Effects Of Lipopolysaccharide Exposure During Embryonic Stage And Early Stage After Birth On Bone Development And Bone Homeostasis

Bone system is composed of bone tissue and bone marrow.In recent years,it has been found that bone not only plays important roles such as exercise,protecting the internal organs,supporting human body,participating in the calcium and phosphate metabolism and other basic functions,but also been involved in a variety of physiological and pathological processes.For example,osteoblasts participate in the formation of hematopoietic stem cell niche and enhance the proliferation of hematopoietic stem cells,while osteoclasts have an important functionon hematopoietic stem cell mobilization.Bone can also produce a variety of factorssuch as Osteocalcin,IGF-1,FGF23,thus regulating energy metabolism,affecting the brain cognition and the reproductive system,etc.The role of the bone in hematopoiesis,energy metabolism and other systems depends on the maintenance of bone homeostasis.Bone formation mediatedby osteoblast couples bone resorption mediated by osteoclast to regulate bone homeostasis.The imbalance of bone formation and bone resorption can lead to osteoporosis.Osteoporosis is a kind of chronic metabolic bone disease caused by a variety of reasons leading to bone strength decreasing.Smoking,drinking,menopause,genetic factors are all related to the increased incidence rate of osteoporosis.With the trend of population aging in China,the incidence of osteoporosis is increasing year by year,which has become a serious impact on the health of the elderly in China.Bone mineral density reduction caused by osteoporosis can significantly increase the risk of fracture.Hip fracture is one of the most serious complications of osteoporosis which related with high mortality and disability rate.However,the mechanism of osteoporosis has not been fully clarified.The developmental origins of health and disease hypothesis postulates that the experience of various unfavorable factors during early stage of development will affect the incidence rate of adult chronic non communicable diseases(NCDs),such as osteoporosis,diabetes,asthma,cancer,cardiovascular disease,mental disease,etc.But the specific effect and mechanism about unfavorable factors exposure in early life on bone development and bone homeostasis in adulthood is still unknown.These series of experiments are helpful to further reveal the pathogenesis of osteoporosis and to clarify the relationship between the exposure of adverse factors during early stage of development of life and the originof the development of chronic non-communicable diseases in adulthood.Objective:We take advantage of two kinds of mice models---LPS exposure during the embryonic stage and LPS exposure during the early stage after birth to dissect the effect on the early development period and bone mass of the adulthood and their mechanism by measuring growth plate length,physical parameters,bone mass,osteoclast activity,osteoblast activity and other parameters.Methods:Part I:1.The metatarsal and tibia of mice on E15.5 were isolatedand divided randomly into the blank group and the LPS group,treated with vehicle and LPS 10 ? g/m L(0111:B4)respectively for 7 days.2.Measurement of the length of total length(TL)and calcified zone(CZ)of the metatarsal and tibia,calculate TL-rate,CZ-rate,CZ%.3.Tissues sampling and trimming;measurement of hypertrophic zone;detection of the chondrocytes differentiation by type II collagen and type X collagen immumohistochemical staining;observation of the apoptosis of chondrocytes by TUNEL staining.4.The pregnant mice were randomly divided into 2 groups:(1)LPS group,given 25ug/kg LPS i.p on E15.5;(2)control group,administered equal dose of physiological saline on E15.5;Tissues sampling and trimming on 2m,4m and 8m.5.Phenotype observation: measurement of body weight and tail length.6.Body X-ray,femur X-ray,femur Micro-CT scan and reconstruction.7.Safranin-O and Fast Green staining of tibia sections was used to observe the growth plate,HE staining to observe the trabecular bone,TRAP staining to observe the osteoclasts.8.RT-PCR was used to detect the expression levels of Osteogenic related gene(OP?OC)and the osteoclast related gene(Tracp?Ctsk?Mmp9).Part II:1.The C57 mice on the fifth day after birth were randomly divided into 2 groups:(1)LPS group,given 2mg/kg LPS(0111:B4)i.p;(2)control group,administered equal dose of physiological saline;Tissues sampling and trimming on 2m,4m and 8m.2.The same as 5-8 in Part IResults:Part I:1.TL-rate and CZ-rate of tibia and metatarsal of LPS group decreased compared with the control group;the hypertrophic zone in LPS group was significantly shorter than that in control group(P<0.001);the expression of Col II,Col X in LPS group decreased;the apoptosis of chondrocytein LPS group was increased.2.Compared with the control group,the tail length in LPS group was shorter and the body weight of LPS group was lower in 0d~2m(P<0.05).3.Bone mineral density decreased in LPS group.CT results show that the related indexes of trabecular bone BV/TV,TB.N,TB.Th were decreased(P<0.05)and TB.SP,SMI were increased(P<0.05);the related indexes of cortex BV/TV,C.Th were also decreased(P<0.05).4.In LPS group,the growth plate became narrow(P<0.05),the trabecular bone volume was fewer,indexes related to the function of osteoclasts---Oc.S/BS,N.Oc/t.Ar and N.Oc/B.Pm were higher than that of control group(P<0.05).5.The expressions of osteogenic related genes were decreasedin LPS group(P<0.05),while the expressions of osteoclast related genes were increased in LPS group(P<0.05).Part II:1.Tail length and body weight of mice in the LPS group were significantly lower than the control group(P<0.05).2.Bone mineral density of LPS group decreased compared with control group.Micro-CT results showed that in LPS group the related indexes of trabecular bone BV/TV,TB.N,TB.Th were decreased(P<0.05)and TB.SP,SMI were increased(P<0.05);the related indexes of cortex BV/TV,C.Th were decreased(P<0.05).4.Pathological analysis found growth plate of the LPS group became narrow,trabecular bone was fewer,whilethe number of osteoclast increased and Oc.S/BS,N.Oc/t.Ar,N.Oc/B.Pm were higher(P<0.05).5.Osteogenic related genes expression decreased in the LPS group(P<0.05),while osteoclast related genes expression was increased(P<0.05).Conclusion:Part I:LPS effected the development of bone in embryonic period by inhibiting the proliferation,differentiation and up-regulating apoptosis of hypertrophic chondrocytes.The stimulation of LPS in the embryonic stage led to decrease of bone mass in adult mice which may due to the decreased expression levels of osteogenesis related genes,up-regulaed osteoclast expression levels of osteoclast related gene,increased osteoclast numbers and enhanced osteoclast activity.Part II:LPS stimulation in the early stage after birth could inhibite the development of mice.LPS also induced low bone mass in the adulthood of mice which may related with depressed bone formation and enhanced bone absorption.