| Purpose:Based on the theory of " spleen kidney correlation",Bushen Jianpi Huoxue Prescription was used to intervene tail suspension rats.It was proved that Bushen Jianpi Huoxue Prescription had preventive and therapeutic effects on bone loss and muscle atrophy in tail suspension rats,and its mechanism of intervention by regulating muscle synthesis and bone formation metabolism was revealed.Material and method:Forty healthy Wistar rats,female,2 months old,SPF grade,body mass(250±10)g.All rats were fed adaptively for 7 days,and then randomly divided into blank control group,model group,low dose group of Bushen Jianpi Huoxue Prescription,middle dose group of Bushen Jianpi Huoxue Prescription and high dose group of Bushen Jianpi Huoxue Prescription according to body weight,with 8 rats in each group.Rats in model group,high dose group of Bushen Jianpi Huoxue Prescription,middle dose group of Bushen Jianpi Huoxue Prescription and low dose group of Bushen Jianpi Huoxue Prescription were modeled by tail suspension method to simulate weightlessness.Equivalent dosage of Bushen Jianpi Huoxue Prescription was converted according to the ratio of human to rat body surface area(Chen Qi,Editor-in-Chief,Pharmacological Experiments of Traditional Chinese Medicine,People’s Health Publishing House,1994).From the first day of the experiment,the corresponding concentration of Bushen Jianpi Huoxue Prescription was given to each group intervened by traditional Chinese medicine,and the model group and the blank control group were given the same volume distilled water solution.The rats were given intragastric administration once a day,2 hours after the last intragastric administration on the 28 th day of the experiment.The weight of the rats was weighed and recorded before the sampling.After abdominal anesthesia,abdominal aorta blood was taken and collected into the blood vessel with vacuum serum.After 2 hours,the upper serum was collected by centrifugation and packaged,and stored in a cryogenic refrigerator.The soleus muscle of rat’s right hind limb was carefully separated,weighed and recorded quickly,then put into the labeled EP tube,and stored in the refrigerator at-80℃ for testing.The right femur was wrapped with gauze soaked with normal saline.The outer layer was wrapped with tin paper and labeled.The left femur was placed in EP tube and labeled.The left femur was stored in a refrigerator at-80 ℃.Bone mineral density of right femur was measured and the ratio of wet weight to body weight of left soleus muscle was calculated.Detection of serum myostatin in rats by ELISA.Western Blot method was used to detect the expression of AKT,GSK-3β,mTOR and P70S6 K in soleus muscle of rats.Western Blot method was used to detect the expression of DKK-1,GSK-3β,β-catenin and Runx-2 protein in femur of rats.Results: 1.Effect of Bushen Jianpi Huoxue Prescription on Bone Mineral Density and Soleus Muscle in Tail Suspension Rats 1.1 Effect of Bushen Jianpi Huoxue Prescription on Bone Mineral Density of Femur in Tail Suspension RatsCompared with the blank control group,the bone mineral density of femur in the model group decreased significantly(P<0.01);Compared with the model group,the bone mineral density of femur in each dose group of Bushen Jianpi Huoxue Prescription increased,and the difference was statistically significant.;Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the femoral bone mineral density of the high dose group of Bushen Jianpi Huoxue Prescription increased(P<0.05).1.2 Effect of Bushen Jianpi Huoxue Prescription on the Wet Weight to Weight Ratio of Soleus Muscle in Tail Suspension RatsCompared with the blank control group,the weight ratio of soleus muscle in the model group decreased significantly(P<0.01);The ratio of wet weight to body weight of soleus muscle of rats in different dosage groups of Bushen Jianpi Huoxue Prescription increased(P<0.01).Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the wet weight ratio of soleus muscle in the low dose group of Bushen Jianpi Huoxue Prescription was lower than that in the middle dose group,and there was no significant difference in the wet weight ratio of soleus muscle in the high dose group(P<0.05).1.3 Effect of Bushen Jianpi Huoxue Prescription on the Content of Myostatin(MSTN)in Serum of Tail Suspension RatsCompared with the blank control group,the content of serum MSTN in the model group was significantly increased(P<0.01);Compared with the model group,the content of serum MSTN in the middle and high dose groups of Bushen Jianpi Huoxue Prescription decreased(P<0.05);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,there was no significant difference in the expression of serum MSTN between the low dose group of Bushen Jianpi Huoxue Prescription and the high dose group of Bushen Jianpi Huoxue Prescription(P>0.05),but with the increase of concentration,the content of serum MSTN showed a downward trend.2.Mechanisms of Bushen Jianpi Huoxue Prescription on Muscle Atrophy and Bone Loss in Tail Suspension Rats by Regulating Muscle Synthesis and Bone Formation 2.1 Effects of Bushen Jianpi Huoxue Prescription on the AKT/mTOR/P70S6 K and AKT/GSK-3β signal pathways in soleus muscle of tail suspended rats 2.1.1 Effect of Bushen Jianpi Huoxue Prescription on the Expression of AKT Protein in Soleus Muscle of Tail Suspension RatsCompared with the blank control group,the expression of AKT protein in soleus muscle of rats in model group decreased(P<0.01);Compared with model group,the expression of AKT protein in soleus muscle of rats in different dosage groups of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of AKT protein in soleus muscle of rats in low dose group of Bushen Jianpi Huoxue Prescription was lower than that in middle dose group(P<0.01),and the expression of AKT protein in soleus muscle of rats in the high dose group of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01).2.1.2 Effect of Bushen Jianpi Huoxue Prescription on the Expression of mTOR Protein in Soleus Muscle of Tail Suspension RatsCompared with the blank control group,the expression of mTOR protein in soleus muscle of the model group was significantly lower(P<0.01);Compared with model group,the expression of mTOR protein in soleus muscle of rats in low dose group of Bushen Jianpi Prescription was lower than that in model group,the expression of mTOR protein in soleus muscle of rats in high dose group of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,The expression of mTOR protein in soleus muscle of rats in low dose group of Bushen Jianpi Huoxue Prescription was lower than that in middle dose group(P<0.01),while the expression of mTOR protein in soleus muscle of rats in the high dose group of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01).2.1.3 Effect of Bushen Jianpi Huoxue Prescription on P70S6 K Protein Expression in Soleus Muscle of Tail Suspension RatsCompared with the blank control group,the expression of P70S6 K protein in soleus muscle of model group rats was significantly increased(P<0.01);Compared with model group,the expression of P70S6 K protein in soleus muscle of rats in different dose groups of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of P70S6 K protein in soleus muscle of rats in low-dose group and high-dose group of Bushen Jianpi Huoxue Prescription was lower than that in medium-dose group,and the difference was statistically significant.2.1.4 Effect of Bushen Jianpi Huoxue Prescription on the Expression of GSK-3β Protein in Soleus Muscle of Tail Suspension RatsCompared with the blank control group,the expression of GSK-3β protein in soleus muscle of rats in model group increased(P<0.01);Compared with model group,the expression of GSK-3β protein in soleus muscle of rats in middle and high dose groups of Bushen Jianpi Huoxue Prescription decreased(P<0.01),while the expression of GSK-3β protein in soleus muscle of rats in low dose of Bushen Jianpi Huoxue Prescription had no significant difference(P>0.05);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of GKS-3β in soleus muscle of rats in low dose group of Bushen Jianpi Huoxue Prescription was higher than that in middle dose group(P<0.01),and the expression of GSK-3β protein in soleus muscle of rats in the high dose group of Bushen Jianpi Huoxue Prescription decreased significantly(P<0.01).2.2 Effect of Bushen Jianpi Huoxue Prescription on Classical Wnt Signal Pathway Related Proteins in Femur of Tail Suspension Rats 2.2.1 Effect of Bushen Jianpi Huoxue Prescription on the Expression of GSK-3β Protein in Femur of Tail Suspension RatsCompared with the blank control group,the expression of GSK-3β protein in femur of model group rats increased(P<0.05);Compared with the model group,The expression of GSK-3β protein in femur of rats in different dosage groups of Bushen Jianpi Huoxue Prescription was decreased,and the difference was statistically significant;Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of GSK-3β protein in femur of rats in low-dose group and high-dose group of Bushen Jianpi Huoxue Prescription was higher than that in middle-dose group,and there were significant differences.(P<0.01).2.2.2 Effect of Bushen Jianpi Huoxue Prescription on DKK-1 Protein Expression in Femur of Tail Suspension RatsCompared with the blank control group,the expression of DKK-1 protein in the femur of the model group increased(P<0.01);Compared with model group,the expression of DKK-1 protein in femur of rats in different dosage groups of Bushen Jianpi Huoxue Prescription decreased(P<0.01);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of DKK-1 protein in femur of low-dose rats treated with Bushen Jianpi Huoxue Prescription was higher than that of middle-dose rats treated with Bushen Jianpi Huoxue Prescription(P<0.01).2.2.3 Effect of Bushen Jianpi Huoxue Prescription on the Expression of β-catenin Protein in Femur of Tail Suspension RatsCompared with the blank control group,the expression of β-catenin protein in the femur of the model group was decreased(P<0.01);Compared with the model group,the expression of beta-catenin protein in femur of rats in middle-dose group and high-dose group of Bushen Jianpi Huoxue Prescription increased,and the difference was statistically significant;Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of beta-catenin protein in femur of rats in low-dose and high-dose groups of Bushen Jianpi Huoxue Prescription was lower than that in middle-dose group(P<0.01).2.2.4 Effect of Bushen Jianpi Huoxue Prescription on Runx-2 Protein Expression in Femur of Tail Suspension RatsCompared with the blank control group,the expression of Runx-2 protein in the femur of the model group was decreased(P<0.01);Compared with model group,the expression of Runx-2 protein in femur of rats in different dosage groups of Bushen Jianpi Huoxue Prescription increased significantly(P<0.01);Compared with the middle dose group of Bushen Jianpi Huoxue Prescription,the expression of Runx-2 protein in femur of rats in low-dose group and high-dose group of Bushen Jianpi Huoxue Prescription was significantly lower than that in middle-dose group(P<0.01).Conclusion:1.Based on the theory of "correlation between spleen and kidney",Bushen Jianpi Huoxue Prescription can nourish spleen and kidney,nourish and promote blood circulation,it also can increase femoral bone density and soleus muscle mass of tail-suspended rats,improve bone loss,muscle atrophy and muscle mass reduction of tail-suspended rats,prevent bone loss of tail-suspended rats and prevent disused osteoporosis.2.The mechanism of Bushen Jianpi Huoxue Prescription on bone loss in tail suspension rats: Activating AKT to activate AKT/GSK-3β and AKT/mTOR/P70S6 K signaling pathway,regulating myosin synthesis,increasing muscle mass,improving muscle stress stimulation to bone,regulating the expression of GSK-3β in skeleton and muscle,activating classical Wnt signaling pathway,thereby counteracting bone loss caused by tail suspension. |
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