| 1.ObjectiveThe main objective of this research was to provide the evidence of the treatment of Qing’e prescription on the Primary Osteoporosis, which through observing the function of Qing’e prescription on the bone metabolism from the perspective of LEP and LEP-R. The experiment was based on observing the difference of LEP、 LEP-R、T、E2 and bone metabolic markers in the natural aged rats with age increasing under feeding the Qing’e prescription.2. Method2.1 material preparationThe amount of 6 months old health female and male rats was 81, respectively. After fed one week,9 of female and male rats were random picked up with death treatment, respectively. The remaining 1446 months old SD rats (male and female each 72) were randomly selected 36 (male and female each 18) and divided by four groups, which were 9 female rats with Qing’e prescription,9 female rats with natural saline,9 male rats with Qing’e prescription, and 9 male rats with natural saline. Furthermore, the remaining rats were grouped by the same method as 6 months old SD rat group at 9 months old,12 months old and 15 months old with normal feeding, respectively. The groups with Qing’e prescription were fed 1.9g/kg/d Qing’e prescription with 2ml natural saline and the groups with natural saline were only fed 2ml natural saline. The sample with death treatment was picked after fed 3 months.Then, the blood of ventral artery in the rats was stored in EP tube at the -80℃. In addition,the shinbone、lumbar of rats were stored at the-80℃.2.2 sample determinationThe concentration of LEP、LEP-R、Cbfa1、Col-Ⅰ、OC、OPG、RANKL、T and E2 in the serum was detected by the method of ELISA.The bone tissue morphology of the shinbone slices and Masson staining were analysed by Motic Med 6.0 digital medical image analysis system.BMD of the shinbone was detected by Dual-energy X-ray absorptiometry. Biomechanical test of the shinbone was based on the Three-point bend test. Protein and gene expression levels of LEP、LEP-R were detected by Western blot. Real time PCR.3. Result3.1 As the growth of the age. the LEP-R,E2,T,Cbfa-1.COL-Ⅰ. OC levels in serum and the expression of mRNA and protein of LEP and LEP-R in bone tissue in physiological saline group are decreasing in both male and female with increase of age,but the LEP levels in serum in physiological saline group are rising. The physiological saline group from 6 months to 9 months comes a increase about OPG and RANKL and the BALP level in serum with the grows of age both in male and female (P< 0.05 or P< 0.01), a significant reduction in 9 months to 18 months of age (P< 0.05 or P< 0.01), TRACP-5b level in serum decrease with increase of age.BMD reached the peak in the 15th month in male rat and in the 12th month in female rat. The BMD and bone trabecular area percentage of the physiological saline group was decreased (P< 0.01),compared with the same gender in the 6th months in both male and female, but in males are higher than females with the same age and intervention.In the physiological saline group,the load and stress of tibia of female can withstand reached the maximum in the 12th months and there was great difference between the 18th and 6th months(P< 0.05);but there was a slowly decreasing in males without significant differences. In the same months age and intervention group the maximum load and stress of male are higher than females.3.2 In the group of Qing’e prescription compared with the physiological saline group the LEP, LEP-R, E2, T, OPG and RANKL, Cbfa-1, COL-I,OC and BALP levels in serum,and the expression levels of mRNA and protein and LEP-R and LEP in bone tissue were increased in the same age of months and sex groups, in both female and male.The levels of LEP, OPG and RANKL, serum level of Cbfa-1 had significant difference in different months (P<0.05, P<0.01),the level of TRACP-5b is a little down without significant.In the group of Qing’e prescription compared with the physiological saline group the BMD, bone trabecular area percentage,the maximum load and stress are significant raised in the same age of month and sex group. The group of Qing’e prescription of the same months of age and sex had a better integrity of shape and structure of trabecular bone, rising in the numbers, smaller spacing, and less fracture.4. Conclusion4.1 With the age growing, the level of LEP-R in serum of natural aged rats are decreasing while the level of LEP are rising, which may result in by leptin resistance. In addition, the function of osteoclast is enhanced and the function of osteoblast is weakened as age growing, the bone resorption and the bone formation are losing coupling, bone quality is decreasing.4.2 Qing’e prescription has function on prevention and cure osteoporosis by controlling the bone metabolism of natural aged rat, adjusting the absent coupling of bone resorption and the bone formation with aging, improving bone quality and slowing down the bone senescence through improving the leptin resistance by increasing the level of LEP、LEP-R in natural aged rats. |
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