The Effect of "Horror Hurt Pregnant Rat" on the Offspring Rat Early Long Bone Development and Its Mechanism
According to Chinese medicine theory,the main function of kidney is the possession of Shenjing,which is the essence of birth and also the main essence associated with growth,development and reproduction of our bodies.Kidney is in charge of bone,while Shenjing can made Sui,which can provide nutrition to bone.Therefore,we have put forward the main opinion of kidney controlling bone growth and development,and separately from the theoretical and experimental aspects to study.ObjectiveThe study was designed to investigate the mechanism that" horror hurt pregnant rat "had effect on the long bone development.Methods①3-month-old SD(Sprague-Dawley) female rats 30,male 15 to be combined according to the proportion of 2:1 matching cage.Female after conception using a random access method is divided into normal group,model group and bu-shen group,10 in each group.During gestational period,the rats of the model and bushen group were frighented by cat four hours(2 hours Am,2 hours Pm) a day until the birth.The results were examined at postnatal day 20.Bushen group model since the first day,a day to the Chinese side Bushen Tianjing 2ml-/100g weight decoction orally,the normal group and model group also based on body weight given normal saline orally.Time model for gestational.②Record the number of pregnant rats given birth and assess their fertility.Record the eye,the teeth,the ear,the hair growth time;measurement of rats from birth up to 20 days once every four days in length and weight to calculate the progress in the growth period,compared among the three groups of rats to determine the growth and development of the situation.③20- day-old rats were randomly selected when the litter is only 1-2,broken to death.From the hip,cutting the right lower limb,carefully remove all the muscles and connected organization,remove the fibula,and the remaining tibia. Put right proximal tibia from 1/3 removal of soft tissue into 10%neutral formaldehyde fixed one week,decalcified,paraffin-embedded conventional,5 urn thick con- tinuous vertical sections;20-day-old rats were randomly selected, decapitated executed quickly separated from the right tibia,1 mm~3 cut into the bone tissue fixative glutaraldehyde fixed.Separately in the optical microscope and electron microscope observation of the growth plate of rats in each group metaphyseal tibia morphological changes of the organization.④20-day-old rats were randomly selected at each of five specimens of the right proximal tibia,the detection section.Determination of in situhybridizal pups born tibial metaphysis expression of TGF-PmRNA.The average optical density were determined by photo analysis technique.⑤Rats 20 days after birth,1-2 litter were randomly selected,each group collected a total of 15,collectting blood,enzyme-linked immunosorbent assay serum IGF-1,BALP content.⑥Rats 20 days after birth,litter three or six randomly selected,collectting blood. Chemilum inescence detection of three groups of serum FT_3?FT_4 and T-SH levels.⑦Each taking 30 right proximal tibia biopsy specimens by immunohistoche-mical SABC,growth plate chondrocytes were observed the expression of Bcl -2/bax.The positive ratio were determined by photo-analysis technique.All the results were showed by mean value±SEM.Results①The number of neonatal rats and their conditions of growth and development :the model group of pregnant rats produce lower than normal or bushen group (P<0.01).From postnal day 1 to day 20,the average weight of the model g -roup rats have significantly reduced campared with normal group and bushen group;from the 8th day after birth,the model group rats average length compared with the normal group and bushen group have great decrease;In model group,the average weight and length of the major time were obviously lagging behind.At the same time,their teeth and hair growing out of time slightly later than the bushen group and normal group.②Rats 20 days after birth,the morphological changes of growth plate:In the model group,the structure of rat growth plate disorders,proliferative zone chondrocytes are stunted and weakened the bone formation,bone volume decreased significantly.Form electron microscope,in model group rats,a number of vision in growth plate cartilage cells have abnormal structure,some nuclear pyknosis.Normal and bushen rats showed no significant change in morpholog -y.③Rats 20 days after birth the TGF-βexpression in the metaphysis:positive signals in cells,dark blue,located in cytoplasm;positive expression intensity of the use of quantitative analysis of optical density.The average brightness of m odel group has a decline while the normal group has no difference compared with bushen group.④Rats of 20 days blood serum IGF-1,BALP concentration:a significant reduction in the model group(P<0.05),the normal group has no difference compared with bushen group.⑤Rats of 20 days after birth,blood serum FT_3,FT_4,TSH levels:Compared with the other two groups,the model group rats FT_4 serum levels lower than normal group(P<0.05),while serum FT3,TSH levels has no difficence.Bushen rats serum FT3,FT4,TSH levels compared with normal group have no statisstical difference.⑥Immunohistochemical positive reaction for the brown-yellow granules,Bcl-2 expression is located in cytoplasm;bax expression located in cytoplasm and nucleus.Bcl-2 mainly in the static and the proliferation areas;bax expression mainly in the hypertrophic zone;the rate of positive cells compared each other: the model group rats Bcl-2 expression than normal and bushen group has decreased significantly(p<0.05)while bax expression has increased(p<0.05).Conclusion①Model group of pregnant rats have lower fertility than normal and bushen group;rats from birth to the first 20 days,the average weight,average length, average weight and length growth of each period,the teeth,the hair growth time are significantly behind.Combined with fear of injury etiology and pathogenesis of the kidney,the model group of pregnant rats showed decreased reproductive function,at the same time its existence essence deficiency of the kidney resulting to their offspring body growth retardation.②Model group rats tibial growth plate histology changes and the ultrastructture of proliferative zone delicate that chondrocytes of cartilage cell proliferation prompting change does not meet the normal level,together with reducing the growth rate of growth plate cartilage into bone.③The expression of TGF-βin model rats tibial growth plate(Prolierative and Hypertrophic zone)was reduced,which causing the formation from cartilage into bone slow and affecting the growth of long bones.④Bone developmental disorder caused by deficiency of the kidney of the molecular mechanisms were likely to be:the existence of the model group rats congenital deficiency of the kidney,long bones stunted growth,decreased serum levels of thyroid ho- rmones and IGF-1.Kidney deficiency of modern research studies suggest that the existence of hypothalamic-pituitary -thyroid axis dysfunction,kidney deficiency and IGF-1 is closely related,consider the impact of thyroid hormone to IGF-1 expression causing the development of the growth plate;model group congenital deficiency of the kidney,stunting of long bone,growth plate decreased the expression of TGF-β,while serum IGF 1 content,accompanied by Bcl-2 gene expression declines.There are close relationship between kidney deficiency and the expression of IGF-1,TGF-P with Bcl-2.We can conclud that IGF-1,TGF-P have synergies and their adjustment on expression of Bc-1-2/bax is another molecular regulatory mechanism.⑤The topic of "kidney is bone growth decision " was raised for the first time, and from the angles of the impacts of kidney to the bone structure and function,we made an in-depth study.Experiments using "horror hurt pregnant rats" approach successfully replicated the essence deficiency model and found that the model group rats with the performance of a congenital deficiency of the kidney,existing an obvious obstacle of the long bone development,whiile Chinese medicine can reversed the abovementioned abnormal and effectively block the impacts of kidney deficiency on bone development.