| ObjectiveThe present study was to explore the therapeutic mechanism that how the Zuogui pill treat osteoporosis using cell culture technics from the cellular and molecular point. Hope to get further to know the pathogenesis of "The kidney dominates the bones" in the aspect of couple signal conduction between osteoblast and osteoclast.Methods1 Preparation of serum containing Zuogui pillFifteen female Wister rats were selected and allocated to three groups randomly: normal control group, ovariectomy (OVX) group, and ovariectomy (OVX) group with Zuogui pill treated group. After anesthesia, both ovaries of all the rats in the OVX group and OVX with Zuogui pill treated group were extirpated. After three months of the operation, the rats of OVX with Zuogui pill treated group were administrated orally Zuogui pill twice one day, the rats in other two groups were fed with the same volume diluted water. The dosage of Zuogui pill to rat is eight times of clinical dosage for human according to measurement. The blood of all the rats was collected by abdominal aorta one hour later after those rats were administrated in the fifth time. The blood was placed quietly 1 hour, and then centrifuged at 2500r/min, 25min. The serum was isolated and conserved at -20℃. We called the serum of rats in the OVX with Zuogui pill treated group the serum containing Zuogui pill.2 The detection of the protein and mRNA expression of IL-1, IL-6, COX-2 of osteoblastsThere are three groups in the experiment: Osteoblasts+ normal serumgroup, Osteoblasts+ serum of OVX model group and Osteoblasts+ serum of OVX model treated with Zuogui pill group, and eight samples per group. The third passage osteoblasts were digested by trypsin, and the cell blendings were inoculated in the petridish as lx104/ml. After the cells adhered to the bottom of the petridish placed on the glass slides in the bottom, the culture medium was removed, and replaced by the culture medium containing 10% serum of three groups respectively. After the cells were cultured 72 hour later, then the glass slides were removed, washed by PBS, followed by acetone for lOmin, and saved at -20°C for use.The immunohistochemistry methods and in situ hybridization methods were used to detect the protein and mRNA expression of IL-1, IL-6, COX-2. The extent of expression of three cytokines was displayed by the intensity of grey by Leica image analysis system. 3 The detection of the amounts and areas of bone resorption lacuna in thin thighbone slices of cattle induced by obsteoclast There are six groups in this experiment: Osteoclasts +normal serum group; Osteoclasts + serum of OVX group; Osteoclasts +serum of OVX model treated with Zuogui pill group; Osteoblasts + Osteoclasts +normal serum group; Osteoblasts + Osteoclasts + serum of OVX group; Osteoblasts + Osteoclasts +serum of OVX model treated with Zuogui pill group, and eight bone slices in one group. The osteoclasts in the former three groups that didn't contain osteoblasts were cultured according to the methods as 2.3.1.1 described. Every well was added to 0.2ml osteoclasts blendings, and was changed to the culture medium containing 10% serum of three groups respectively, 24 hour once. As to the later three groups that contained osteoblasts and osteoclasts, the third passage osteoblasts were inoculated in the 96-well plates as 1 x 104/ml in advance. After 24 hour the osteoblasts were adhered to the bottom of the well, the bone slices containing osteoblasts were added to the well next to the well containing osteoblasts and the culture medium was replenished to 200ul. Both kinds of cells were incubated in 37 °C\ 5%CO2 air atmosphere. The culture medium was changed to the culturemedium containing 10% serum of three groups respectively 24 hourslater, then slipped the plates every hour to promote the liquid of twowells to exchange and changed the culture medium every 24 hour. Thebone slices were removed 7 days later, washed 5mininxthird times in0.25mol/l ammonia ultrasonic. After a series of alcohol dehydration, thebone slices were dyed in 1% toluidine blue, and observed in thetelescope.The amounts of bone resorption lacuna were measured in a piece of bonesection using telescope and the magnified multiple was 100 times, theresult was showed by the amounts of bone resorption lacuna per bonesection. At the same time, the areas of bone resorption lacunas wereassessed by Leica image analysis system and the magnified multiple was200 times.4 Statistical analysisResults are presented as mean+SE. Differences between groups wereanalyzed using q test by one-way ANOVA.Results1 Effect of the serum containing Zuigui pill on the protein and mRNA expression of IL-1, IL-6, COX-2 of osteoblasts. 1.1 Effect of the serum containing Zuogui pill on the protein and mRNA expression of IL-1 of osteoblasts.Compared with the normal serum group, the value of the grey extent of the protein and mRNA expression of IL-1 of osteoblasts in the serum of OVX model group decreased significantly, the serum of OVX treated with Zuogui pill group increased significantly compared with the serum of OVX model group, but have no difference from the normal serum group. The results indicates that the protein and mRNA expression of IL-1 of osteoblasts in the serum of OVX model group increased compared with the normal serum group, and the protein and mRNA expression of IL-1 of osteoblasts in the serum of OVX treated with Zuogui pill group decreased significantly compared with the serum of OVX model group, but have no difference form the normal serum group.1.2 Effect of the serum containing Zuogui pill on the protein and mRNA expression of IL-6 of osteoblasts.The value of the grey extent of the protein and mRNA expression of IL-6 of osteoblasts in the serum of OVX model group decreased significantly compared with the normal serum group; the value of the grey extent of the serum of OVX treated with Zuogui pill group increased significantly compared with the serum of OVX model group, but have no difference from the normal serum group. The results indicates that the protein and mRNA expression of IL-6of osteoblasts in the serum of OVX model group increased compared with the normal serum group, and the protein and mRNA expression of IL-6 of osteoblasts in the serum of OVX treated with Zuogui pill group decreased significantly compared with the serum of OVX model group, but have no difference form the normal serum group.1.3 Effect of the serum containing Zuogui pill on the protein and mRNA expression of COX-2 of osteoblasts.Compared with the normal serum group, the value of the grey extent of the protein and mRNA expression of COX-2 of osteoblasts in the serum of OVX model group decreased significantly, the serum of OVX treated with Zuogui pill group increased significantly compared with the serum of OVX model group, but have no difference from the normal serum group. The results indicates that the protein and mRNA expression of COX-2 of osteoblasts in the serum of OVX model group increased compared with the normal serum group, and the protein and mRNA expression of COX-2 of osteoblasts in the serum of OVX treated with Zuogui pill group decreased significantly compared with the serum of OVX model group, but have no difference form the normal serum group. 2 Effect of the serum containing Zuogui pill on the amounts and areas of bone resorption lacuna induced by osteoclast There are some bone resorption lacuna appeared in the thin bone slices cultured in the first day. With the time going, the amounts and areas of bone-resorption lacuna increased accordingly. In the seventh day, the amounts and areas of bone-resorption lacuna of Osteoclasts+ serum of...
|
PDF Full Text Request