Cholesterol Accumulation Mediates Increased Osteoarthritis Susceptibility In Male Adult Offspring Rat Induced By Prenatal Dexamethasone Exposure

Osteoarthritis(OA)is chronic joint disease featured with degenerative articular cartilage,which is the most common cause of joint pain in middle-aged and elderly people.However,the etiology and pathogenesis of OA are still unclear.Recent studies have revealed that OA was closely involved in metabolic syndrome(MS).More and more scholars believed that OA belonged to the category of MS.Large-scale epidemiological survey results suggested,people with intrauterine growth retardation(IUGR)had an increased incidence of MS.Recent epidemiological data also showed that the incidence of hand OA was considerably higher in men with low birth weight,and a similar trend was observed in women.Previous studies in our laboratory also showed that adverse factors exposure during pregnancy(dietary restriction,caffeine,nicotine,and ethanol)could lead to IUGR and increased susceptibility to MS and OA in offspring rats.All these studies indicate OA has fetal origin.Studies have shown that biological processes including cholesterol synthesis,uptake,outflow and transformation also exist in the cartilage to maintain the physiological homeostasis of local cholesterol metabolism.Under pathological conditions,abnormal imbalance of cholesterol uptake,synthesis,output and transformation occured in the local cartilage of adult patients with OA,accompanied with abnormal accumulation of cholesterol.Accumulation of cholesterol could damage chondrocytes through multiple ways and reduce the cartilage quality.Other studies have shown that hypercholesterolemia is closely correlated with the occurrence of OA and has become an independent risk factor for OA.At the same time,a large number of epidemiology data suggest that hypercholesterolemia also has intrauterine origin.Our previous study confirmed that prenatal exposure to adverse factors(e.g.,caffeine,nicotine and ethanol)could cause hypercholesterolemia and accumulation of articular cartilage cholesterol in IUGR offspring on a high-fat diet after birth.The above indicated that the disorder of blood circulation and cartilage cholesterol metabolism may be involved in the occurrence of fetal originated OA.Synthetic glucocorticoid(GC),such as dexamethasone,is widely used clinically for the premature pregnancy related diseases.Numerous studies have shown that prenatal use of dexamethasone could lead to low birth weight of newborns,multiple organ developmental toxicity and susceptibility to a variety of chronic diseases in adulthood.Dexamethasone is known to regulate the expression of a series of genes related to cholesterol metabolism in hepatocytes.These results suggest that prenatal dexamethasone exposure may affect local cholesterol metabolism in fetal cartilage."Intrauterine programming" refers to the adverse environment encountered by the fetus during the intrauterine period,which can cause changes in the development and function of the fetal organs and can last until after birth.Previous studies in our laboratory have found prenatal dexamethasone exposure(PDE)can cause IUGR and multiple organ dysplasia in offspring.These results suggest that PDE might induce abnormal changes of local cholesterol metabolism in cartilage of IUGR offspring through intrauterine programming,which mediated local cholesterol accumulation and OA susceptibility in adult.Epidemiology showed that fetal originated OA was more susceptible in males.Therefore,this project selects male offspring as the research object.The following work is proposed in this study:(1)IUGR model in male offspring rats induced by PDE,OA model injected with articular cavities papain and three-dimensional culture model of adult chondrocytes with sodium alginate microspheres are utilized to verify the cholesterol accumulation and low quality of cartilage in offspring caused by PDE and OA susceptibility in adverse environment;(2)The changes of articular cartilage development quality and local cholesterol metabolism pathway expression in intrauterine and postnatal cartilage of PDE offspring rats are further observed to explore the intrauterine programming mechanism of cartilage cholesterol accumulation induced by PDE in adult male offspring rats.(3)Based on primary fetal chondrocytes,the effect of dexamethasone on CYP27A1 expression of fetal chondrocytes and its molecular mechanism are investigated.The implementation of this project will help us to have a more comprehensive understanding of cholesterol accumulation,OA susceptibility and its intrauterine programming mechanism induced by PDE,which is better understand fetal originated OA,and also playes a guiding role in the further exploration of early prevention and treatment of OA.Part One Prenatal dexamethasone exposure induces articular cartilage cholesterol accumulation and osteoarthritis susceptibility in male offspring adult ratsObjective: IUGR rat model induced by PDE,OA model induced by papain injection into the knee cavity and the three-dimensional culture model of adult chondrocytes combined with sodium alginate microspheres,were utilized to observe cholesterol accumulation,low cartilage quality and OA susceptibility in male offspring induced by PDE.Methods: In utero: Wistar maternal rats were randomly divided into Control group and PDE group after natural conception.From gestational day(GD)9 to GD20,0.2mg /kg.d dexamethasone was injected subcutaneously in the PDE group,and equal volume normal saline was given to the control group.On GD20,pregnant mice were anesthetized with phenobarbital and removed by laparotomy.Fetal weight and sex were recorded.Male fetal blood and femur specimens of both lower limbs were collected.After birth: some pregnant mice were randomly retained and allowed to give birth naturally.Newborn mice with 8-15 pups per litter were kept and weaned for 4 weeks.Normal diet was given until 8 weeks after birth(postnatal week,PW8).At the time of PW8,the knee joint was injected with papain to make OA model.The rats in the control group and the PDE group were injected with 4% papain solution 100 μl in the left knees joint at the 1st,4th,7th,10 th and 13 th day of the modeling respectively.At the same time,the right knees were injected with normal saline at the above time points.In PW12,rats were sacrificed under ether anesthesia,and blood was taken from the lower limbs and knee joints.In vitro: adult chondrocytes were isolated and proliferated,and sodium alginate microspheres were combined to establish a three-dimensional culture system.Cholesterol was treated with different concentrations(0,5,15,45 g/ml).The supernatant and cells were harvested.Results:(1)Before papain stimulation,cartilage matrix and Col2a1 immunohistochemical staining was slightly weakened in PDE group compared with the control group.After stimulation by papain,cartilage surface of PDE group showed obvious roughness,cartilage matrix structure disorder,cartilage thickness became thinner,matrix staining became lighter,and chondrocyte number decreased significantly.In addition,modified Mankin’s score of PDE group increased more considerably than that of control group,and optical density value of Col2a1 was considerably lower than that of corresponding control group.(2)Before papain stimulation,the number of Tunel positive cells in the PDE group was considerably higher than that in the control group.The m RNA expressions of Col2a1 and Aggrecan were considerably decreased,and the expressions of matrix degrading enzymes MMP3 and MMP13 were considerably increased.(3)Before the stimulation of papain,the cholesterol content in the PDE group was considerably higher than that in the control group.(4)In vitro three-dimensional culture condition,different concentration of cholesterol in the group of adult cartilage cell vitality than the control group considerably reduced,m RNA expression of apoptosis related gene abnormality(increased Bax,reduced the Bcl-2 and higher ratio of Bax/Bcl-2),microspheres section by safranin O stain becomes shallow,cells was considerably reduced,supernatant GAG content decreased,Col2a1 and the expression of aggrecan lower,the expressions of MMP3 and MMP13 were increased.Conclusion: PDE caused cholesterol accumulation in cartilage of adult male offspring rats,resulting in low quality of cartilage,which showed inreased susceptibility to OA after stimulation with papain in the knee joint cavity.Part Two Intrauterine programming mechanism of cholesterol accumulation in cartilage of male offspring rats induced by prenatal dexamethasone exposureObjective: The changes of articular chondrogenesis quality in intrauterine period and the expression changes of local cholesterol metabolism pathway in intrauterine and postnatal cartilage of PDE offspring rats were observed so as to explore the intrauterine programming mechanism of PDE induced cartilage cholesterol accumulation in adult male offspring rats.Methods: Same with the part one.Results:(1)the fetal weight of PDE group was considerably lower than that of the control group,and the IUGR rate was considerably increased.(2)fetal cartilage quality: safranin-o staining was weakened in the articular cartilage area of fetal rats in PDE group,and the content of GAG was considerably reduced by optical density analysis.The thickness of articular cartilage became thinner and the number of cells was decreased.Col2a1 immunohistochemical staining became lighter,and its content was reduced by optical density analysis.The m RNA expressions of Col2a1 and aggrecan were lower than those of the control group.(3)cartilage cholesterol metabolism pathway: In utero,the m RNA expression of SR-B1 in PDE group’s offspring local cholesterol uptake related genes had no significant change,but the m RNA expression of LDLR was considerably reduced.The m RNA expression of cholesterol synthesis related genes HMGCR was considerably decreased.The m RNA expression of cholesterol eflux-related genes ABCA1,ABCG1 and Apo A-1 showed no significant changes.The m RNA expression of cholesterol transformation related genes CYP27A1 and cholesterol hydroxylase CH25 H were considerably reduced.At week 12,m RNA expressions of SR-B1 and LDLR were considerably increased.m RNA expressions of SREBP-2 and HMGCR were not considerably changed,m RNA expressions of ABCA1,ABCG1 and Apo A1 were considerably increased,m RNA expression of CYP27A1 was considerably decreased,and m RNA expression of CH25 H had no changes.(4)epigenetic modification changes: In utero,the m RNA expressions of GR and HDAC1 were increased in the fetal cartilage in the PDE group.The H3K9 ac and H3K27 ac levels of promoter region in CYP27A1 in the PDE group were considerably lower than those in the control group.At PW12,the level of H3K9 ac in the promoter region of CYP27A1 decreased significantly,and there was no significant change in the level of H3K27 ac.Conclusion: PDE,on one hand,inhibited fetal chondrogenesis in offspring rats,reduced the H3K9 ac level and expression of CYP27A1 in local cartilage(low-function programming).On the other hand,the uptake of cholesterol in adult cartilage was increased by PDE.The combined effect of the above caused the local cholesterol accumulation in the cartilage of the offspring in adulthood,which further leads to low cartilage quality and increased susceptibility to OA.Part Three Effect of dexamethasone on CYP27A1 expression in primary fetal chondrocytes and its molecular mechanismObjective: Effects of dexamethasone on CYP27A1 expression of fetal chondrocytes and the molecular mechanism were investigated.Methods: Fetal articular cartilage were obtained to isolate the original generation of fetal cartilage cells,then the following treatments were listed as follows:(1)fetal cartilage cells were treated with 500 n M dexamethasone for different time points(1,2,3,4,5 days)or with different concentrations(0,20,100,500 n M)dexamethasone for 3 days,and the MTS method was used to detect cell activity;(2)fetal chondrocytes were treated with dexamethasone at different concentrations(0,20,100,500 n M),Or the fetal chondrocytes were pretreated with GR antagonist RU486(5 μM)for 2 hours,and then treated with dexamethasone at 500 n M;or the cells were co-treated with TSA and dexamethasone.After continuous treatment for 72 h,cells were collected.Results:(1)Cell viability was not considerably changed after 1～3 days of administration of dexamethasone at 500 n M,but considerably decreased after 4 and 5 days of administration.Fetal chondrocytes treated with dexamethasone at different concentrations(0,20,100,500 n M)showed no significant changes in cell viability after 3 days.(2)in the fetal chondrocytes of dexamethasone group,the m RNA and protein expression of CYP27A1 were considerably lower than those of the control group.(3)Under the effect of dexamethasone,the expression of GR and HDAC1 increased,activation of GR into the nucleus increased,and then GR recruited more HDAC1 to form a complex,which binded to the CYP27A1 promoter region.(4)Dexamethasone could increase the m RNA expression of HDAC1 and reduce the level of H3K9 ac in the promoter region of CYP27A1.RU486 could cancel the promoting effect of dexamethasone on HDAC1 expression.RU486 or TSA could cancel the inhibitory effect of dexamethasone on the level and expression of H3K9 ac in CYP27A1.Conclusion: this part confirmed that dexamethasone could activate GR and promote GR into the nucleus,and then GR recruited more HDAC1 to form a complex,which binded to the CYP27A1 promoter region,causing a decrease in the level of H3K9 ac in the CYP27 A promoter region,and thereby leading to a decrease in the expression of CYP27A1.