Prenatal Dexamethasone Exposure Induce The Trans-generational Inheritance Of Poor Articular Cartilage In Female Rats

Osteoarthritis(OA)is a chronic joint disease characterized with the degeneration of articular cartilage.OA is traditionally thought to be caused by age,trauma and strain.However,recent studies showed that OA belongs to metabolic syndrome.Barker first reported that the incidence of metabolic syndrome increase in adults with low birth weight and suggested the theory of "the development origin of adult disease".Epidemiological studies have also shown that men with low birth weight have a higher risk in hand OA,and women have the similar trends.These suggested that OA might originate from the fetal development.Studies have shown that the occurrence of OA is closely related to the quality of articular cartilage.The main components of articular cartilage are extracellular matrix(ECM)and a small number of chondrocytes.Cartilage matrix,which composed of al chain of type ? collagen(COL2A1)and aggrecan(ACAN),is important for lubrication and buffering due to its smooth and toughness.Due to the lack of blood vessel in cartilage,the metabolism of chondrocyte is slow.It's hard for the cartilage to repair when damaged.The development of cartilage is extremely important to cartilage's disease resistance.Most of the articular cartilage has matured during the fetal period;the environmental factors during this period may affect the quality of cartilage in adulthood.Transformation growth factor beta(TGF?)signaling pathway was involved in the process of cartilage matrix synthesis.Briefly,TGF? combined to the transformation growth factor beta receptor(TGF?R),which activates the classical TGF?/Smads signaling pathway.The activated Smads dissociate from the receptor complex and form a heteropolymer complex with Smad4 into the nucleus,and then bind to other DNA binding proteins to regulate the transcription of the target gene.SRY-type high mobility group box9(Sox9)is the initial transcription factor of cartilage formation,which bind to the enhancer regions of cartilage phenotypic genes,promote the gene expression of chondrocyte and maintain the characteristics of chondrocyte.The TGF?-Smads-SOX9 pathway played a key role in the synthesis of cartilage matrix.Dexamethasone,as a synthesis glucocorticoid(GC),can pass through the placenta easily.Dexamethasone is widely used in pregnant women with the risk of preterm birth to reduce the risk of respiratory distress syndrome.However,more and more evidence showed that prenatal exposure to dexamethasone can not only lead to intrauterine growth restriction,but also lead to the susceptibility to multiple metabolic diseases.Fetal over exposed to GC was related with adult hypertension,diabetes,mental illness and metabolic syndrome.Did the prenatal dexamethasone exposure(PDE)have toxicity effect on the development of articular cartilage?Trans-generational inheritance refers to parental generation(FO)exposed to adverse environmental factors during pregnancy,the germ line of F1 and F2 generations were directly exposed,but the F3 generation was not exposed,thus the phenotype changes in F3 generation was trans-generational inheritance.Recent epidemiology investigation and laboratory research showed that the adverse environment(such as restricted feeding,nicotine exposure)during pregnancy may not only cause the islet change in morphology or susceptibility to asthma in F1 generation,but also cause the similar change in F2 generation or even the F3 generation.Previous studies in our laboratory have also found that the neuroendocrine metabolism programming changes caused by prenatal caffeine exposure can be inherited to F2generation.Does PDE have a trans-generational inheritance effect on the development of articular cartilage and what is the mechanism of this inheritance?MicroRNAs(miRNAs)are small regulatory non-coding RNAs of 21-25 nucleotides.Mature miRNAs mediate mRNA degradation or suppress mRNA translation by binding to the 3'-untranslated region(3'-UTR)of target mRNAs.It was known that histone deacetylase 2(HDAC2)was the target gene of miR-92a.Compared with normal articular cartilage,the expression of miR-92a in OA cartilage decreased significantly while the expression of HDAC2 was significantly increased.Based on the above research,we speculated that PDE may inhibit the miR-92a expression in germ line,thus mediate the high expression of HDAC2 to reduce the acetylation of TGFP signaling pathway,leading to the low expression of TGF?signaling pathway and the extracellular matrix(ECM)of articular cartilage.The change of miR-92a in germ line was inheritable,thus lead to the trans-generational inheritance of poor cartilage quality.This study will clarify the effect of PDE on the cartilage quality of female offspring and point out the possible mechanism of the trans-generational inheritance.To this end,the study will be carried out as following:?To confirm the trans-generational inheritance of poor articular cartilage caused by PDE in vivo.?To confirm that miR-92a mediated the dexamethasone's inhibition effect on the expression of TGF? signaling pathway and ECM in fetal chondrocyte in vitro.?To confirm that the lower expression of miR-92a in germ line mediated the trans-generational effect of poor articular cartilage in vivo.Part ONEPDE causes the trans-generational inheritance of poor quality of articular cartilage in female offspring.Objective To investigate the effect of PDE on the cartilage quality of F1/F2/F3 female offspring and explore the role of TGF? signaling pathway in this process.Methods Wistar rats were randomly divided into control group and PDE group after conception.The PDE group rats were injected with 0.8 mg/kg/d dexamethasone subcutaneously from gestational day 9(GD9)to GD20,and the control group were injected with the same volume of saline.The serum and knee joint specimens of female offspring were obtained after isoflurane anesthesia in GD20 and postnatal week 12(PW12).The rest offspring were given a normal diet to produce the F2 and F3 generations.High performance liquid chromatography(HPLC)was used to detect the concentration of dexamethasone in F0 and F1 serum.Part of the knee joint of F1/F2/F3 offspring was used for H&E staining and safranine O green staining.Immunohistochemical staining and RT-qPCR were used to detect the expression of COL2A1 and TGF? signaling pathway.Results The concentration of dexamethasone in PDE-FO rats was 0.864?mol/L,and in PDE-F1 fetal rats was 0.260?mol/L,suggesting that dexamethasone could pass through the placental barrier easily.H&E staining and safranine O green staining showed less cells and reduced staining matrix in F1-GD20/F1-PW12/F2-PW12/F3-PW12 in PDE group.The mRNA and protein expression of ECM and TGF? signaling pathway were significantly reduced in PDE group in the three following generations.Conclusions PDE can lead to the poor cartilage quality in F1,F2 and F3 generations.The lower expression of TGF? signaling pathway might mediate the poor quality of articular cartilage.Part TWOThe epigenetic mechanism of dexamethasone's effect on chondrocyte Objective To explore the effect of dexamethasone on the matrix synthesis of chondrocyte and the epigenetic mechanism.Methods The fetal rat chondrocyte were treated with 0,20,100,500,2500nM dexamethasone respectively,and the GR inhibitor,mifepristone,the miR-92a-mimic and anti-miR-92a were used respectively.The cell proliferation rate,miR-92a,HDAC2,TGF? signaling pathway,COL2A1 and ACAN expression were detected.Results MTS showed no change in cell proliferation rate after treated with different concentration of dexamethasone.Compared with control group,the expression of miR-92a in dexamethasone treatment group was significantly decreased,and the expression of HDAC2 was significantly increased.Mifepristone could eliminate this effect of dexamethasone.The expression of miR-92a was increased and HDAC2 expression was inhibited after the treatment with miR-92a-mimic,and after treated with anti-miR-92a,the expression of miR-92a was decreased and HDAC2 was increased.The TGF? signaling pathway in dexamethasone treated group was reduced in concentration dependent manner and the following expression of COL2A1 and ACAN were reduced.Dexamethasone can significantly reduce the H3K9ac of TGF?R1 and COL2A1,while mifepristone could eliminate this inhibitory effect.Conclusions Dexamethasone could inhibit the expression of miR-92a,thus enhance the expression of HDAC2,leading the low acetylation of TGF?R1 and COL2A1 and the inhibition of TGF? signaling pathway and matrix synthesis.Part THREEThe low expression of miR-92a in oocyte mediated the trans-generational inheritance of poor cartilage quality in female offspring Objective To investigate the epigenetic modification of oocyte on the trans-generational inheritance of poor cartilage quality.Methods Pregnant horse serum gonadal hormone-human chorionic gonadotropin(PMSG-HCG)was used to stimulate the F1 and F2 female rats to collect oocytes,and detect the expression of miR-92a.The expression of HDAC2 and the acetylation of TGF? signaling pathway in the articular cartilage of F1/F2/F3 offspring were detected.Results Compared with the control group,the expression of miR-92a in oocytes of PDE group was significantly decreased.The expression of HDAC2 in the cartilage of F1/F2/F3 offspring was significantly increased,and the acetylation of TGF? signaling pathway and COL2A1/ACAN were reduced.Conclusions PDE induced the low acetylation of TGF? signaling pathway in articular cartilage by inhibiting the expression of miR-92a,which results in the trans-generational inheritance of poor quality of articular cartilage.