Mechanism Of Fetal Programming In Adult Osteoporosis Induced By Prenatal Caffeine Exposure

Osteoprosis is a common metabolic bone disease with characteristics of bone loss, weaker biomechanics of bone strength and higher fracture incidence, high bone absorption or low bone synthesis is the main causes.Pathology peculiarity showed thin bone cortex, trabecular bone loss, deformation, thinner and disordered arrangement. According to statistics, in 1980-2008, steoporosis incidence in China is 6.6%to 19.3%. Our country population base of osteoporosis is big, According to the trend of population development, predictions showde that by 2020 the number of osteoporosis disease in our country will be more than 150 million, which will bring huge economic burden to the society. Epidemiological investigation showed that dexamethasone exposure during pregnancy, smoking, nutrition cause reduce of children’s bone mass, in addition, low birth weight is directly related to adult bone loss and osteoporosis. Prompt that osteoporosis with fetal origins.A variety of adverse environment (such as exogenous, alcohol, drugs, etc.) can cause fetal low birth weight and organ dysplasia. Laboratory results confirmed that caffeine exposure during pregnancy can cause maternal glucocorticoid exposure, leading to fetal developmental delays and fetal rats long bone dysplasia, showing shorter body length and delays in the primary ossification center. So, what about the intrauterine programming mechanism caused by caffeine exposure of maternal high GC during pregnancy caused by exposure of caffeine? Can the performance of the intrauterine dysplasia continue to after birth and eventually cause the occurrence of osteoporosis in adult?In 2014, Nat Rev Endocrinol series report that maternal source of high GC exposure may be the key factors mediating intrauterine programming. Renin angiotensin system is an important humoral regulation system in the human body. A growing body of evidence showed that the RAS is involved in regulation process of the development of bone, RAS activation cause delayed osteoblastic differentiation and even osteoporosis. Prompting that intrauterine maternal GC exposure may cause chronic activation of RAS, and osteogenetic differentiation inhibiting, changes of the intrauterine programming may continue into adulthood, leading high osteoporosis susceptibilitis.Therefore, this topic will establish a IUGR rats model caused by caffeine exposure during pregnancy and its adult before and after castrate stimulation, focusing on the study of IUGR in intrauterine fetus and adult bone development, RAS and osteogenesis after castration treatment and differentiation ability of change;Further from the perspective of cells in the experiment, this paper discusses on the high GC model of BMSCs and the relationship between RAS and osteogenesis ability of differentiation, caffeine to clarify the mechanisms of the changes in the fetal RAS function development, parsing the fetal development origins of osteoporosis, fully understand adult new risk factors for osteoporosis and guiding eugenics.PART ONEOsteoporosis susceptibility phenomenon and mechanisms in adult offsprings caused by Caffeine exposure in IUGR RatsObjective To demonstrate caffeine exposure during pregnancy could lead to IUGR adult bone RAS activation and susceptibility to osteoporosis on rat model. Methods The animals were divided into four main groups, control group, caffeine group, castration group, caffeine +castration group. Rats were used to establish IUGR model induced by caffeine exposure during trimester of gestation (120 mg/kg-d), from day 9 to day 20, the females were anesthetized with isoflurane and the fetuses were removed from uteri by cesarean section, dried of amniotic fluid, weighed and examine the length of body as well as tail. After castration group of young rats born 5.5 month (PM5.5) to remove bilateral ovaries processing, all rats 7 month executed, normal control group and caffeine group under the knee joint tibial cancellous bone, parts used for detecting content of angiotensin Ⅱ part used to extract RNA, real time quantitative PCR technology, real-time detection of tibia cancellous bone RAS (ACE, AT1R and AT2R) mRNA expression.Castration group and castration group take the femur length, caffeine MicroCT changes of the bone strength, fragments of volume fraction (BV/TV), bone trabecular number (TB. N), trabecular bone porosity (TB. SP), bone trabecular thickness (TB. TH), the density of bone is connected (Conn. D.) and trabecular bone connection degree (SWI).Take part under the knee joint tibial cancellous bone extract RNA, using real-time quantitative PCR technology, real-time detection of the tibia cancellous bone RAS (ACE, AT1R and AT2R) mRNA expression.All the needful organs or tissues were isolated and stored at -80℃ immediately. Results Caffeine exposure during pregnancy (120 mg/kg-d) adult female rats exposed to foundation condition angiotensin Ⅱ content increased significantly (P< 0.05), bone ACE expression (P< 0.05).Compared with castration group, caffeine during pregnancy (120 mg/kg·d) exposure in castrated female adult rats model induced by reduced bone strength and bone 3 d scanning showed osteoporosis degree aggravating, the main index for BV/TV value, TB. N number is reduced, and TB. SP increased (P< 0.05), TB. Th, Conn. D, SWI value did not change significantly, the tibia cancellous bone ACE mRNA expression increased (P< 0.05).Conclusion Pregnancy middle-late caffeine (120 mg/kg-d) exposure of IUGR caused by young adult castrated rats before and after the treatment of bone RAS are in a state of activation, increased susceptibility to osteoporosis.PART TWOPrenatal caffeine exposure induced maternal GC overexposure to fetus and abnormality of fetal long bone developmentObjective On caffeine caused IUGR fetal rats model to ivestigate the effect of maternal sourced high GC on bone marrow mesenchymal stem cells (BMSCs) proliferation and the influence of the dispersion of osteogenesis and bone tissue local RAS, confirmed fetal bone is the primary ossification center developmental delay and the phenomenon of long bone dysplasia and cellular mechanisms. Methods Wistar rats fed a week later, a night at 6pm by female:male= 2:1 or cage, rick observe female Yin chuan or vaginal smear microscopy, for the day of conception to Yin chuan or sperm 0 d.Pregnant rats were randomly divided into two groups:normal control group and caffeine during pregnancy during pregnancy treatment groups, each group of 12.Caffeine during pregnancy treatment group in pregnant 11 d, with 120 mg/kg · d caffeine to fill the stomach, once per day, continuous dosing to was born the day before (GD20), under isoflurane anesthesia was executed to pheromones, selected IUGR female fetal rats, ossification center part in liquid nitrogen frozen after the transfer, to-80 degrees c cryopreserved for Elisa method to detect the contents of angiotensin Ⅱ;Part of the long bone in a fixed in 4% paraformaldehyde, used to make pathological section;Long bone ossification center part put in 1 ml TRIZOL in-80-c cryopreserved, used to extract total RNA;HE staining and Von kossa staining techniques, detection of prenatal fetal rats (GD20) primary ossification center, relative length;Real time quantitative PCR technology, real-time detection of fetal bone primary ossification center RAS (ACE, AT1R and AT2R) and osteogenetic differentiation (Runx2, BGLAP, BSP, ALP) the mRNA expression of related indicators;The rest of the normal female fetal rats and caffeine group IUGR female fetal rats, to two weeks after birth (PW2) executed young mice, extract the original generation of BMSCs for counting cell clones (CFU-F) detection.The methyl violet CFU-F clone dyeing technology, testing two weeks after birth (PW2) of the rat BMSCs colony formation.Results Caffeine (120 mg/kg·d) treatment group, fetal rat primary ossification center relative length significantly shortened (P< 0.05);Angiotensin Ⅱ content increased obviously (P< 0.05);Fetal bone primary ossification center ACE expression increased obviously, AT1R/AT2R expression than rise significantly (P< 0.01), the osteogenetic differentiation related gene (Runx2, BGLAP, BSP, ALP) express lower (P< 0.05);Compared with normal control group, two weeks after the birth of animals osteogenic functions decreased significantly (P< 0.05).Conclusions Caffeine exposure during pregnancy can pass the "maternal high GC exposure" to RAS activation in bone, reduce fetal bone osteogenesis ability of differentiation, the primary ossification center retardation, characterized by long bone palace dysplasia.Based decreased fetal BMSCs pool, reduce proliferation ability.PARTTHREEGR/ACE induced the changes of high GC caused bone mesenchamal stem cells osteogenic retardationObjective At a cellular level, bone marrow mesenchymal stem cells (BMSCs) as the representative, studying the different concentrations of corticosterone processing-GR expression increasing-ACE expression-RAS activation-osteogenesis differentiation ability to reduce the inner link between, confirm GR/ACE expression mediated by caffeine fetal bone GC activation and osteogenesis ability is reduced, and through the GR inhibitors mifepristone (5 um) and angiotensin converting enzyme inhibitors enalaprilat (5 um) use, discusses high GC RAS activation and reduce osteogenetic differentiation mechanism.Methods Establish the original generation of BMSCs in vitro culture system to 0-250 mM corticosterone deal with BMSCs,7 days a real-time quantitative rt-pcr technique to detect the differentiation of BMSCs development under the condition of the ACE, AT1R and AT2R and osteogenetic differentiation related Runx2, ALP, the mRNA expression of BSP and osteocalcin. Results (1)different concentrations of corticosterone treatment can significantly increase the bone marrow mesenchymal stem cells between the ACE mRNA expression (P< 0.01, P< 0.05), and at the same time make the AT1R/AT2R expression than raised (P< 0.01, P< 0.05), and inhibits osteogenesis differentiation related gene (Runx2, BGLAP, BSP, ALP) expression (P< 0.05).(2) the GR inhibitors and the effect of ACEI:GR inhibitors can significantly reduce the use of BMSCs GR and ACE mRNA expression (P< 0.01, P< 0.05);The use of ACEI can reverse corticosterone treatment of BMSCs ACE mRNA expression increased (P< 0.01, P< 0.05), and to reverse the related genes of corticosterone on osteogenesis differentiation Runx2, ALP, BSP and lower osteocalcin mRNA expression effect (P< 0.01, P< 0.05). Conclusions High GC environment (cortisone) may by increasing the activation of the GR expression of BMSCs, cause the increase of the ACE, this effect can be osteoblastic differentiation related genes they Runx2, ALP, BSP, and reduce the expression of osteocalcin.High caffeine exposure caused by GC environment on fetal bone RAS activation may be the cause of fetal long bone dysplasia, and ultimately caused IUGR and adult osteoporosis susceptibility of intrauterine origin.