Activation Of Renin-angiotensin System By Prenatal Ethanol Exposure Induce The Dysplasia Of Long Bone In Offspring Rats:the Intrauterine Programming Alternation

Osteoporosis is a common disease,which is age-related,and characterized by low bone mass,damaged bone microstructure.The main manifestation is increased bone fragility and fracture risk.The patients with osteoporosis are prone to fracture even if suffering from mild external forces.In recent years,based on a large number of researches among the correlation of the pregnancy adverse environment,fetal birth weight and adult chronic diseases,scholars proposed the concept of the origin of human diseases-"the developmental origin of health and disease"(DOHaD).Epidemiological studies indicate that the intrauterine growth retardation(IUGR)individuals suffere significantly lower peak bone mass and higher risk of osteoporosis in adulthood.It suggests that osteoporosis exists developmental origin and may be originated from bone dysplasia in the embryonic and early postnatal period.'Previous studies showed that the directional differentiation from bone marrow mesenchymal stem cells(BMSCs)to the osteoblast involved in the formation of bone ossification center and the maintenance of bone mass during the process of development.Renin angiotensin system(renin angiotensin system,RAS),as an important endocrine regulation system of the body,participated in the directional differentiation of BMSCs to the osteoblast,which played an important role in the process of bone development.The activated local RAS was observed in the adult patients with osteoporosis,as well as the inhibited osteogenetic differentiation of BMSCs.But the correlation among RAS,osteogenetic differentiations of BMSCs,and the formation of ossification center was unknown in the embryonic and early postnatal period.Drinking is common daily life behavior and a portion of pregnant women still drink during pregnancy.Studies showed that prenatal alcohol exposure(PEE)can cause abnormal fetal development and a series of the development-related diseases in adulthood,such as adult neuropsychiatric diseases and metabolic syndrome.This kind of disease was collectively known as fetal alcohol syndrome(FAS)and IUGR is the main common characteristic.The researches in our and other labs showed that maternal GC overexposure was a commonality in the IUGR individuals caused by prenatal adverse environment.Studies suggest,the bone local RAS in patients with is glucocorticoid induced osteoporosis was activated and the osteogenetic differentiation ability was inhabited.However,whether PEE can cause developmental restriction of fetal long bone?Whether this effect could be sustained to the adulthood?Whether the mechanism is involved in the chronic activation of the RAS caused by maternal high GC level?These issues has not been reported.Therefore,this research focused on the following works:1,to observe the phenomenon of long bone dysplasia in the embryonic and early postnatal period on the basis of IUGR model on PEE offspring rats.2,to demonstrate the variation of the local RAS components of long bone at different time points after birth and to explore the intrauterine programming mechanism of long bone dysplasia on PEE offspring rats.3,to explore osteogenetic differentiation inhibition effect of high GC level induced by RAS activation on BMSCs.4,to explore the direct effects of ethanol to the osteogenetic differentiation of BMSCs.This study will to be beneficial for understanding the phenomenon of susceptibility to adult OP induced by PEE and its intrauterine programming mechanism,and better understand the intrauterine developmental origin of adult OP.The study also provides the theoretical foundation for early prevention and treatment of OP.PART1Prenatal ethanol exposure cause to long bone dysplasia in offspring rats in the embryonic and early postnatal periodObjective In recent years,based on a large number of researches among the correlation of the pregnancy adverse environment,fetal birth weight and adult chronic diseases,scholars proposed the concept of the origin of human diseases-"the developmental origin of health and disease"(DOHaD).The epidemiological survey results show that the birth weight is closely relative to the peak bone mass in adulthood,low birth weight adults present significantly increased risk to osteoporosis.Our laboratory found that PEE offspring rats presented lower body weight and body length than the control group in the stabile established IUGR model.Following,this study attempt to observe the phenomenon of long bone dysplasia in the embryonic and early postnatal period on the PEE offspring rats.Methods After natural conception,Wistar rats were randomly divided into control group and PEE group.From gestational day(GD)9,the pregnant rats in PEE group were given ethanol(4 g/kg,d)on gavage until the natural delivery,correspondingly,the control group were given isometric saline.The body weight,body length and the femur length of offspring rats were measured on the time point of GD 20,post week(PW)2 and PW6 respectively.The ossification center and growth plate was observed using HE staining,the morphological quantitative analysis of femoral epiphysis was evaluated by Micro CT scan,and the osteopsathyrosis was tested by biomechanical detecting instrument.Results 1,comparing to the control group,the PEE offspring presented lower body weight,shorter body length and femoral length at the time point of GD20,PW2,PW6,respectively.2,HE and the von Kossa staining showed the PEE offspring rats had shorter primary ossification center length on GD20,as well as growth plate length on PW2 and PW6,comparing to the control group.3,Micro CT quantitative analysis showed that PEE offspring rats had sparser and thinner bone trabecular,lower connectivity and bone mineral density(BMD)but higher porosity at PW2 and PW6 compared with the control group.4,bone biomechanical test showed that the PEE offspring rats had significantly reduced femur load and degrees than the control group at PW2 and PW6.Conclusion This study proposed and confirmed the phenomenon of long bone dysplasia in the embryonic and early postnatal period on the PEE offspring rats,which manifest as the shorten 'length of femur,the primary ossification center and growth plate,reduced cancellous bone mass,decreased bone elasticity and increased brittleness.PART 2Activation of the bone local RAS caused by maternal glucocorticoid overexposure mediated long bone dysplasia in PEE offspring ratsObjective A large number of studies suggest that patients with corticosteroid-induced osteoporosis(GIOP)present activated bone local RAS(increased mRNA expression of angiotensin converting enzyme(ACE)and angiotensin receptor AT1R,at the same time accompanied by osteogenetic differentiation inhibition.Our previous researches suggest that IUGR fetal rats caused by PEE exposed to the high maternal GC level,which is a recognized adverse intrauterine environment.The present study attempt to demonstrate the RAS intrauterine programming mechanism of lone bone dysplasia induced by PEE.Methods After natural conception,Wistar rats were randomly divided into control group and PEE group.From GD 9,the pregnant rats in PEE group were given ethanol(4 g/kg,d)on gavage until the natural delivery,correspondingly,the control group were given isometric saline.The femoral metaphysis containing cancellous bone area was respectively collected at different time points(GD20 PW2 PW6)and the bone tissue RNA was extracted the extraction by Trizol,osteogenetic and GC activation system related genes were detected.With hair testing offspring rat DNA methylation of ACE gene promoter region at cancellous bone area was detected by direct gene sequencing.Results 1.Osteogenetic differentiation related gene expression was depressed on the PEE offspring rats.2.The bone tissue local RAS of PEE offspring rats was activated in the embryonic and early postnatal period,manifested as:higher ACE mRNA expression,lower AT1R and AT2R expression and increased AT1R/AT2R ratio.3.The bone tissue GC activation system of PEE offspring rats was activated in the embryonic and early postnatal period,manifested as higher GR and C/EBPa mRNA expression levels.4,DNA of the bone local ACE gene promoter region was decreased on PEE offspring rats.Conclusion The present research demonstrated that the intrauterine programming mechanism of lone bone displysia induced by PEE may be involved in the bone local RAS activation(elevated ACE expression and suppressed ATRs expression)mediated by maternal GC overexpress for the first time.PART3High level GC inhibits BMSCs osteogenic differentiation through increased ACE expression mediated by GR/C/EBP alpha/DnmtsObjective Our research on Wistar rats demonstrated that PEE offspring rats presented a phenomenon of long bone dysplasia in the embryonic and early postnatal period,the intrauterine programming mechanism may be related to the bone local RAS activation mediated by maternal GC overexpress.However,there is no direct evidence to confirm this potential mechanism.The present study attempt to confirm the relationship between high GC level-RAS activation-the osteogenic differentiation depression of BMSCs using BMSCs osteogenic differentiation model.Methods On the stable established BMSCs osteogenic differentiation model,we cultured cells with different concentration of corticosterone(0,50,250,1250 ng/ml)for 5 days and observed its mineralization nodules staining.Cell RNA were extracted with Trizol,the genes expression related with osteogenetic RAS components were detected.On the BMSCs osteogenic differentiation model,after treated with the GR inhibitor Mifepristone(10 mM)or ACE inhibitor Enalapril(10 mm),the cells were cultured with corticosterone(1250 mM),the genes expression related with RAS components were detected.At the same time DNA methylation of ACE promoter region was detected with direct gene sequencing.Results 1.Treated with high concentration of corticosterone,osteogenetic markers mRNA expression of BMSCs decreased significantly,containing BSP,BGLAP,Runx2 and ALP.2.Treated with high concentration of corticosterone,the mRNA expression of GR/C/EBPa increased significantly,RAS are activated(increased ACE mRNA expression and decreased AT1R and AT2R expression).The total DNA methylation rate of ACE promoter region decreased and the DNA methyltransferase Dnmtl mRNA expression was increased significantly.3.After cultured with GR inhibitor BMSCs osteogenic ability was reversal compared with corticosterone only,as well as the ACE inhibitor.Conclusion The present study confirm that high level corticosterone decreased DNA methylation rate,increased ACE mRNA expression and depression osteogenic differentiation by GC activating system for the first time.PART4Effect of ethanol on osteogenic differentiation of rat BMSCs Objective To investigate the direct effect of ethanol on osteogenic differentiation of BMSCs in vitro,and to elucidate the potential mechanism.Methods The primary BMSCs was isolated and subcultured to the third generation.On the BMSCs osteogenic differentiation model,cells was treated by ethanol(0,0.8,4,20,100 mm).The proliferation was detected with MTT and related gene expression was tested.Results 1,Ethanol with different concentrations had no significant toxic effects on proliferation of BMSCs.2,After treated with ethanol(100 mM),genes expression related with osteogenic differentiation decreased but GR/GRC/EBPa.3,After treated with ethanol(100 mM),BMSCs present increased Egr-1 expression but decreased 11 ?-HSD2 expression.Conclusion Ethanol could directly inhibit osteogenic differentiation of BMSCs.Its underlying mechanisms may be associated with inhibition of the expression of 11-?-HSD2 by upregulating cAMP/PKA/Egr-1,which could result in the GC barrier effect decrease.