A Comparative Study On The Pathological Mechanism Of "flesh And Bone Incompatibility" In Rats With Glucocorticoid-induced Osteoporosis And Different Treatment Methods Of Traditional Chinese Medicine

Purpose:This study took the expression of collagenⅠand the enzymes related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis as research core by molecular biology approach to expound the effect of the coordination of skeletal muscles contraction and bones on the formation of osteoporosis and observe the preventing and treating effect of reinforcing the kidney method,strengthening the spleen method and promoting blood circulation to remove blood stasis method on osteoporosis and their mechanisms.This study provides scientific experimental evidence for increasing the preventing and treating effect on osteoporosis and choosing the best therapeutic schedule.Materials and methods:This experiment established the osteoporosis animal models by intramuscular injection of dexamethasone, prevented and treated the model rats with reinforcing the kidney method,strengthening the spleen method and promoting blood circulation to remove blood stasis method,chose GUSHUKANG granula as positive control drug,took the normal rats as standard comparison and the model rats as blank comparison and the time of establishing models and administrating was 9 weeks. After 9 weeks,we put all the rats to death and get materials to detect the following indicators:①We detected the vitro femur bone mineral density（BMD） by using the dual energy X-ray absorptiometry;②We detected the tartrate-resistant acid phosphatase（TRAP）,alkaline phosphatase（ALP）, calcium and phosphorus in blood serum and troponin and Ca²⁺-Mg²⁺-ATP enzyme of skeletal muscles by biochemistry method;③We detected the mRNA and protein expression of bone gla protein, collagenⅠand Na⁺-K⁺-ATP enzyme in the bone tissue and skeletal muscles of each group rats by PCR and Western blot.Results:1. Rats’vitro upper 1/3 femur bone mineral density （BMD）:compared with normal group,blank model group decreased significantly（P <0.01）;compared with blank model group,all the treating groups increased at different degrees,reinforcing the kidney herbs group increased the most significantly（P<0.01）.2. Rats’biochemistry indicators of bone metabolism:①compared with normal group,blank model group rats’ALP in blood serum trended to increase,but the difference had not statistical significance; compared with blank model group,all the treating groups rats’ALP in blood serum increased, promoting blood circulation group increased the most significantly（P<0.05）.②compared with normal group, blank model group rats’TRAP in blood serum increased significantly（P<0.01）;compared with blank model group, all the treating groups rats’TRAP in blood serum decreased significantly, reinforcing the kidney group and GUSHUKANG group decreased the most significantly, compared with other groups, the difference had statistical significance（P < 0.01）.③compared with normal group, blank model group rats’calcium in blood serum increased,but the difference had not statistical significance; compared with blank model group,all the treating groups rats’calcium in blood serum increased significantly, reinforcing the kidney group and GUSHUKANG group increased the most significantly, compared with other groups, the difference had statistical significance（P<0.01）.④Phosphorus in blood serum had no obvious variation. compared with normal group,GUSHUKANG group rats’phosphorus in blood serum increased significantly（P<0.01）.3. Rats’calcium metabolism indicators in bones and skeletal muscles:①compared with normal group,blank model group rats’bone gla protein mRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’bone gla protein mRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）. Compared with normal group, blank model group rats’bone gla protein protein expression in bones decreased ,but the difference had not statistical significance; compared with blank model group, all the treating groups rats’bone gla protein protein expression increased,reinforcing the kidney group increased the most significantly（P<0.01）.②Compared with normal group, troponin of the rats’skeletal muscle of the other groups decreased significantly（P<0.01）; compared with blank model group, troponin of the rats’skeletal muscle of all the treating groups increased significantly（P<0.01）; troponin of the rats’skeletal muscle of reinforcing the kidney group increased the most significantly,higher than other groups and the difference had statistical significance（P<0.01）;the increasing degree of troponin of the rats’skeletal muscle of promoting blood circulation group was the lowest and had statistical significance（P<0.01）, comparing with normal group, blank model group and reinforcing the kidney group.4. Rats’collagenⅠin bones and skeletal muscles:①compared with normal group,blank model group rats’collagenⅠmRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’collagenⅠmRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）, secondly, strengthening the spleen group（P<0.05）, thirdly, promoting blood circulation group, had statistically significant difference, comparing with reinforcing the kidney group（P<0.05） ;the increasing degree of GUSHUKANG group was the lowest and had statistically significant difference （P < 0.01）, comparing with reinforcing the kidney group. Compared with normal group, collagenⅠprotein expression of the rats’bone of the other groups decreased significantly（P<0.01）; compared with blank model group, collagenⅠprotein expression of the rats’bone of all the treating groups increased, reinforcing the kidney group and GUSHUKANG group increased the most significantly（P<0.01）.②compared with normal group,blank model group rats’collagenⅠmRNA expression in skeletal muscles decreased significantly（P<0.01）; compared with blank model group,all the treating groups rats’collagenⅠmRNA expression increased at different degrees,reinforcing the kidney group and strengthening the spleen group increased the most significantly（P<0.01）. Compared with normal group, collagenⅠprotein expression of the rats’skeletal muscles of the other groups decreased significantly（P<0.01）; compared with blank model group, collagenⅠprotein expression of the rats’skeletal muscles of all the treating groups increased significantly（P<0.01）, reinforcing the kidney group increased the most significantly.5. Rats’energy metabolism indicators in bones and skeletal muscles:①compared with normal group,blank model group rats’Na⁺-K⁺-ATP enzyme mRNA expression in bones decreased significantly（P<0.05）; compared with blank model group,all the treating groups rats’Na⁺-K⁺-ATP enzyme mRNA expression increased at different degrees,reinforcing the kidney group increased the most significantly（P<0.01）, secondly, strengthening the spleen group（P<0.01）,2.43 times of normal group; the increasing degree of promoting blood circulation group was the lowest and had statistical significance（P<0.01）, comparing with reinforcing the kidney group and strengthening the spleen group.②compared with normal group,blank model group rats’Na⁺-K⁺-ATP enzyme mRNA expression in skeletal muscles decreased significantly（P<0.01）; compared with blank model group,all the treating groups rats’Na⁺-K⁺-ATP enzyme mRNA expression increased at different degrees, strengthening the spleen group increased the most significantly（P<0.01）,1.00 times of normal group, secondly, reinforcing the kidney group（P<0.01）,0.68 times of normal group; the increasing degree of promoting blood circulation group and GUSHUKANG group was the lowest.③Compared with normal group, Ca²⁺-Mg²⁺-ATP enzyme of the rats’skeletal muscles of the other groups decreased significantly（P<0.01）; compared with blank model group, Ca²⁺-Mg²⁺-ATP enzyme of the rats’skeletal muscles of all the treating groups increased significantly（P<0.01）, reinforcing the kidney group increased the most significantly,higher than GUSHUKANG group, promoting blood circulation group and strengthening the spleen group significantly and the difference had statistical significance （P<0.01）.Conclusions:1. The method of hind limb intramuscular injection of dexamethasone（2.5mg/kg,twice a week,9 weeks continuously） can induce osteoporosis rat models successfully.2. Dexamethasone can cause gene and protein expression level of bone gla protein, collagenⅠand Na+-K+-ATP enzyme in rats’bones and troponin,Ca²⁺-Mg²⁺-ATP enzyme, collagenⅠand Na+-K+-ATP enzyme of skeletal muscles decreasing.This suggests that maybe the decline of energy metabolism and coordination of bones and skeletal muscles is one aspect of the important pathogenesis of glucocorticoid-induced osteoporosis.3. The reinforcing the kidney herbs have obvious preventing and treating effect on glucocorticoid-induced osteoporosis by increasing the genes expression level of collagenⅠand others related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis effectively and increasing energy metabolism in bones and skeletal muscles of rats with osteoporosis to promote bone formation and inhibit bone resorption.4. The strengthening the spleen herbs have some preventing and treating effect on glucocorticoid-induced osteoporosis by increasing the genes expression level of collagenⅠand others related to calcium and energy metabolism of skeletal muscles to increase energy metabolism of skeletal muscles of rats with osteoporosis to protect bones effectively and increase the coordination of bones and muscles to promote bone formation.5. The comparation of the preventing and treating effect of reinforcing the kidney herbs,strengthening the spleen herbs and promoting blood circulation to remove blood stasis herbs on osteoporosis by the genes expression change of collagenⅠand others related to calcium and energy metabolism in bones and skeletal muscles of rats with osteoporosis shows reinforcing the kidney herbs better than strengthening the spleen herbs and strengthening the spleen herbs better than promoting blood circulation to remove blood stasis herbs.This suggests that reinforcing the kidney method is the main treating method to prevent and treat osteoporosis and strengthening the spleen method is an important treating method to assist reinforcing the kidney method to treat osteoporosis.