| Osteoarthritis(OA)is the chronic joint disease characterized by a major pathological feature of articular cartilage degeneration.OA is the most common cause of joint pain in the elderly.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 is characterized as a metabolic disorder.Based on a great deal of epidemiological evidences,British scholar Barker found that the incidence of adult MS with intrauterine growth retardation(IUGR)is higher than that of normal fetus,and proposed the theory of "the intrauterine origin of adult disease".Epidemiological data showed that hand OA was significantly associated with lower birth weight in male,and 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.Many studies have confirmed cartilage quality is significantly associated with the onset of OA.The formation of articular cartilage occurs mostly in embryogenesis.The quality of articular cartilage in adult life is closely related with the development of cartilage in embryonic period.Fetal articular cartilage dysplasia may be an important reason for the susceptibilety to OA.Some studies showed that OA was significantly associated with abnormal cholesterol metabolism.Hence,the underlying mechanisms of fetal originated OA may include cholesterol accumulation in articular cartilage.The physiological function of articular cartilage is dependent on the molecular composition of its extracellular matrix,which consists mainly of proteoglycans and collagens ?.Transforming growth factor-?(TGF?)signaling pathway play an important role in the development and differentiation of cartilage.TGFP-Smad2/3 pathway initiate the transcription of the extracellular matrix synthesis genes,which is the key pathway in the development of cartilage.Cartilage cholesterol efflux pathway disorder also participates in the cartilage cholesterol accumulation in adult OA.TGF?also could enhance the expression of cholesterol efflux associated proteins.Caffeine is a methylxanthine alkaloid that widely present in coffee,tea,soft drinks,food and some drugs.Many studies have revealed that prenatal caf'feine exposure(PCE)correlates with IUGR.Our previous studies showed that PCE could over-expose the fetus to maternal glucocorticoids(GC),resulted in IUGR and increased susceptibility to MS(e.g.hypercholesteremia)in later life,which may be associated with the "neuroendocrine metabolic programming mechanism".Moreover,we found that PCE resulted in retardation of chondrogenesis.Some studies have confirmed that prenatal exposure to GC results in the inhibition of longitudinal growth in fetal skeleton.Treatment with high level GC reduces the proliferation and matrix synthesis in chondrocytes.Basing on the above,we propose a series of questions as follow:whether PCE can lead to increased susceptibility to OA by "over-expose to maternal GC"?Whether it is related to changes in TGF? signaling pathway and cholesterol efflux system?Whether the hypercholesterolemia in PCE offspring aggravated OA susceptibility?These questions has not been clearly demonstrated.Therefore,the present study aimed to carry out the following studies:?Verify the phenomenon of susceptibility to OA and cartilage cholesterol accumulation in PCE adult offspring rats,and explore its cartilage development situation in utero and after birth;? Further observe the changes of TGF? signaling pathway and cholesterol efflux pathway in PCE offspring cartilage,and to explore the intrauterine programming mechanism;?To investigate the effect of GC on TGF? signaling pathway and cholesterol efflux pathway in fetal rat chondrocytes;? The effect of hypercholesterolemia on the cartilage was investigated by the administration of pravastatin.The present study allow us to fully understand the phenomenon of susceptibility to adult OA induced by PCE 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 ONE Prenatal caffeine exposure induces articular cartilage cholesterol accumulation and osteoarthritis susceptibility in offspring adult ratsObjective:To investigate the phenomenon of susceptibility to adult OA in IUGR adult offspring rats induced by PCE,and developmental condition of articular cartilage in utero and in different postnatal periods,and to discuss the potential mechanisms.Methods:In utero:Used different dose of caffeine(30,60 and 120 mg/kg day)to establish PCE-induced IUGR model.On GD20,pregnant rats were sacrificed were anesthetized with ether and decapitated,live fetuses were quickly removed to weigh,IUGR rates were also calculated.Take blood and knee tissue.After birth:Pregnant rats were randomly divided into control and PCE groups.From gestational day(GD)9 to GD20,rats in the PCE group were each administered caffeine 120 mg/kg day by oral gavage.Rats in control group were given the same volume of distilled water.On postnatal week(PW)1,we weighed the litter sizes and recorded the weight gain.The pups in each litter were randomly divided into 4 batchs,according to the postnatal week respectively named as PW2,PW6,PW8 and PW12.On PW2,PW6 and PW12,the corresponding batchs of rats were anesthetized with ether and decapitated to collect blood and knee tissus.OA model:The PW8 male 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 PCE 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.Half 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 staining were used for obvsering pathological changes,and making modified OA Mankin's score.Results:? After accepting intra-articular injection of papain-induced OA,compared with control group,PCE offspring rats showde a rougher articular surface,appeared serious wear and tear.?Pathological change and OA score:after accepting intra-articular injection of papain to induce OA,Safranin O-fast green staining found that the cartilage matrix mild lightly stained and the surface was mild rough in control group;in PCE group,cartilage surface defects,cartilage matrix structure disorders were observed,reduced cartilage thickness,matrix lightly stained and chondrocytes were also significantly reduced.The cartilage pathological modified Mankin's score in PCE group was significantly higher than that of control group(P<0.01).?After accepting intra-articular injection of papain-induced OA,the content of TCH in the cartilage was significantly increased in PCE group than that in the control(P<0.05),and the mRNA expression of Col2al and Aggrecan were lower(P<0.01).? PCE groups cartilage matrix Safranin O staining and Col2al immunohistochemical staining were shallow than control group in utero and in different postnatal periods,as well as the mRNA expression of Col2al and Aggrecan were lower(P<0.05,P<0.01).Conclusion:PCE can cause offspring fetal rat articular cartilage dysplasia,and can be extended to after birth,leading to increased susceptibility to OA;We also found that,PCE can cause offspring articular cartilage cholesterol accumulation.PART TWO Effect of prenatal caffeine exprosure on increasing the susceptibility to osteoarthritis and its intrauterine programming mechanismsObjective:Observe the changes of TGF? signaling pathway and cholesterol efflux pathway in PCE offspring cartilage,and to explore the intrauterine programming mechanism.Methods In utero:Used different dose of caffeine(30,60 and 120 mg/kg day)to establish PCE-induced IUGR model.On GD20,pregnant rats were sacrificed were anesthetized with ether and decapitated,live fetuses were quickly removed to weigh,IUGR rates were also calculated.Take blood and knee tissue.After birth:Pregnant rats were randomly divided into control and PCE groups.From gestational day(GD)9 to GD20,rats in the PCE group were each administered caffeine 120 mg/kg day by oral gavage.Rats in control group were given the same volume of distilled water.On postnatal week(PW)1,we weighed the litter sizes and recorded the weight gain.The pups in each litter were randomly divided into 3 batchs,according to the postnatal week respectively named as PW2,PW6 and PW12.On PW2,PW6 and PW12,the corresponding batchs of rats were anesthetized with ether and decapitated to collect blood and knee tissus.Results:?compare to control group,the bodyweights of fetuses in different dose of PCE group were lower while corresponding IUGR rates were higher,and serum CORT levels were higher(P<0.05,P<0.01);?compare to control group,the mRNA and protein expression of PCE group TGF?R1 and Smad2/3 in articular cartilage were decreased in different degree in utero and different time points after birth(P<0.05,P<0.01);? compare to control group,the mRNA and protein expression of PCE group LXRa in articular cartilage were decreased in different degree in utero and different time points after birth(P<0.05,P<0.01),and the mRNA expression of PCE group SR-B1,ABCA1,ABCG1 in articular cartilage were decreased in different degree in utero and different time points after birth(P<0.05,P<0.01).Conclusion:PCE may lead to low-functional programming of TGF? signaling pathway and cholesterol efflux pathway in offspirng cartilage through maternal GC,leading to poor offspring cartilage development and accumulation of cholesterol.PART THREE The effect of corticosterone on the fetal cartilage matrix synthesis and its molecule mechanismObjective:To investigate the effects of CORT and caffeine on cartilage matrix synthesis function and TGF? signaling pathway expression in primary rat fetal chondrocytes in vitro,and to investigate whether GC can regulate cholesterol efflux via TGFp-Smad2/3.Methods:Dissect for articular cartilage tissue in the fetal rats(GD20),and obtain and culture primary chondrocytes.(1)Treated with CORT:The primary rat fetal chondrocytes were treated with CORT with different concentrations(0,0.35,0.7,1.4,2.8 ?M)for 5 days,or treated with 0.35?M CORT with different time(0,1,3,5,7 days);the primary rat fetal chondrocytes were treated with 5 ?M glucocorticoid receptor(GR)inhibitor Mifepristone for 0.5 hour,than treated with different concentrations(0,0.35,0.7,1.4?M)CORT for 5 days;the primary rat fetal chondrocytes were treated with Smad2/3 siRNA or TGF?1(5 ng/ml),than treated with 1.4?M CORT for 5 days;Real-time fluorescent quantitative PCR detected gene expression levels of matrix synthesis function,TGF? signaling pathway and cholesterol efflux pathway.(2)Treated with caffeine:The primary rat fetal chondrocytes were treated with caffeine with different concentrations(0,0.1,1,10?M)for 5 days,Real-time fluorescent quantitative PCR detected gene expression levels of matrix synthesis function,TGF? signaling pathway and cholesterol efflux pathway.Results:(1)Effect of CORT:?Compared with control,the treatment of CORT for 1-3 days had no significant effect on cell viability,but treatment of CORT for 5-7 days obviously reduced the number and viability of primary rat fetal chondrocytes(P<0.01);the treatment of 2.8pM CORT for 5 dyas obviously reduced the number and viability of primary rat fetal chondrocytes(P<0.01).? Compared with control,in primary chondrocytes treated with CORT for 5 days,gene expression levels matrix synthesis function,TGF? signaling pathway and cholesterol efflux pathway showed dose-dependent reduction(P<0.05,P<0.01),treated with Mifepristone reversedly increased the gene expression levels;? Compared with 1.4?M CORT group,Smad2/3 siRNA +1.4?M CORT group LXRa mRNA level has no significant change,as well as the matrix synthesis genes;Compared with Smad2/3 siRNA group,after treat with TGF?1,TGF?1?1.4?M CORT group Smad2,Smad3 and LXRa mRNA levels were increased(P<0.01),and the matrix synthesis genes mRNA levels were also increased(P<0.01).(2)Effect of caffeine:?In primary chondrocytes treated with caffeine for 5 days,gene expression levels of Aggrecan and Col2al showed dose-dependent reduction compare with the control(P<0.05,P<0.01),but gene expression levels of TGF? signaling pathway and cholesterol efflux pathway had no obviously consistency changes.Conclusion:CORT could directly inhibit matrix synthesis function in fetal primary chondrocytes and the mechanism is associated with GC can regulate cholesterol efflux via TGF?-Smad2/3;furthemore,caffeine could directly inhibit matrix synthesis function,but its mechanism still needs further study.PART FOUR Hypercholesterolemia induced by prenatal caffeine exprosure could aggravate cartilage cholesterol accumulationObjective:The effect of hypercholesterolemia on the cartilage was investigated by the administration of pravastatin,and by extracting PW6 offspring rat chondrocytes to administration with exogenous cholesterol,observing the effect of hypercholesterolemia on the cartilage quality and cholesterol efflux pathway.Methods Pregnant rats were randomly divided into control and PCE groups.From gestational day(GD)9 to GD20,rats in the PCE group were each administered caffeine 120 mg/kg day by oral gavage.Rats in control group were given the same volume of distilled water.On postnatal week(PW)8,given pravastatin(20mg/kg.d)or equal volume of saline to PW12.On PW12,the rats were anesthetized with ether and decapitated to collect blood and knee tissus.Results:?In the saline group,compare to control group,the serum TCH and LDL-C levels were higher in PCE group,as well as the TCH content of cartilage(P<0.05,P<0.01).In the pravastatin group,the serum cholesterol levels and TCH content of cartilage have no significant change in PCE group;? In the saline group,compare to control group,the cholesterol efflux pathway mRNA expressions were decreased in PCE group,as well as the matrix synthesis genes(P<0.05,P<0.01).In the pravastatin group,the cholesterol efflux pathway and matrix synthesis genes mRNA expressions have no significant change in PCE group;? At PW6,the serum cholesterol levels have no significant change in PCE group compare to control group;At PW6 offspring rat chondrocytes,with the increase in exogenous cholesterol levels,the cholesterol efflux pathway and matrix synthesis genes mRNA expressions were decreased more obviously in PCE group(P<0.05,P<0.01).Conclusion:PCE can cause hypercholesterolemia in adult offspring rats,and the latter may further increase chondrocyte cholesterol accumulation by further inhibiting chondrocyte cholesterol efflux pathway in the offspring.In addition,statins may be able to reduce cartilage localized cholesterol accumulation by improving chondrocyte cholesterol efflux pathway inhibition,thereby reducing cartilage local cholesterol accumulation. |
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