| Osteoarthritis (osteoarthritis, OA) is a kind of chronic joint disease characterized by a major pathological feature of articular cartilage degeneration. Which is the most common cause of joint pain in the elderly. OA affects the quality of life in patients, and has brought great economic burden to the families and society. Currently, the etiology and pathogenesis of OA are inconclusive. Generally, OA is reported to be related to age, mechanical friction, inflammation and matrix-degrading enzymes. The relationship between metabolic syndrome (MS) and OA has been highlighted in the aetiology study of OA, and more and more researchers' opinion about OA was that it was characterized as a metabolic disorder. Increasing evidence shows that MS (such as hypertension, hyperglycemia and dyslipidemia) have an intrauterine origin. Barker found that intrauterine growth retardation (IUGR) fetuses are prone to cardiovascular disease and type ? diabetes through retrospective epidemiological surveys, and proposed the theory of "the intrauterine origin of adult disease." An increasing number of reports have identified a positive correlation between OA and MS and between MS and IUGR; however, the mechanism by which IUGR increases the susceptibility to OA in adults has not been clearly interpreted. Epidemiological evidences showed that hand OA was significantly associated with lower birth weight in male, female had the similar trend. The analogeous conclusion was also found in another epidemological research that focused on the relation between low birth weight and lumbar spine OA. These reports indicate that OA may be originated from intrauterine stage.Prenatal ethanol exposure (PEE) is one of the most definite and dangerous causes of IUGR. Studies indicate that children with PEE show growth retardation in the womb, infancy, and adulthood, and have some long-term consequences on endocrine and metabolic function in offspring. Our previous studies and other studys showed that PEE gives rise to the fetus being over-exposed to maternal glucocorticoid (GC) and finally results in IUGR.Articular cartilage is the core of OA pathological changes tissue, its quality is closely related to the onset of OA. The formation of articular cartilage occurs mostly in embryogenesis. Fetal articular cartilage dysplasia may be an important reason for the susceptibilety to OA. Articular cartilage is a uniquely ordered tissue that is avascular, alymphatic and non-innervated, and chondrocytes is the only cell components. The physiological function of articular cartilage is dependent on the molecular composition of its extracellular matrix, which consists mainly of proteoglycans (aggrecan) and collagens ?. Transforming growth factor-? (TGF?) signaling pathway play an important role in the development and differentiation of cartilage. TGF?-Smad2/3-SRY-type high mobility group box 9 (Sox9) pathway initiate the transcription of the extracellular matrix synthesis genes, which is the key pathway in the development of cartilage. Some studies have confirmed that prenatal exposure to GC results in the inhibition of longitudinal growth in fetal skeleton. Treatment with high level GC reduce the proliferation and matrix synthesis in chondrocytes. Therefore, GC may influence the development and factional differentiation of fetal articular cartilage via TGF? signaling pathway. Cell experiments confirmed, that excessive levels of GC can inhibit the expression of TGF?R in osteoblast.Basing on the above, we propose a series of questions as follow:Is there the existence of intrauterine developmental origin of adult osteoarthritis? Is the increased susceptibility to OA in IUGR adult offspring induced by articular cartilage dysplasia? Colud PEE-induce maternal excessive GC exposure in fetus relate to inhibited TGF? signaling pathway? Is the increased susceptibility to OA in IUGR adult offspring induced by PEE related with low-fuctional TGF? signaling pathway programming mechanism? And are the alterations come from the indirect effect of over-expose the fetus to maternal GC or direct effect of ethanol? These questions has not been clearly demonstrated.Therefore, the present study aimed to carry out the following studies: ? select three OA-induced model to verify the phenomenon of the intrauterine origin of adult OA in rats; ? Investigate the effects of PEE on dysplasia and inhibited functional extracellular matrix synthesis of fetal articular cartilage, and explore its inhibited TGF? signaling pathway mechanism; ?Explore the effect of the effects of CORT on chondrogenic differentiation of BMSCs and TGF? signaling pathway expression in the differentiating BMSCs in vitro, and to elucidate the potential mechanism;? Explore the effect of ethanol on chondrogenic differentiation of BMSCs and TGF? signaling pathway expression in the differentiating BMSCs in vitro, and to elucidate the potential mechanism. The present study allow us to fully understand the phenomenon of susceptibility to adult OA induced by PEE and its intrauterine programming mechanism, and better understand the intrauterine developmental origin of adult OA. The study also provides the theoretical foundation for early prevention and treatment of OA.PART ONEPregnancy ethanol exposure increases the adult IUGR offspring rats' susceptibility of OAObjective To confirm that the pregnancy ethanol exposure could increase the susceptibility of adult IUGR offspring's OA in rats.Methods After natural conception, pregnant rats were given ethanol (4 g/kg·d) from 9 days of gestation until natural delivery. On postnatal week (PW) 1, we weighed the litter sizes and recorded the weight gain. Retained the litter pups 8-15 in newborn rats. after weaning (postnatal week 4, PW4),?treadmill running-induced OA model: The offspring rats were fed with normal diet to adult. At PW18, the offspring rats were given the long distance running to induced OA until the rats were running for six weeks with a total running mileage of 30 Km. Rats were sacrificed under ether anesthesia.?high-fat diet (HFD) induced OA model:all female pups from the control and PEE group were fed with HFD until being sacrificed on PW24. The HFD was provided 18.9% of its energy content as protein,61.7% as carbohydrate, and 19.4% as fat. The body weights of the offspring rats were measured weekly until PW24, the corresponding growth rates were calculated. ? Intra-articular injection of papain induced-OA model:The PW8 offspring rats were given intra-articular injection of papain to induce OA. A solution of 4%(w/v) papain solution in saline 100 ?L was sterilized and then injected into the left knee of rats from control group and PEE group on days 1,4,7,10 and 13 of the experimental period. As a normal control, the same volume of sterile saline was injected into the right knee of rats in the both groups on the same days. On PW12, rats were sacrificed under ether anesthesia. Portion of knee cartilage samples were ink stained for obvsering gross morphological changes; the remaining knee samples were fixed, embedded, decalcified and sectioned, Safranin O-fast green, and/or hematoxylin and eosin (HE), and/or toluidine blue (TB) staining were used for obvsering pathological changes, and making OA Mankin's score.Results ?PEE resulted in the decreased body weights of the PEE female pups related to the control pups at PW1 (P<0.01). After weaning, the body weights of female offspring rats in PEE group were close to that of control when fed with HFD (P<0.01, P<0.05), but the corresponding body weight growth rate were all significant higher related to control at each time piont (P<0.01, P<0.05, respectively).?After accepting treadmill running, HFD or Intra-articular injection of papain to induce OA, PEE offspring rats'cartilage surface, especially tibial plateau cartilage surface, appeared serious wear and tear. ? Safranin O-fast green staining found, after different OA induced model, compared to the respective control group, the PEE group showed the articular cartilage became thinner, varying degrees of cartilage surface defects were observed, chondrocytes were also significantly reduced, cartilage matrix structure disordersand, cartilage matrix Safranin O staining and Col2a1 immunohistochemical staining were shallow. After different model to induced OA, the cartilage pathological Mankin's score in PEE group was significantly higher than that of control group (P<0.01).Conclusion Pregnancy ethanol exposure could increase the susceptibility of adult IUGR offspring's OA in rats.PART TWOPregnancy ethanol exposure leads to retardation of fetal rats' articular cartilage development and its Programming AlterationsObjective Our previously study demonstrated that PEE increased the susceptibility to OA, The present study aimed to investigate the effects of pregnany ethanol exposure on fetal rats' articular cartilage development and its Programming Alterations.Methods Pregnant rats were randomly divided into control and PEE group. From gestational day (GD) 9 to GD20, pregnant rats of PEE group were given ethanol (4 g/kg-d). Rats in control group were given the same volume of distilled water. On GD20, Portion of pregnant rats from the two groups were sacrificed and live fetuses were quickly removed to weigh and distinguish gender. IUGR rates were also calculated. Take blood and knee tissue. Blood CORT concentration were measured by ELISA kit. fetal right femurs were separated under a dissecting microscope and collected for PCR. Fetal left knee joints were randomly selected and fixed, embedded, decalcified and sectioned for further analysis. Furthermore, The other pregnant rats were kept until normal delivery on GD21. On postnatal week (PW) 1, we retained the litter pups 8-15 in newborn rats. The pups in each litter were randomly divided into 4 batchs, according to the postnatal week respectively named as PW2, PW6 and PW12. For each batchs 10 male pups for control or PEE group were selected randomly, and all of the pups were weaned to an adl ibitum diet before being sacrificed. On PW2, PW6 and PW12, the corresponding batchs of rats were anesthetized with ether and decapitated to collect knee tissus. Portion of knee cartilage samples were fixed, embedded, decalcified and sectioned, Safranin O-fast green staining were used for obvsering pathological changes. The remaining knee samples were used for extract total RNA for detecting the mRNA expression.Results ? Body weights, IUGR rates and serum CORT level:the body weights of fetuses in PEE group were lower than control, while corresponding IUGR rates were higher(P<0.05, P<0.01); Fetal serum endogenous CORT levels in PEE group were significantly higher (P<0.01) than that of control group. ?Morphological synthesis of articular cartilage:In PEE group, fetal rats'articular cartilage resting zone became thinner, the number of chondrocytes were reduced, cartilage matrix Safranin O staining and Col2al immunohistochemical staining were shallow; Optical density analysis for Safranin O staining of articular cartilage GAG and the Col2a1 immunohistochemical staining found that PEE significantly decreased fetal rat articular cartilage matrix GAG and Col2a1 levels (P<0.01). Furthermore,these changes can continue to postnatal week 2,6,12.?The expression of TGF? signaling pathway in fetal articular cartilage:Compared with control group, the TGF? signaling pathway mRNA and proteins expressions of fetal articular cartilage in PEE group were significantly decreased (P<0.05, P<0.01). As with the morphological changes, these gene expression patterns also can continue to postnatal week 2,6,12.Conclusion PEE lead to the developmental retardation of IUGR fetal rats' articular cartilage. The potential mechanism is that PEE induced over-expose the fetus to maternal GC, which caused intrauterine low-functional TGF? signaling pathway programming in articular cartilage, than resulted in continuely decreased articular cartilage matrix synthesis function through life.PART THREEEffect of Corticosterone on chondrogenic differentiation of rat bone marrow mesenchymal stem cellsObjective To investigate the effects of CORT on chondrogenic differentiation of BMSCs and TGF? signaling pathway expression in the differentiating BMSCs in vitro, and to elucidate the potential mechanism.Methods Wistar male rat were purchased from the Laboratory Animal Center of Wuhan University (Wuhan, China). Bone marrow was collected by flushing femur with medium. Monolayer cultures were trypsinized, washed, and centrifuged. The isolated BMSCs were suspended in a 1.25% alginate (Sigma-Aldrich, USA). The beads with approximately 25000 cells/bead were cultured with a medium: high-glucose DMEM containing,1% ITS,100 nM dexamethasone (Sigma-Aldrich, USA) and 10 ng/mL TGF-?3 (PeproTech, USA). After chondrogenic differentiation of 4 weeks, to investigate the capability of BMSCs to induce chondrogenesis, alginate beads sections were stained for glycosaminoglycan (GAG) with safranin-O (Saf-O). During the period of chondrogenic differentiation the culture medium with or without CORT at concentrations of 125,250,500 and 1000 ng/ml was replaced every other day. MTT test was used to measure the cell viability in bead treated with the indicated concentration of CORT for 4 weeks. After treatment of 4 weeks with CORT, the beads were fixed and dehydrated through alcohols and embedded in paraffin. The sections were routinely stained with Saf-O. The mRNA expression of chondrogenesis related genes, including collagen type 2 alpha 1 (Col2A1), GR/CEBP?and TGF? signaling pathway (TGF?R ?, Smad2/3 and SOX9) genes expressions were detected by quantitative real-time PCR.Results ? Cell viability:Compared with control, the treatment of CORT for 4 weeks, CORT did not affect viability of cells at any indicated concentration during chondrogenic differentiation of BMSCs; ? Matrix synthesis function:After 4 weeks cultured in chondrogenic differentiation conditions, alginate beads sections were positively stained for GAG with safranin-O, which identified a dense GAG-rich matrix. Continuous exposure to CORT for 4 weeks did not cause significant changes in BMSCs cell numbers. However, CORT significantly decreased the area stained with safranin-O in a concentration-dependent manner, compared with the control (P< 0.05).? Matrix synthesis function and expression of GR/CEBP?, TGF?signaling pathway:After BMSCs treated with CORT (125,250,500and 1000 ng/ml) for 4 weeks, could dose-dependently upregulate the expression of GR and CEBP?, while gene expression levels of TGF?, TGF?R ?, Smad2, Smad3, Sox9, aggrecan and Col2al showed dose-dependent reduction (P<0.05, P<0.01) compare with the control; treated with Mifepristone reversedly decreased the gene expression levels of GR, and reversedly increased the gene expression levels of TGF?R ?, Smad2 and Smad3 (P<0.05, P<0.01).Conclusion CORT could directly inhibit matrix synthesis function in chondrogenic differentiation of BMSCs and the mechanism is associated with inhibition of TGF? signaling pathway by upregulating GR/CEBPa.PART FOUREffect of Ethanol on chondrogenic differentiation of rat bone marrow mesenchymal stem cellsObjective To investigate the effects of ethanol on chondrogenic differentiation of BMSCs and TGF? signaling pathway expression in the differentiating BMSCs in vitro, and to elucidate the potential mechanism.Methods Wistar male rat were purchased from the Laboratory Animal Center of Wuhan University (Wuhan, China). Bone marrow was collected by flushing femur with medium. Monolayer cultures were trypsinized, washed, and centrifuged. The isolated BMSCs were suspended in a 1.25% alginate (Sigma-Aldrich, USA). The beads with approximately 25000 cells/bead were cultured with a medium: high-glucose DMEM containing,1% ITS,100 nM dexamethasone (Sigma-Aldrich, USA) and 10 ng/mL TGF-?3 (PeproTech, USA). After chondrogenic differentiation of 4 weeks, to investigate the capability of BMSCs to induce chondrogenesis, alginate beads sections were stained for glycosaminoglycan (GAG) with safranin-O (Saf-O). During the period of chondrogenic differentiation the culture medium with or without ethanol at concentrations of 0.8,4,20 and 100 mM was replaced every other day. MTT test was used to measure the cell viability in bead treated with the indicated concentration of ethanol for 4 weeks. After treatment of 4 weeks with ethanol, the beads were fixed and dehydrated through alcohols and embedded in paraffin. The sections were routinely stained with Saf-O. The mRNA expression of chondrogenesis related genes, including collagen type 2 alpha 1 (Co12A1), EGR1,11-?-HSD2, GR/CEBPaand TGF? signaling pathway (TGF?R I, Smad2/3 and SOX9) genes expressions were detected by quantitative real-time PCR.Results ? Cell viability:Compared with control, the treatment of ethanol for 4 weeks, ethanol did not affect viability of cells at any indicated concentration during chondrogenic differentiation of BMSCs;? Matrix synthesis function:After 4 weeks cultured in chondrogenic differentiation conditions, alginate beads sections were positively stained for GAG with safranin-O, which identified a dense GAG-rich matrix. Continuous exposure to ethanol for 4 weeks did not cause significant changes in BMSCs cell numbers. However, ethanol significantly decreased the area stained with safranin-O in a concentration-dependent manner, compared with the control (P< 0.05). ? The expression of GR and CEBPa:the gene expression levels of GR and CEBPahad no obviously consistency changes. ? Matrix synthesis function and expression of, TGFPsignaling pathway:After BMSCs treated with ethanol for 4 weeks, gene expression levels of TGF?, TGF?R I, Smad2, Smad3, Sox9, aggrecan and Col2al showed dose-dependent reduction (.P<0.05, P<0.01) compare with the control; ? The expression of EGR1 and 11-?-HSD2:The treatment of ethanol for 4 weeks could dose-dependently upregulate the expression of EGR1, but dose-dependently downregulate the expression of 11-?-HSD2.Conclusion Ethanol could directly inhibit matrix synthesis function in chondrogenic differentiation of BMSCs, Its underlying mechanisms may be associated with two aspects:one is directly inhibite the expression of TGF? signaling pathway, the other is the ethanol could inhibite the expression of 11-?-HSD2 by upregulating cAMP/PKA/EGRl,which will result in the GC barrier effect decrease. |
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