Zuo Gui Pills Factors On Osteoclast Differentiation Regulation Of Opg, Rankl Expression

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 the change of the expression of osteoclast differentiation and regulation factors: OPG and RANKL.Materials and methods1 Preparation of serum containing Zuogui pillFifteen female Wistar 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 amounts and areas of bone resorption lacuna in thin thighbone slices of cattle induced by obsteoclast2.1 The co-culture of osteoblast and osteoclastRefitting the 96-well plate,drilling in the middle of the side wall of the wells to communicate the adjiont wells.One well is osteoblast culture room and the other is osteoclast culture room,then irradiated by ultraviolet radiation for 2 hours as a preparation for co-culture of osteoblast and osteoclast.The third passage osteoblasts were inoculated in the osteoblast culture rooms of the 96-well plate as 1×x104/ml in advance. After 24 hours the osteoblasts were adhered to the bottom of the well, the bone slices containing osteoblasts were added to the osteoclast culture rooms adjoining to the wells containing osteoblasts and the culture medium was replenished to 200ul. Both kinds of cells were incubated in 37℃, 5%CO2 air atmosphere.2.2 Grouping and adding of the serum respectivelyThere 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 2.3.2.1 in "materials and methods "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 hours once. As to the later three groups that contained osteoblasts and osteoclasts were cultured according to the above methods of 2.1 described. The culture medium was changed to the culture medium containing 10% serum of three groups respectively 24 hours later, then slipped the plates every hour to promote the liquid of two wells to exchange and changed the culture medium every 24 hours. The bone slices were removed 7 days later, washed 5mininxthird times in 0.25mol/l ammonia ultrasonic. After a series of alcohol dehydration, the bone slices were dyed in 1% toluidine blue, and observed in the telescope. The amounts of bone resorption lacuna were measured in a piece of bone section using telescope and the magnified multiple was 100 times, the result was showed by the amounts of bone resorption lacuna per bone section. At the same time, the areas of bone resorption lacunas were assessed by Leica image analysis system and the magnified multiple was 200 times.3 The detection of the protein and mRNA expression of OPG> RANKL of osteoblastsThere are three groups in the experiment: Osteoblasts+ normal serum group, 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 steapsin, and the cell blendings were inoculated in the culture utensile as l><104/ml. After the cells adhered to the bottom of the culture utensile ( glass slides placed in it beforehand), the culture medium was removed, and replaced by the culture medium containing 10% serum of three groups respectively. After the cells were cultured 72 hours , the glass slides were removed, washed by PBS, followed by acetone for lOmin, and saved at -20 °C for immunocytochemistry detection. The specimen for detection of the mRNA should be fixed by 4% polyformaldehyde containing 1/1000 DEPC, then washed enough by dilute water and saved at -20 °C for use.The immunocytochemistry methods and in situ hybridization methods were used to detect the protein and mRNA expression of OPG> RANKL. The extent of expressionof two factors was displayed by the MOD by Leica image analysis system.4 Statistical analysisResults are presented as Mean±SE. Differences between groups were analyzed using q test by one-way ANOVA.Results1 Effect of the serum containing Zuogui pill on the amounts and areas of bone resorption lacuna induced by obsteoclastThere are some bone resorption lacuna appeared in the thin bone slices cultured at the first day. With the time going, the amounts and areas of bone-resorption lacuna increased accordingly. At the seventh day, the amounts and areas of bone-resorption lacuna of OC+ serum of OVX model group was much more than OC+normal serum group;the amounts and areas of bone-resorption lacuna of the serum of OC+OVX model treated with Zuogui pill group were much smaller than the serum of OC+OVX model group, but there was still more than OC+normal serum group. Compared with OC+OB+normal serum group, the amounts and areas of bone-resorption lacuna of OC+ OB+OVX model group increased remarkably;OC+ OB+OVX model treated with Zuogui pill group decreased evidently compared with OC+ OB+OVX model group,but there was nothing difference between the serum of OC+ OB+OVX model treated with Zuogui pill group and OC+OB+normal serum group .Compared with the serum of OC+OVX model group, the amounts and areas of bone-resorption lacuna of OC+ OB+OVX model group increased remarkably;while the amounts and areas of bone-resorption lacuna of the serum of OC+ OB+OVX model treated with Zuogui pill group decreased evidently compared with the serum of OC+OVX model treated with Zuogui pill group.2 Effect of the serum containing Zuigui pill on the protein and mRNA expression of OPG ? RANKL of osteoblasts.2.1 Effect of the serum containing Zuogui pill on the protein and mRNA expression of OPG of osteoblasts.Compared with the normal serum group, the value of the MOD of the protein and mRNA expression of OPG of osteoblasts in the serum of OVX model group decreased remarkably, the value of the serum of OVX treated with Zuogui pill group increased remarkably compared with the serum of OVX model group, but have no evident difference from the normal serum group. The results indicates that the protein and mRNA expression of OPG of osteoblasts in the serum of OVX model group decreased compared with the normal serum group, and the protein and mRNA expression ofOPG of osteoblasts in the serum of OVX treated with Zuogui pill group increased significantly compared with the serum of OVX model group, but have no remarkable difference form the normal serum group.2.2 Effect of the serum containing Zuogui pill on the protein and mRNA expression of RANKL of osteoblastsThe value of the MOD of the protein and mRNA expression of RANKL of osteoblasts in the serum of OVX model group increased remarkably compared with the normal serum group;the value of the MOD of the serum of OVX treated with Zuogui pill group decreased remarkably compared with the serum of OVX model group, but have no evident difference from the normal serum group. The results indicates that the protein and mRNA expression of RANKL of osteoblasts in the serum of OVX model group increased compared with the normal serum group, and the protein and mRNA expression of RANKL of osteoblasts in the serum of OVX treated with Zuogui pill group decreased remarkably compared with the serum of OVX model group, but have no evident difference form the normal serum group.ConclusionThe serum containing Zuogui pill could inhibit the activity of obsteoclasts significantly by regulating the expression of osteoclast differentiation and regulation factors : OPG and RANKL. The serum containing Zuogui pill could not only inhibit the secretion of RANKL to repress the activity of osteoclast but also stimulate osteoblasts to secret OPG, then the combination of OPG and RANKL increased ,which could check the activity of RANKL and decrease the activity of the bone resorbing activity of obsteoclasts consequently.