| Osteoarthritis(OA)is one of the most common chronic diseases in today’s society.Its morbidity and prevalence rate will increase with age and the people over 65 years old can even reached 50%,which is the primary cause of joint pain and chronic disability in the elderly.It is an important public health problem in the world with not only causes the decline of limb function and seriously affects people’s quality of life,but also consumes expensive medical care and social costs.In recent years,it was found that adult diseases are associated with low birth weight of the fetus,and the hypothesis of "the fetal origin of adult diseases " was proposed.Epidemiology shows that the incidence of osteoarthritis in the joints of the hands,knees and hips of adults with low birth weight increases significantly.It is suggested that osteoarthritis exist fetal origin.The development of osteoarthritis is complicated,which is related to various factors such as age,gender,trauma,genetics and metabolism.Although osteoarthritis was long regarded as a major disease of articular cartilage,the role of subchondral bone is drawing increasing attention,and it plays a crucial role in the pathogenesis of osteoarthritis.Epidemiological survey shows that osteoporosis(OP)is closely associated with the development of osteoarthritis.Many patients with osteoarthritis have osteoporosis,and postmenopausal women are more prone to develop osteoarthritis.Some scholars proposed the concept of osteoporotic osteoarthritis,suggesting that osteoporotic changes in subchondral bone could lead to microfracture and accelerate the progression of osteoarthritis.Bone is a highly vascularized connective tissue and bone vessels play an important role in the process of bone development,regeneration and remodeling.There are two forms in the process of bone formation.One is endochondral osteogenesis,which is a typical feature of the development of long tubular bones(e.g.,femur or tibia),and it mainly occurs in cartilage.The other is intramembranous osteogenesis,which is the main mechanism of flat(e.g.,craniofacial)bone development,and it is related to the direct differentiation of bone mesenchymal stem cells into osteoblasts.Regardless of the osteogenesis method,angiogenesis plays a key role in its process.The growth of bone vessels is coupled with osteogenesis,and is closely related to the formation of new bone.H-type blood vessels are newly discovered blood vessel subtype found in bone tissue(a small amount exist in liver)in recent years,which can promote the proliferation and differentiation of bone progenitor cells.The abundance of H-type blood vessels in bone tissue has a significant positive correlation with bone mass,that is,the more H-type blood vessels,the higher the bone mass,and vice versa.It is suggested that the reduction of H-type blood vessels generation may be one of the mechanisms of low subchondral bone mass.Yes-associated protein(YAP)is a key effector protein of the Hippo signaling pathway,which can enter the cell nucleus to participate in the regulation of gene transcription.It can regulate the homeostasis of tissues,organ size,cell death,and stem cell differentiation.Recent studies found that YAP is closely related to angiogenesis,and it plays a role in regulating angiogenesis as a key factor mediating VEGF-VEGFR signaling pathway.At the same time,YAP can promote the expression of CTGF,CYR61,Ang-2,CD31 and other genes after nuclear import,thereby promoting the proliferation,migration and tube formation of endothelial cells,which plays an important role in regulating angiogenesis.It is indicated that YAP may be involved in the generation of H-type blood vessels in bone tissue.Caffeine is a purine-like alkaloid that naturally exists in nearly 60 plants,as well as cocoa,soda and a variety of energy drinks.As a result,caffeine intake is an inevitable part of our diet.Epidemiology and animal experiments show that caffeine can result in reproductive and developmental toxicity,including intrauterine growth retardation(IUGR).Our previous studies also demonstrated that prenatal caffeine exposure(PCE)can lead to delayed skeletal development in offspring,characterized by delayed endochondral ossification and restricted development of growth plate.Basic level of glucocorticoids(GC)are essential for the growth,development and functional maturation of fetal bone.However,long-term use of dexamethasone and excessive exposure to endogenous glucocorticoids in pregnant women can lead to abnormal development of fetal bone.Our previous studies shown that PCE could increase the level of corticosterone in fetal blood of rats and result in the dysplasia of long bone in offspring,which will eventually lead to the reduction of long bone mass production and the decrease of bone mass in adulthood.At the same time,PCE could lead to susceptibility to osteoarthritis caused by poor articular chondrocyte differentiation in adult offspring rats.However,it was not studied whether PCE can cause continued subchondral bone dysplasia and reduction of bone mass production in offspring after birth and lead to susceptibility to osteoarthritis in adulthood.Micro RNA regulation is one of the main epigenetic modifications and has became an important post-transcriptional regulator in various physiological and pathological processes.Micro RNA can regulate the angiogenesis by regulating the proliferation and migration of endothelial cells in various tissues and organs(heart,brain,kidney,tumor,etc.).Studies suggests that micro RNA can also regulate the generation of H-type blood vessels and osteogenesis of bone tissue.While involved in regulating the expression of various genes,mi RNA’s own expression is also affected by various factors.Glucocorticoids mainly activate the glucocorticoid receptor(GR),and then bind to the promoter region of related genes to complete the regulation of these genes.P300 is a acetyltransferase that can cause histone acetylation by cooperating with GR to participate in the regulation of downstream genes.In summary,glucocorticoids may regulate key micro RNAs in the process of the generation of H-type blood vessels through GR and P300.In summary,does PCE cause the reduction of subchondral bone mass generation in offspring after birth,which in turn leads to increased susceptibility to osteoarthritis?Is its mechanism related to the reduction of H-type blood vessels generation in the subchondral bone of the offspring? Is the reduction of H-type blood vessels generation associated with decreased expression of YAP? Does glucocorticoid regulate the expression of YAP in bone tissue through mi RNA? Does glucocorticoid regulate the expression of key mi RNAs through GR and P300? These issues have not yet been reported.Based on these questions,the following work is proposed in this study:(1)We established the IUGR model of the offspring induced by PCE,and observed the development of ossification center/subchondral bone in female offspring rats at gestational day 20(GD20)and postnatal week 6(PW6)before and after birth.At the same time,the female offspring rats were bilaterally ovariectomized to make a castration-osteoporosis model at PW22,and the subchondral bone mass and osteoarthritis phenotype were detected at PW28.We further analyzed the correlation between the subchondral bone mass and osteoarthritis phenotype.(2)We further observed the changes of the bone mass and the abundance of H-type blood vessel in the ossification center/subchondral bone of PCE female offspring at GD20,PW2,PW6 and PW12.And the intrauterine programming mechanism of susceptibility to osteoarthritis of PCE female offspring in adult was investigated by observing the expression changes of related mi RNA and its target genes of bone tissue at different time points.(3)In the model of rat bone marrow endothelial progenitor cells,the effect of corticosterone on the differentiation and function of tube formation of bone marrow endothelial progenitor cell was explored.And we confirmed the molecular mechanism of decreased H-type blood vessel generation of subchondral bone in female offspring induced by PCE through detecting the expression of related mi RNA and its target genes,GR and P300.The implementation of this topic helps us to comprehensively understand the susceptibility to osteoarthritis of PCE offspring and its intrauterine programming mechanism,which provides a theoretical basis for further exploration of early prevention and treatment of fetal-originated adult osteoarthritis.PART ONE Subchondral bone dysplasia mediates susceptibility to osteoarthritis in female adult offspring rats induced by prenatal caffeine exposureObjective This part of the study intends to use female offspring IUGR rat model induced by PCE and subchondral bone osteoporosis model induced by ovariectomy to investigate whether the increased susceptibility to osteoarthritis is associated with decreased bone mass production due to subchondral bone dysplasia in adult female offspring of PCE by observing the changes of the development and osteogenic differentiation function of ossification center/subchondral bone before and after birth and subchondral bone mass after birth and analyzing the correlation between subchondral bone mass and osteoarthritis phenotype in adulthood.Methods Wistar female rats were randomly divided into control group and PCE group after naturally conceived.From GD9 to GD20,the rats in PCE group were administered caffeine(120 mg/kg.d)intragastrically,and the control group was given equal volume of saline.Some pregnant rats were euthanized after being anesthetized with 3% pentobarbital sodium at GD20.The fetuses were removed by laparotomy and one female offspring rat per litter was randomly selected and sacrificed.At same time,fetal blood,femurs and tibias of both lower limbs were collected.The remaining pregnant rats went on to spontaneous labor to produce offspring which were fed normally until PW6.One female offspring rat per litter was randomly selected and euthanized after being anesthetized with 3% pentobarbital sodium to collect the femurs and tibias of both lower limbs.The rest offspring continued to be fed normally to PW28,and one female offspring rat per litter and euthanized after being anesthetized with 3% pentobarbital sodium to collect the femurs and tibias of both lower limbs.In addition, one female offspring rat per litter was randomly selected to undergo bilateral ovariectomy at PW22 and euthanized after being anesthetized with 3% pentobarbital sodium at PW28 to collect the femurs and tibias of both lower limbs.The related indexes of the above femur samples were tested.Results(1)Compared with the control group,the articular cartilage phenotype of the female offspring rats in the PCE group showed significant changes in PW28,which were manifested by the shallow coloration of articular cartilage,decreased articular cartilage thickness(P<0.05)and increased Mankin score(P<0.05),which was aggravated after ovariectomy(P<0.01).At the same time,the expression of Sox9 decreased both in non-ovariectomy and ovariectomy rats(P<0.05,P<0.01).The expression of Col2α1 did not change in non-ovariectomy rats but decreased after ovariectomy(P<0.05).The expression of MMP13 and ADAMTS5 increased in nonovariectomy and ovariectomy rats(P<0.05,P<0.01).The similarly increased expression of Caspase3 existed both in non-ovariectomy and ovariectomy rats(P<0.05,P<0.01).(2)Compared with the control group,the subchondral bone mass of the PCE group was significantly decreased,which was reflected by the decrease of bone volume/tissue volume(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)(P<0.05,P<0.01)and the increase of trabecular separation(Tb.Sp)(P<0.05),and that was aggravated after ovariectomy(P<0.05,P<0.01).(3)Mankin score or articular cartilage thickness was not correlated with subchondral bone mass in the control group.Whereas in the PCE group,the Mankin score was negatively correlated with BV/TV,Tb.N and Tb.Th(P<0.05,P<0.01)and positively correlated with Tb.Sp(P<0.05).The articular cartilage thickness was positively correlated with BV/TV,Tb.N and Tb.Th(P<0.05)and negatively correlated with Tb.Sp(P<0.05).(4)Compared with the control group,the female fetal rats in the PCE group showed a delayed development of the primary ossification center,which was shown by the shortening of the length and the decrease of the area of the primary ossification center(P <0.01).The development of the secondary ossification center was delayed of the female offspring rats in the PCE group at PW6,which was shown by the shortening of the length and the decrease of the area of secondary ossification center(P<0.05,P<0.01).At the same time,the bone mass of secondary ossification center/subchondral bone in PCE female offspring rats was lower than that of the control group,in which BV/TV,Tb.N,Tb.Th,decreased(P<0.05,P<0.01)and Tb.Sp increased(P<0.01).(5)The number of osteoblasts of the rats in the PCE group was decreased both in GD20 and PW6 compared with the control group(P<0.05,P<0.01).Meanwhile,the m RNA levels of transcriptional activator Runx2,osteogenic differentiation genes BSP,ALP,and OCN were all decreased(P<0.05,P<0.01).Conclusion PCE can induce susceptibility to osteoarthritis in adult female offspring.The potential mechanism is likely to be related to the reduction of subchondral bone mass production caused by the dysplasia of the primary and secondary ossification centers due to osteogenic differentiation disability induced by PCE.PART TWO Decreased H-type blood vessels generation mediates low subchondral bone mass in female offspring rats induced by prenatal caffeine exposureObjective This part of the study intends to observe the changes of bone mass and the abundance of H-type blood vessels of ossification center/subchondral bone in PCE female offspring rats at different periods through the animal experiment.And further detect the changes of the expression of related mi RNA and its target genes,GR,P300 in order to explore the intrauterine programming mechanism of the decreased H-type blood vessels generation of subchondral bone in female offspring induced by PCE.Methods Wistar female rats were randomly divided into a control group and a PCE group after naturally conceived.From GD9 to GD20,the rats in PCE group were administered caffeine(120 mg/kg.d)intragastrically,and the control group was given equal volume of saline.Some pregnant rats were euthanized after being anesthetized with 3% pentobarbital sodium at GD20.The fetuses were removed by laparotomy and one female offspring rat per litter was randomly selected and sacrificed.At same time,fetal blood,femurs and tibias of both lower limbs were collected.The rest offspring including the control group and the PCE group went on to spontaneous labor to produce offspring which were fed normally until PW2.One female offspring rat per litter was randomly selected and euthanized after being anesthetized with 3% pentobarbital sodium to collect the femurs and tibias of both lower limbs.The remaining offspring were fed normally until PW6,and one female offspring rat per litter was randomly selected and euthanized after being anesthetized with 3% pentobarbital sodium to collect the femurs and tibias of both lower limbs.The rest offspring continued to be fed normally to PW12,and one female offspring rat per litter was randomly selected and euthanized after being anesthetized with 3% pentobarbital sodium to collect the femurs and tibias of both lower limbs.The related indexes of the above femur samples were tested.Results(1)Compared with the control group,the bone mass of the primary ossification center in GD20 of the female offspring in the PCE group were decreased,which was manifested as the decrease in BV/TV,TB.N and trabecular bone width(TB.Wi)(P<0.05,P<0.01)and the increase in TB.Sp(P<0.01).The bone mass of the secondary ossification center in PW2 continued to decrease with decrease of BV/TV,TB.N and TB.Wi(P<0.05,P<0.01)and the increase in TB.Sp(P<0.05).The subchondral bone mass of female offspring rats in the PCE group was still reduced in PW6 and PW12 compared with the control group,which showed the decrease of BV/TV,TB.N and TB.Th(P<0.05,P<0.01)and the increase of TB.Sp(P<0.05,P <0.01).(2)Compared with the control group,the number of H-type blood vessels in the primary ossification center of female fetal rats in the PCE group decreased(P<0.01),and the number of H-type blood vessels in the secondary ossification center of the PCE female offspring at PW2 was still decreased(P<0.05),and the number of H-type blood vessels in subchondral bone of the PCE female offspring continued to decreased at PW6 and PW12(P<0.05,P<0.01).(3)Compared with the control group,the expression of mi R-6215 in the primary ossification center of PCE female fetal rats increased through microarray analysis(P<0.01).Compared with the control group,the expression level of mi R-6215 in the secondary ossification center of PCE female offspring rats continued to increase in PW2(P<0.01).Compared with the control group,the expression level of mi R-6215 in the subchondral bone of the PCE female offspring rats was still elevated in PW6 and PW12(P<0.01).(4)YAP1 was predicted and verified to be the target gene of mi R-6215.Compared with the control group,the expression of YAP1 in the blood vessels of primary ossification center was decreased in the PCE group(P<0.01).Compared with the control group,the expression of YAP1 in the blood vessels of secondary ossification center in PCE offspring rats continued to decrease in PW2(P<0.01).Compared with the control group,the expression of YAP1 in the blood vessels of the subchondral bone in PCE offspring rats was still decreased in PW6 and PW12(P<0.05).(5)Compared with the control group,the expression of GR and P300 of the primary ossification center of female fetal rats in the PCE group increased(P <0.01).However,there was no significant change in the expression of GR and P300 of the secondary ossification center in PW2 in the PCE female offspring rats compared with the control group.Compared with the control group,the expression of GR and P300 in subchondral bone of PCE female offspring rats remained unchanged in PW6 and PW12.At the same time,the histone acetylation(H3K27ac)of the mi R-6215 promoter region in the primary ossification center of the fetal rats in the PCE group increased compared with the control group(P<0.01).Conclusion PCE can cause decreased H-type blood vessels generation and low bone mass in the primary ossification center of female offspring fetal rats,and the mechanism may be related to the decreased expression of YAP1 inhibited by increased expression of mi R-6215 caused by elevated H3K27 ac in the promoter region of mi R-6215 which was upregulated by GR and P300 in bone tissue.At the same time,the high expression of mi R-6215 continued after birth,which eventually led to the sustained suppression of H-type blood vessels generation and low bone mass in the secondary ossification center/subchondral bone of the offspring.PART THREE Effects of corticosterone on differentiation and tube formation function of rat bone marrow endothelial progenitor cells and its molecular mechanismObjective This part of study intends to explore whether corticosterone could inhibit the differentiation and tube formation function of rat bone marrow endothelial progenitor cells and confirm that the change of mi R-6215/YAP1 caused by corticosterone is the main mechanism of decreased H-type blood vessels generation in the subchondral bone of PCE female offspring rats,and deeply explore whether corticosterone could upregulate mi R-6215 through GR and P300 to inhibit the expression of YAP1.Methods The tibia and femur of PW4 Wistar rats were taken to extracted the primary bone marrow endothelial progenitor cells by gradient centrifugation.The following treatments were carried out:(1)Corticosterone treatment: cells were treated with corticosterone at different concentrations(0,250,500,1000,1500 n M)for 72 hours,and the cell viability,differentiation ability to mature endothelial cells and tube formation ability on Matrigel of bone marrow endothelial progenitor cells were observed.Meanwhile,the expressions of mi R-6215/YAP1,GR and P300,as well as the histone acetylation of mi R-6215 were detected.(2)Bone marrow endothelial progenitor cells was treated with corticosterone(1000 n M)and mi R-6215 inhibitor or YAP1 overexpressing agent or GR antagonist Mesperidone(RU486)or P300 inhibitor (P300-si RNA)after 72 hours respectively.The differentiation and tube formation function of bone marrow endothelial progenitor cells,the expressions of mi R-6215/YAP1,GR,P300 and the histone acetylation of mi R-6215 were detected.(3)Bone marrow endothelial progenitor cells were treated with corticosterone(1000 n M)to detect whether the combination of GR to P300 was changed.Results(1)The cell viability of bone marrow endothelial progenitor cells did not change significantly after treatment with different concentrations(0,250,500,1000,1500 n M)of corticosterone.Compared with the control group,corticosterone treatment at different concentrations(500,1000,1500 n M)can significantly promote the expression of mi R-6215 and inhibit the expression of YAP1,which exhibited a doseeffect relationship(P<0.05,P<0.01).(2)Compared with the control group,corticosterone treatment with different concentrations(500,1000,1500 n M)could significantly inhibit the differentiation and tube formation function of bone marrow endothelial progenitor cells(P<0.05,P<0.01).(3)Compared with the control group,the differentiation and tube formation function of the bone marrow endothelial progenitor cells decreased after being treated with corticosterone(1000 n M)(P<0.01).At the same time,compared with the corticosterone treatment group,the differentiation and tube formation function increased in the corticosterone and YAP1 overexpression co-treatment group after the administration of overexpression of YAP1(P<0.05).(4)Compared with the control group,the differentiation and tube formation function and YAP1 expression and nuclear import of bone marrow endothelial progenitor cells decreased after being treated with corticosterone(1000 n M)(P<0.01).Meanwhile,compared with corticosterone treatment group,the differentiation and tube formation function and YAP1 expression and nuclear import of bone marrow endothelial progenitor cells increased in corticosterone and mi R-6215 inhibitor co-treatment group after administration of mi R-6215 inhibitor(P<0.05,P<0.01).(5)Compared with the control group,there was no significant change in the expression of GR in the corticosterone(1000 n M)treatment group,but the expression of P300 increased(P<0.01),and both GR and P300 increased in the nucleus(P<0.05).Compared with corticosterone treatment group,the differentiation and tube formation function of bone marrow endothelial progenitor cells increased in corticosterone and RU486 cotreatment group(P<0.05).Meanwhile,the expression of mi R-6215 decreased(P<0.05),YAP1 expression and nuclear import increased(P<0.05),the expression of P300 did not change significantly,but its nuclear import decreased(P<0.05).Compared with corticosterone treatment group,the differentiation and tube formation function increased(P<0.05),the expression of mi R-6215 elevated and YAP1 expression and nuclear import increased(P<0.05),but GR expression and nuclear import did not change significantly of bone marrow endothelial progenitor cells in corticosterone and P300-si RNA co-treatment group.(6)Compared with the control group,the combination of GR and P300 in the corticosterone(1000 n M)treatment group increased significantly.At the same time,compared with the control group,there was no significant change in the H3K9 ac,H3K14ac,H3K27 ac of the IFFO2 promoter region and in H3K9 ac,H3K14ac of the mi R-6215 promoter region,but there was a significant increase in H3K27 ac of the mi R-6215 promoter region(P<0.01),and the H3K27 ac both showed a decrease after the administration of RU486 or P300-si RNA(P<0.05).Conclusion Corticosterone can inhibit the differentiation and tube formation function of rat bone marrow endothelial progenitor cells.The molecular mechanism is related to corticosterone binds to the mi R-6215 promoter region through GR and recruited P300,which leads to the up-regulation of the histone acetylation and expression of H3K27 of mi R-6215 promoter region,thereby promoting the expression of mi R-6215,and then inhibiting the expression and nuclear import of YAP1.In conclusion,we confirmed the susceptibility to osteoarthritis in female offspring in the rat IUGR model induced by PCE and the ovariectomy-induced adult rat osteoporosis model as well as the bone marrow endothelial progenitor cell model.The mechanism is related to the decreased H-type blood vessel generation and low bone mass of subchondral bone caused by continued increase of mi R-6215 and decrease of YAP1 before and after birth which was the result of the increased GR,P300 and mi R-6215 acetylation of primary ossification center in utero induced by PCE.In brief,this study elucidated the H-type dysplasia of subchondral bone mechanism of the female offspring’s susceptibility to osteoarthritis induced by PCE,and provided a new direction for early prevention of the fetal-originated adult osteoarthritis. |
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