% was of intron-derived, and 5% was of intergenic-derived circRNA. The most obvious 15 circRNAs are increased and decreased by each selection, and further the PCR verification shows that the circGtdc1 change multiple is most obvious on tissues. Further RT-qPCR verification shows that the reduction of circGtdc1 is most pronounced. RNase R and AcD results show that the circGtdc1 has more obvious stability than the linear mGtdc1, and Sanger sequencing proves the ring formation phenomenon. The above results indicate that prednisone exposure during pregnancy leads to dysplasia of the cartilage, the mechanism of which may be associated with reduced expression of circGtdc1.
Meanwhile, the invention proves that prednisolone (but not prednisone) inhibits proliferation of chondrocytes (EdU shows that the number of positive cells is reduced) and mRNA and protein expression of matrix synthesis genes (Col 2a1 and Acan) at the cellular level, and has good concentration dependence, and has no obvious influence on expression of cartilage matrix degradation related genes (MMP 13 and ADAMTS 5) and cartilage apoptosis. On fetal chondrocytes, prednisolone may decrease circGtdc1 expression in a concentration-dependent manner, while glucocorticoid receptor (glucocorticoid receptor, GR) inhibitor RU486 (Sigma-Aldrich, cat#M8046) may reverse the above. Taken together, prednisolone is suggested to inhibit the expression of circGtdc1 by GR.
Furthermore, the present invention indicates that pLC-ciR-Gtdc1 promotes chondrocyte proliferation and increases matrix synthesis gene (Col 2a1 and Acan) mRNA and protein expression when the circGtdc1 over-expression plasmid (pLC-ciR-Gtdc 1) or siRNA (siRNA-circGtdc 1) is administered to fetal chondrocytes, whereas siRNA-circGtdc1 has the opposite effect. In addition, pLC-ciR-Gtdc1 and prednisolone are combined to treat the fetal chondrocytes, so that pLC-ciR-Gtdc1 can reverse the reduction of chondrocyte proliferation and the reduction of mRNA and protein expression of matrix synthesis genes (Col 2a1 and Acan) caused by prednisolone. The above results indicate that circGtdc1 mediates chondrocyte proliferation inhibition and reduced extracellular matrix synthesis by prednisolone.
Finally, the invention utilizes an animal model of the cartilage dysplasia of a offspring rat caused by the exposure of prednisone during pregnancy to inject an adeno-associated virus (AAV) -entrapped circGtdc1 over-expression plasmid (AAV-circGtdc 1) into the joint cavity 8-12 weeks after birth. The results indicate that AAV-circGtdc1 can reverse reduced red-green staining of cartilage from prednisone during pregnancy, increased RSI scores for osteoarthritis, and increased mRNA and protein expression of matrix synthesis genes (Col 2a1 and Acan).
In conclusion, the circGtdc1 can be used as an intervention target for child-derived cartilage dysplasia and susceptibility to adult osteoarthritis caused by prednisone exposure in pregnancy, and provides a new approach for treating the child-derived osteoarthritis.
Compared with the prior art, the invention has the following advantages and effects:
1. the present invention discovers and identifies a novel circular RNA, namely, circGtdc1.
2. The invention discovers that circGtdc1 mediates the low quality of offspring cartilage and susceptibility to osteoarthritis after adulthood caused by prednisone exposure during pregnancy.
3. Based on the application of the circGtdc1 in the fetal adult osteoarthritis caused by the prednisone during pregnancy, a target is provided for developing the medicine for treating the fetal osteoarthritis.
4. The invention finds that the joint cavity injection circGtdc1 mimic AAV-circGtdc1 can be used for preparing a therapeutic drug for treating fetal osteoarthritis.
Drawings
Figure 1. Prednisone exposure during pregnancy resulted in poor cartilage quality in offspring fetal mice.
In fig. 1: (A) Fetal mouse knee safranin fast green staining (R, C and H represent cartilage circles, columns and mast cell layers, respectively), scale = 500 μm, n = 5; (B) Quantitative statistics of mean optical density of safranin fast green staining (Mean optical density, MOD), n=5; (C-E) cartilage round, columnar and mast cell layer area statistics, n=5; (F) RT-qPCR detects fetal cartilage tissue Col2a1, acan, mp3, mp13 and adams 5 mRNA expression, n=12; (G-H) immunohistochemical detection of Acan and Col2a1 protein expression, scale = 300 μm, n = 5; (I) Immunohistochemical staining average optical density (MOD) quantitative statistics, n=5. Results are expressed as mean ± standard error. In comparison with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001。
figure 2. Prednisone during pregnancy exposing adult offspring with low cartilage quality and susceptibility to osteoarthritis.
In fig. 2: (A) Postnatal day 12 rats were stained for safranin and green, scale = 1000 μm, n = 5; (B) Safranin fast green staining average optical density (MOD) quantitative statistics and osteoarthritis RSI score, n=5; (C) RT-qPCR detects fetal cartilage tissue Col2a1, acan, mp3, mp13 and adams 5 mRNA expression, n=12; (D) Immunofluorescence and histochemical analysis Col2, acan and mmap 13 protein expression, fluorescence scale = 100 μm, histochemical scale = 50 μm, n = 5; (E-G) Col2, acan and mmap 13 protein expression quantitative statistics, n=5×6; (H-I) safranin-fast green staining and Mean Optical Density (MOD) quantitative analysis of articular cartilage of 28 weeks postnatal rats, scale = 1000 μm, n = 5; (J) Postnatal 28 week ratsThe articular cartilage osteoarthritis RSI score, n=5; (L) immunohistochemical detection of Col2 and mmap 13 protein expression, scale = 50 μm; (M) Col2 and mmap 13 proteins expressed average optical density (MOD) quantitative analysis, n=5×6. Results are expressed as mean ± standard error. In comparison with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001。
fig. 3, prednisone exposure during pregnancy resulted in reduced expression of circGtdc1.
In fig. 3: (a) volcanic plot represents circular RNA sequencing expression; (B) differential circular RNA gene profile; (C) RT-qPCR verifies the most variable circular RNA in sequencing results, n=12; (D) RT-qPCR detects the expression of circGtdc1, circRab31 and circMlip in postnatal 12 week cartilage tissue, n=12; (E) Sanger sequencing verifies that circGtdc1 loops; (F) Northern blot detection of circGtdc1 and mgdc 1 expression; (G) the expression of circGtdc1 and mgdc 1 after Rnase R treatment, n=3; (H) The expression of circGtdc1 and mGtdc1 after actinomycin treatment, n=3; (I) circGtdc1 nuclear mass distribution, n=3; (J) RT-qPCR detects Acan and Col2a1mRNA expression, n=3; (K) EdU detection chondrocyte proliferation and quantitative statistics, scale = 500 μm, n = 3. Results are expressed as mean ± standard error. In comparison with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001。
figure 4.Circgtdc1 mediated decrease in proliferation of chondrocytes in fetoprotein and decrease in extracellular matrix synthesis by prednisolone.
In fig. 4: (A) Detecting the expression of an and Col2a1 in the chondrocytes of the fetus after the prednisolone treatment by RT-qPCR, wherein n=6; (B-C) EdU detection chondrocyte proliferation and quantitative statistics, scale = 500 μm, n = 3; (D) Safranin assay chondrocyte matrix content and average optical density quantitative statistics, scale = 500 μm, n = 6; (F) Immunofluorescence detection of the expression of the Acan and Col2 proteins after prednisone treatment of the chondrocytes, scale = 25 μm, n = 6; (G) EdU detects chondrocyte proliferation and quantitative statistics, n=3; (H-I) safranin assay chondrocyte matrix content and average optical density quantitative statistics, scale = 500 μm, n = 6; (J) RT-qPCR detects the expression of Acan and Col2a1 in strong-fetoprotein chondrocytes, n=3; (K-L) Western blot detection of Acan and Col2 protein expression and quantitative statistics, n=3;(M) RT-qPCR detection of circGtdc1 expression in prednisolone treated fetal chondrocytes, n=6. Results are expressed as mean ± standard error. In comparison with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001。
fig. 5.Circgtdc1 reverses in vivo fetal cartilage dysplasia caused by prednisone exposure during pregnancy.
In fig. 5: (A) A mode diagram and a living body imaging diagram of the cartilage dysplasia of a offspring rat caused by the prednisone exposure during the intervention of the injection of the adeno-associated virus-entrapped circGtdc1 into the joint cavity; (B) Safranin solid green staining of articular cartilage, scale = 50 μm, n = 5; (C, D) quantitative analysis of the RSI score for osteoarthritis and the mean optical density of safranin fast green staining, n=5×6; (E, F) RT-qPCR to detect Acan and Col2a1mRNA expression in cartilage tissue, n=8; (G-I) immunohistochemical detection of articular cartilage Col2 and Acan protein expression and quantitative analysis, scale = 50 μm, n = 5 x 6. Results are expressed as mean ± standard error. In comparison with the respective normal control group, * P<0.05, ** P<0.01, *** P<0.001。
Detailed Description
The above-described aspects of the present invention will be described in further detail by way of specific embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. The technology realized based on the above description of the present invention belongs to the content of the present invention.
All reagents involved in the experiment in the invention can be purchased in domestic and foreign markets or prepared by themselves according to the formula in the specification; experimental methods not specifically described are all conventional methods known in the art.
Example 1 establishment of a model for susceptibility to fetal-derived osteoarthritis
1 Experimental method
1.1 laboratory animals and treatments
The present invention purchased pathogen free (SPF) healthy Wistar rats, animal license number from s Bei Fu (beijing) biotechnology limited: SCXK (Beijing) 2019-0010. All animal protocols involved in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the university of martial arts animal care center (IACUC No. wp20210060). After 7 days of adaptive feeding, all rats were female 18 hours a night: male = 2:1 cage, vaginal secretion objective was taken the next morning and sperm was found to be day 0 (gd0) of gestation. The pregnant mice were respectively given 0.125 and 0.25 mg/kg.d of prednisone and the normal control group was given an equivalent amount of sodium carboxymethylcellulose and the stomach was irrigated. After euthanasia of a portion of pregnant mice (n=12) at GD20, the knee joints of the pregnant mice were obtained. 5 fossa was taken, one right knee joint per fossa was fixed in 4% paraformaldehyde for histological analysis, and its knee joint tissue was immediately frozen at-80 ℃ for subsequent additional analysis. One feeding per litter of remaining rats was selected and only after birth, part of the rats acquired knee joints 12 weeks after birth (postnatal week 12, PW 12) and PW 28. The rest rats were harvested after 24-28 weeks of PW running treatment after knee joint acquisition. The knee joint fraction obtained was fixed in 4% paraformaldehyde and the other was immediately frozen and stored at-80 ℃ for further analysis.
1.2 safranin fast green staining and histological scoring
The cartilage tissue is decalcified and embedded and then cut into 4-6 mu m thick slices. Sections were placed on slides, allowed to dry and fixed at 60℃for 15 minutes. Sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I10 min-absolute ethanol II 10min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5 min-distilled water for washing. The sections were then immersed in a solution of Safranin O and allowed to stand at room temperature for 5 minutes. The sections were gently rinsed with water 2 times for 5 seconds each to remove excess water on the filter paper. The slices are immersed in a solid green dye solution of 0.5% for dyeing for 30-60s. The sections were gently rinsed with water 2 times for 5 seconds each to remove excess water on the filter paper. Finally, the sections were covered with Xylene and blocked. Photographs were taken with an optical microscope, 6 fields of view were taken per sample, 5 samples per group (n=5). Finally semi-quantitative statistical analysis was performed with Image J and an international society of osteoarthritis research (Osteoarthritis Research Society International, OARSI) score was performed. OARSI scoring criteria: grade 1.0: the chondrocytes are intact; grade 1.5: cell death, including apoptosis or necrosis; grade 2.0: surface discontinuities include fibrosis; grade 2.5: surface wear and loss of a portion of the superficial cartilage region; grade 3.0: simple fissures or cracks invade the intermediate zone; grade 3.5: the fracture propagates and becomes a bifurcation or complex fracture; grade 4.0: only the superficial cartilage region is lost (eroded); grade 4.5: the middle zone disappears; grade 5.0 (flaking): there is a bone surface consisting of intact calcified cartilage or hardened bone; grade 5.5: reparative fibrocartilage tissue or new bone formation; grade 6.0: the joint geometry of the joint edge is deformed; grade 6.5: both the joint edges and the weight bearing areas show deformation. Osteoarthritis stage assessment was performed based on the level range of affected cartilage surface. Stage 1: the degree of involvement is less than 10%; stage 2: the affected degree is 10-25%; stage 3: the affected degree is 25-50%; stage 4: the degree of involvement is greater than 50%. Final osteoarthritis RSI score = grade score x stage score. The final score was the average of the independent scores of the two studies.
1.3 quantitative polymerase chain reaction (RT-qPCR) of reverse transcription
Total RNA of cartilage tissue and cells was extracted by TRIzol. Briefly, after 1.5ml of enzyme-free EP tube was added to the sample, 200. Mu.l of TRIzol reagent was added, for cartilage tissue, 200. Mu.l of chloroform was added after grinding, and the cell sample was directly added with 200. Mu.l of chloroform. And (5) standing on ice for 10min after reversing and uniformly mixing. After centrifugation at 12000g for 10min, 400. Mu.l was pipetted into a new EP tube, and after mixing with an equal amount of isopropanol, the mixture was allowed to stand at room temperature for 10min. After centrifugation at 12000g for 10min, washing twice with 1ml of 75% ethanol. After drying, enzyme-free water was added. A ratio A260/280 was then determined to be between 1.8 and 2.1 using a NanoDrop 2000 nucleic acid analyzer. And then reverse-transcribed into cDNA using a reverse transcription kit. Then using StepOneGlus TM Real-Time PCR System detects the expression of Acan, col2a1, mmp3, mmp13, adamts5, circGtdc1, mGtdc1, etc. The reaction conditions were as follows: 95 ℃/30s,95 ℃/10s, and 62 ℃/30s (40 cycles). Primer sequences are shown in Table 1 below:
TABLE 1 primer sequences.
1.4 immunohistochemical staining and tissue immunofluorescent staining
After dewaxing of the knee specimens to water, antigen retrieval was performed with sodium citrate buffer and incubation with BSA for 30min. The primary antibodies (Acan, col2a1 and Mmp13 antibodies at 1:200) were then added overnight at 4 ℃. Biotin secondary antibody was added the next day of immunohistochemistry, while fluorescent secondary antibody was added the next day of immunofluorescence. Immunohistochemistry peroxidase activity was detected with DAB. And observing corresponding index changes under an optical microscope and a fluorescence microscope respectively. Passage of dyed images(v.1.52 q) analysis. The level of staining was determined by calculating the average optical density (mean optical density, MOD) or average gray value of the six fields of view for each sample.
2 experimental results
2.1 prednisone Exposure during pregnancy leads to cartilage dysplasia in the fetal mice
Firstly, the invention detects the influence of prednisone exposure during pregnancy on the cartilage quality of a fetal mouse. Safranin fast green results showed that the pregnancies prednisone exposed group safranin fast green staining was shallower (a in fig. 1), the average optical density was reduced (P <0.001, B in fig. 1), the cartilage in the circular and columnar areas was reduced (P <0.01, P <0.001, C, D in fig. 1), and the cartilage in the hypertrophic areas was increased (P <0.001, fig. 1E) with a clear dose dependency, compared to the normal control group. RT-qPCR results showed dose-dependent decrease in expression of Col2a1, acan, mmp3, mmp13 and Adams 5 mRNA in prednisone-exposed group during pregnancy (P <0.05, P <0.01, F in FIG. 1). Immunization further showed that the expression of Col2a1 and Acan proteins was also significantly reduced in the prednisone-exposed group during pregnancy (P <0.001, G-I in FIG. 1). The above results indicate that prednisone exposure during pregnancy induces dyssynthesis of the cartilage matrix, resulting in dysplasia.
2.2 prednisone exposure during pregnancy leads to dysplasia and susceptibility to osteoarthritis in adult offspring rats
Next, the present invention examined the effect of prednisone exposure during pregnancy on postnatal offspring rat cartilage. The results indicate that prednisone exposure during pregnancy resulted in a poor staining of the cartilage safranin, uneven cartilage surface, reduced thickness, disturbed structure and even loss of tidal line in PW12 rats with reduced OARSI scores for osteoarthritis (P <0.01, P <0.001, a, B in fig. 2). RT-qPCR, immunofluorescence and immunohistochemistry further showed that prednisone exposure during pregnancy dose-dependently reduced the Acan and Col2a1mRNA and protein expression, while Mmp13 and Adamts5 mRNA expression and Mmp13 protein expression were increased (P <0.05, P <0.01, P <0.001, C-G in FIG. 2). The above change may even be continued to PW28 (P <0.05, P <0.01, P <0.001, H-M in FIG. 2). And PW24-28 showed more severe reduction in cartilage safranin staining, cartilage surface destruction and higher OARSI scores (P <0.05, P <0.01, P <0.001, G-J in fig. 2) in the prednisone-exposed animals during pregnancy than in the unexposed animals after the second hit (long-distance running). RT-qPCR and immunohistochemistry also showed that prednisone exposure during pregnancy + Cola1 and Acan mRNA and Cola1 protein expression were lower than in the running alone group, while Mmp3, mmp13 and Adamts5 mRNA and Mmp13 protein expression was also higher (P <0.05, P <0.01, P <0.001, K-M in FIG. 2). The results indicate that prednisone exposure during pregnancy leads to poor cartilage quality and susceptibility to osteoarthritis in adult offspring rats.
The invention is characterized in that the Wistar rats are given 0.125 and 0.25mg/kg of prednisone after 0-20 days of pregnancy, and are given a second striking (long-distance running) treatment 24-28 weeks after birth, so that the prednisone exposure during pregnancy can cause low cartilage quality of the rats and can be continued to the postnatal period and cause osteoarthritis susceptibility. The method for modeling is simple, safranin and fast green dyeing is carried out in the model, cartilage matrix content, cartilage matrix synthesis and degradation gene expression are stable, and obvious pathological changes of osteoarthritis appear after secondary striking (long-distance running). The modeling method is stable and reliable and has strong repeatability.
Example 2circGtdc1 mediates decreased proliferation of chondrocytes and decreased synthesis of extracellular matrix of prednisolone fetoprotein
1 Experimental method
1.1 circular RNA sequencing
After total RNA is extracted from tissues and linear RNA is removed by using exoenzyme such as RNase R to reserve circular RNA, the circular RNA is separated from other RNA by using specific oligonucleotide primer after preliminary quantification of Nanodrop, and then transcribed into cDNA. The circular RNA is transcribed into cDNA using reverse transcriptase, and then the cDNA is secondarily amplified to obtain a sufficient sample size. Qubit 2.0 was initially quantified, then the Agilent 2100 was used to detect the Insert size of the library, and after the Insert size was expected, the effective concentration of the library (library effective concentration higher than 3 nM) was accurately quantified using qRT-PCR to ensure library quality. The cDNA fragments were ligated to the adapters required for the Illumina sequencing chip and PCR amplified to construct a cDNA library. High throughput sequencing was performed on Illumina PE150 platform to obtain sequence information for circular RNAs. Quality control of the sequencing data, removal of low quality reads, removal of rRNA and tRNA sequences, alignment to a reference genome and annotation transcript database, and finally differential expression analysis, functional annotation and other analysis.
1.2RNase R and actinomycin assay
For RNase R experiments, TRIzol or other RNA extraction reagents were used to extract total RNA from the samples. A portion of the RNA was digested by RNase R to remove linear RNA. To one RNase R reaction system, 10. Mu.g of total RNA and an appropriate amount of RNase R (according to manufacturer's recommendations) were added, followed by a reaction (usually at 37℃for 1 hour). Another portion of RNA was left as a linear RNA control group without RNase R treatment. After the treatment, the expression of circGtdc1 and mGtdc1 in the sample was detected by RT-qPCR. For actinomycin experiments, tissue RNA was extracted after treatment of cells 0, 4, 8, 12 and 24h with actinomycin, and RT-qPCR was performed to detect expression of circGtdc1 and mGtdc1 in the samples.
1.3 cell culture
Primary chondrocytes were extracted from GD20 day rat cartilage tissue. Briefly, fresh rat joints were excised, skin and muscle removed, and after washing with sterile PBS, cartilage was cut into small pieces with a sterile scalpel, and digested with 0.25% collagenase solution for 6-8 hours. The cell suspension is then centrifuged with a medium (e.g., DMEM/F12) and the supernatant removed. New medium was added and the procedure repeated to completely disperse the cells. Centrifuging the cell suspension and using a medium containing 10% fetal bovine serum and a diabodyAnd (5) re-suspending. The suspension was transferred to a sterile petri dish and placed at 37℃in 5% CO 2 In a constant temperature incubator, cell culture was performed. After 48 hours of treatment of the expanded cells with prednisone or prednisolone (0, 10, 50 and 250 nM) at different concentrations, relevant indicators were analyzed according to the experimental design.
1.4 quantitative polymerase chain reaction for reverse transcription (real-time quantitative polymerase chain reaction, RT-qPCR) Experimental method 1.3 as described [ example 1]
1.5 experiments for isolation of nucleoplasmic RNA
The nuclear and Cytoplasmic RNA were isolated using the Cytoplasmic & Nuclear RNA Purification kit (Cat.2100) from Norgen Biotek. According to the procedure, for the first time, the nuclear and cytoplasmic fractions were prepared, then the cytoplasmic and cytoplasmic RNA were bound to different columns, respectively, and after washing the columns, the nuclear and cytoplasmic RNA were washed off, respectively, with RNA eluate. The extracted RNA was reverse transcribed and then subjected to RT-qPCR analysis.
1.6RNA interference and overexpression experiments
Three siRNAs to circGtdc1 were synthesized by Guangzhou Ruibo Biotechnology Co., ltd. Briefly, when cells were grown to about 70% in 6-well plates, 5 μl of siRNA and lip3000 were resuspended in 125 μl of serum-free medium per well, respectively, after 5min the two were mixed and incubated for another 15min, then added to 6-well plates, and the serum-free medium was supplemented to 2ml. For the overexpression experiments, the circGtdc1 overexpression plasmid was synthesized from libo. When cells in the 6-well plate grow to about 70%, 5 μl of siRNA and P3000 and lip3000 are respectively resuspended in 125 μl of serum-free medium per well, mixed for 5min, incubated for 15min, then added to the 6-well plate, and the serum-free medium is supplemented to 2ml. The serum culture medium is changed for 8 hours for culture and other treatments are carried out, and after 72 hours, the change condition of the related index is detected according to the experimental design.
1.7 cell EdU and immunofluorescence experiments
The EdU kit (MA 0425) from the Methanen Biotech company was used for cell experiments. After the cells were treated, 20. Mu.M EdU reagent was added to the 6-well plate, and the cells were fixed with 4% paraformaldehyde for 15min and broken with 0.5% Triton X-100 at room temperature for 10min. After three washes with PBS, click solution was added under dark conditions. And then observed with a fluorescence microscope. For cellular immunofluorescence, after cell treatment, cells were fixed with 4% paraformaldehyde for 15min and membrane rupture with 0.5% Triton X-100 at room temperature for 10min. Then 1h after blocking with BSA, 1:200Acan (13880-1-AP, proteintech) and Col2a1 (ab 34712, abclonal) primary antibodies were added overnight. The following day was followed by three PBS washes followed by 1h incubation of the secondary antibody, three TBST washes followed by DAPI. And finally, observing corresponding indexes by using a confocal microscope. Three samples per group, each sample randomly selecting 6 fields of view. Image J was used for EdU count and immunofluorescence gray value statistics.
1.8Western Blotting experiment
After protein extraction, protein concentration was measured using BCA method. The protein was mixed with SDS to have a uniform negative charge, and then placed in a polyacrylamide gel, and the protein was separated into different bands according to size and charge under the action of an electric field. The isolated proteins were transferred to PVDF or NC membranes. Non-specific binding sites were blocked by adding 5% nonfat milk powder or 3% BSA to the membrane and the membrane was gently shaken at room temperature for 1 hour. Specific 1:200Acan (13880-1-AP, proteintech), col2a1 (ab 34712, abclonal) and Gapdh (AC 001, abclonal) primary antibodies were added and allowed to bind to the target protein and incubated overnight. After removing unbound primary antibody by TBST washing, HRP-labeled secondary antibody was added to bind to primary antibody and incubated for 1 hour. Unbound secondary antibody was removed by washing with TBST. Target proteins were shown using chemiluminescence or staining (n=3 per group). Image J is used for gray value statistics.
2 experimental results
2.1 prednisone exposure during pregnancy leads to reduced expression of circGtdc1
To further explore the mechanism of poor cartilage quality and susceptibility to osteoarthritis caused by prednisone exposure during pregnancy, the invention performed circRNA sequencing on cartilage tissue. The results indicate that prednisone exposure during pregnancy resulted in up-regulation of 37 circrnas, down-regulation of 35 circrnas, of which 83% was of exon-derived circrnas, 4% was of intron-derived, and 5% was of intergenic-derived circrnas (a, B in fig. 3). Further PCR validation on tissue showed that the most significant 15 circrnas per selection of the invention increased and decreased, showed that the fold change in circGtdc1 was most significant and could extend to postnatal (P <0.05, P <0.01, P <0.001, C, D in fig. 3). Sanger sequencing showed that circGtdc1 was end to end (E in FIG. 3); northern blot showed that both cDNA and gDNA amplified linear Gtdc1 RNA (mGtdc 1), whereas only cDNA amplified circGtdc1 (F in FIG. 3). RNase R and actinomycin experiments also showed that circGtdc1 had higher stability (P <0.01, P <0.001, G-H in FIG. 3). The nuclear cytoplasmic isolation experiments further showed that circGtdc1 is located mainly in the nucleus (I in FIG. 3). The present invention further interfered with circGtdc1 and found that mRNA expression was significantly reduced in the following siRNA interference by the chondrocytes of fetuses Acan and Col2a1 (P <0.01, J in FIG. 3). EdU results showed that proliferation of fetal chondrocytes was also significantly reduced after siRNA interference (P <0.01, K in FIG. 3). The above results indicate that reduced expression of circGtdc1 may mediate cartilage dysplasia caused by prednisone exposure during pregnancy.
2.2. 2 circGtdcc 1 mediated decrease in proliferation of fetal chondrocytes and decrease in extracellular matrix synthesis by prednisolone
Prednisone is mainly in two forms, prednisone and prednisolone, in vivo after exposure. Thus, the present invention is directed to the administration of prednisone or prednisolone treatments, respectively, on cells. The results showed that prednisone had no significant effect on the Acan and Col2a1mRNA and protein expression, chondrocyte proliferation and matrix content (a-D in fig. 4). Prednisolone significantly reduces chondrocyte proliferation, reduces extracellular matrix content, and reduces both Acan and Col2a1mRNA and protein expression (P <0.01, P <0.001, G-L in FIG. 4). Meanwhile, the invention detects the influence of prednisolone on the expression of chondrocyte circGtdc1. The results indicate that prednisolone can reduce the expression of circGtdc1 in a concentration-dependent manner, while its receptor inhibitor RU486 can reverse the above-mentioned changes (P <0.05, P <0.01, M in FIG. 4). Further, it was confirmed that the circGtdc1 overexpression plasmid pLV-ciR-Gtdc1 could partially reverse the effect of prednisolone (P <0.05, P <0.01, P <0.001, G-L in FIG. 4) after the combined treatment of the chondrocytes with prednisone. In conclusion, prednisolone (but not prednisone) mediates cartilage loss due to prednisone exposure during pregnancy, while circGtdc1 mediates chondrocyte proliferation and matrix synthesis disorders due to prednisolone.
The present invention found that prednisone exposure during pregnancy resulted in a decrease in unidentified expression of circGtdc1 and continued after birth. Sanger sequencing identified the reverse cleavage site of circGtdc1, and RNase R and actinomycin experiments showed cyclization characteristics. The knockdown of the recombinant strain influences the proliferation of chondrocytes and the synthesis of matrixes, and in-vitro overexpression of the circGtdc1 can reverse the proliferation of chondrocytes and the synthesis of matrixes caused by prednisolone.
Example 3 circGtdc1 is useful as an intervention target for poor cartilage quality due to exposure of prednisone during pregnancy
1 Experimental method
1.1 in vivo circRNA intervention and in vitro in vivo imaging experiments
The circGtdc1 plasmid packaged by adeno-associated virus is supplied by the Meta Biomailbox company. Following the breeding of prednisone-exposed animals during pregnancy to PW8 as described above in methodology 1, the joint cavities were injected with empty vector and AAV-circGtdc1, respectively. Knee joints were obtained after euthanasia of animals at PW12, 8 of which were frozen at-80 ℃ for gene detection and 5 samples were fixed with 4% paraformaldehyde for histological analysis. The remaining specimens were examined for expression of circGtdc1 in a small animal in vivo imager.
1.2 safranin fast green staining and OARSI scoring as in [ example 1] experimental method 1.2
1.3 quantitative polymerase chain reaction for reverse transcription (real-time quantitative polymerase chain reaction, RT-qPCR) Experimental method 1.3
1.4 immunohistochemical staining As in example 1 Experimental method 1.4
2 experimental results
In vivo reversal of fetal cartilage dysplasia caused by prednisone exposure during pregnancy by circGtdc1
Finally, the invention detects whether the overexpression of circGtdc1 improves the cartilage quality reduction caused by the prednisone exposure during pregnancy in vivo through adeno-associated virus. First, successful overexpression of circGtdc1 was demonstrated by in vivo imaging (a in fig. 5). Safranin fast green results indicated that overexpression of circGtdc1 reversed the shallowness of cartilage staining in offspring rats resulting from prednisone exposure during pregnancy, thinning of cartilage thickness and increased OARSI scores for osteoarthritis (P <0.05, P <0.01, P <0.001, B-D in FIG. 5). RT-qPCR and immunohistochemistry further showed that overexpression of circGtdc1 reversed reduction of offspring rat cartilage Acan and Col2a1mRNA and protein expression due to prednisone exposure during pregnancy (P <0.05, P <0.01, P <0.001, E-I in FIG. 5). The above results indicate that overexpression of circGtdc1 in vivo can improve cartilage quality deterioration caused by prednisone exposure during pregnancy.
In conclusion, the invention proves that the reduction of circGtdc1 mediates cartilage cell proliferation and matrix synthesis disorder caused by prednisolone and mediates the low quality of offspring cartilage and susceptibility to osteoarthritis caused by prednisolone exposure during pregnancy. The in vitro overexpression of circGtdc1 can obviously reverse the low quality of the filial generation cartilage caused by the exposure of prednisone before pregnancy, and can be used as an intervention target for the dysplasia of the fetal cartilage and the susceptibility of adult osteoarthritis.
Claims (7)
1. A construction method of a fetal osteoarthritis model caused by prednisone exposure during pregnancy is characterized by comprising the following steps: the method comprises the following steps:
s1: normal pregnant rats were given a gastric lavage of prednisone of 0.125, 0.25mg/kg daily for 0-20 days of gestation;
s2: regulating each nest to 14 mice in the first day after the pregnant mice are naturally produced, wherein the sex ratio is 1:1; weaning 4 weeks after birth, and freely obtaining edible water;
s4: running treatment 24-28 weeks after birth;
s5: the method comprises the steps of gestating for 20 days, acquiring articular cartilage tissue of the rat at 12 weeks and 28 weeks after birth; evaluating the quality of knee cartilage, and detecting the change of related indexes of cartilage matrix synthesis and degradation;
the rats in the step S1 are Wistar rats; prednisone is a clinically used prednisone tablet;
the running conditions in the step S3 are as follows: day 1, 10min, speed 10m/min; day 2, 15min, speed 12m/min; day 3, 20min, speed 15m/min; day 4, 30min, speed 18m/min; day 5, 35min, speed 20m/min; on and after day 5, the first 10min includes 12m/min, followed by running for 55 minutes per day at a speed of 20m/min;
the quality of the cartilage in the step S4 comprises the solid green dyeing degree of safranin; cartilage proportion in proliferation area and hypertrophy area of the cartilage tissue of the fetal mouse; cartilage surface smoothness, reduced cartilage thickness, whether cartilage structure is disordered, whether the line is interrupted for hours, and osteoarthritis OARSI scores; detecting mRNA and protein expression of cartilage matrix synthesis indexes including Sox9, acan and Col2a 1; cartilage matrix synthesis indicators include mRNA and protein expression of mp3, mp13, and adamps 5.
2. An intervention target of fetal adult osteoarthritis, characterized in that: the intervention target is circGtdc1 in rat cartilage; overexpression of circGtdc1 in vivo can improve cartilage quality deterioration caused by prednisone exposure during pregnancy.
3. Use of an intervention target as claimed in claim 2 in the screening of a medicament for adult osteoarthritis of fetal origin.
4. A use according to claim 3, characterized in that: the circGtdc1 is a newly identified circular RNA molecule; the expression of circGtdc1 is reduced in the progeny cartilage, resulting in an increased likelihood of developing its cartilage dysplasia and osteoarthritis in adulthood.
5. The use according to claim 4, characterized in that: the circGtdc1 is used as an intervention target, and the medicine for improving the fetal osteoarthritis comprises a circGtdc1 gene mimic.
6. The use according to claim 5, characterized in that: the circGtdc1 gene mimetic is a circRNA over-expression plasmid.
7. The use according to claim 6, characterized in that: the circRNA overexpression plasmid is pLV-ciR-circGtdc1 or AAV-circGtdc1.
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