| Objects: To explore the effect on the mRNA and protein expression of PPARγ2 and TGF-β/Smad2.3 in the bone tissues of osteoporosis rats due to kidney deficiency induced by glucocorticoid, and try to reveal the pathologic mechanism of osteoporosis due to kidney deficiency induced by glucocorticoid, and the mechanism of prevention and treatment of osteoporosis by nanometer calcium combined with tonifying kidney herbs.Materials and methods:Animal models were made by local intramuscular injection with glucocorticoid, fed by nanometer calcium combined with tonifying kidney herbs (low dosage, high dosage) which possess the functions of nourishing kidney and replenishing essence, supplementing calcium and strengthening bone. The test lasted for 8 weeks, animal models fed with granule of Gushukang and calcium tablet as positive control group, health animals as normal control group, and animal models fed without drugs as blank group(placebo). At the end of the test, bone mineral density(BMD) was measured by dual energy X-ray absorbinmetry (DEXA), biochemical methods were applied for the detection of tartrate-resistant acid phosphatase(TRAP), alkaline phosphatase (ALP) and the other indicators like blood calcium and phosphonium, and the mRNA and protein expression of PPARγ2 and TGF-β1/Smad2.3 in the bone tissues were detected by Rt-PCR and Western blot.Main results:1. Comparisons of weight and biological manefestation information of rats: compared with normal control group, the weight of the blank group decreased significantly, and that of the test group and positive control group increased, but there was no significant difference. Compared with the control group, the manifestations of deficiency syndrome could be seen in blank group, test group and positive control group. Compared with the blank group, less manifestations of deficiency syndrome could be seen in test group and positive control group.2. Comparisons of BMD: BMD of blank group was significantly lower than normal group. Compared with blank group, BMD of the other groups with drugs were all increased. Among them, the best improvement was seen in the group fed by high dosage of nanometer calcium combined with tonifying kidney Herbs.3. Comparisons of serum TRAP: TRAP in serum of blank group was significantly higher than that of normal control group. Compared with blank group, the contents of TRAP in the test groups decreased in serum, especially in groups of high dosage of nanometer calcium combined with tonifying kidney herbs and granule of Gushukang.4. Comparisons of serum ALP: ALP in blank group was much higher than in normal control group. Compared with blank group, the other groups all also possessed the ability of increasing contents of ALP in serum. The best one was found in group of high dosage of nanometer calcium combined with tonifying kidney Herbs. There existed no significant deviation with comparison to granule of Gushukang and calcium tablet group in statistics.5. Comparisons of Blood calcium in arterial blood: blood calcium of blank group is much lower than normal group. Compared with blank group, the other groups all possessed the ability of increasing contents of blood calcium. The highest could be seen in group of low dosage of nanometer calcium combined with tonifying kidney Herbs. But there existed no significant deviation among the groups with drugs in statistics.6. Comparisons of blood phosphonium in arterial blood: blood phosphonium of blank group was slightly higher than that of normal group. Compared with blank group, the other groups all possessed the ability of increasing contents of blood phosphonium. The highest could be seen in group of low dosage of nanometer calcium combined with tonifying kidney Herbs. The notable increases were found in groups of high dosage of nanometer calcium combined with tonifying Kidney Herbs and calcium tablet, there existed significant deviation in statistics.7. RT-PCR comparisons: in general, there existed mRNA expression of PPARγ2 and TGF-β1/Smad2.3 in normal bone tissues. Compared with the normal group, the expression of PPARγ2 in blank group was significantly up-regulated, but the expression of TGF-β1/Smad2.3 were down-regulated. After 8 weeks, in all the groups fed with drugs, the expression of PPARγ2 was down-regulated, and the expression of TGF-β1/Smad2.3 were up-regulated to a certain extent. The best effect could be seen in the group of high dosage of nanometer calcium combined with tonifying kidney herbs.8. Western blot comparisons: there existed protein expression of PPARγ2 and TGF-β1/Smad2.3 in normal bone tissues. Compared with normal control group, the expression of PPARγ2 in blank group was significantly increased, but the expression of TGF-β1/Smad2.3 were decreased. After 8 weeks, in all the groups with drugs, the expression of PPARγ2 was decreased, and the expression of TGF-β1/Smad2.3 were increased to a certain extent. The best effect could be seen in group of high dosage of nanometer calcium combined with tonifying kidney herbs.Conclusions:1. The animal model of Osteoporosis rats due to kidney deficiency can be successfully made by local intramuscular injection with glucocorticoid.2. The bone metabolism process of BMD decreasing, and both the absorption and formation of bone accelerating can be seen in osteoporosis rats due to kidney deficiency induced by glucocorticoid.3. The expression of TGF-β1/Smad2.3 can be seen in normal bone tissues, which indicates that there existed signal transduction pathways in normal rat bone tissues, and those pathways can mediate TGF-β1 to induce bone formation, at the same time to express PPARγ2 in the levels of gene and protein, which also indicate that Bone marrow stromal cell (BMS) may differentiate to adipose cell in normal rat bone tissues.4. The findings of the mRNA and protein expressions of PPARγ2 increases, but the expressions of TGF-β1/Smad2.3 decrease, which indicates to be one of the most important mechanisms of the cause of Osteoporosis rats due to kidney deficiency induced by glucocorticoid.5. The nanometer calcium combined with tonifying kidney herbs has the ability of inhibiting adipose cell differentiation on the levels of transcription and translation, meantime, it can also promote Bone marrow stromal cell (BMS) differentiate to skeletogenous cell. It might be expected to be a new effective way for prevent or treat osteoporosis due to kidney deficiency induced by glucocorticoid. 6. The nanometer calcium combined with tonifying kidney herbs plays an important role in regulating the expression of TGF-β1/Smad2.3 in the process of bone formation. It might perform the preventive and treating effects though signal transduction pathways of Smad2.3 in osteoporosis due to kidney deficiency induced by glucocorticoid.7. The nanometer calcium combined with tonifying kidney Herbs may improve the condition of symptoms and other data, which validate the scientific hypothesis. |
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