Mechanism Of Gubao Pill In The Treatment Of Osteoporosis By Targeting BMP-2 Protein

Objective:Studies have proved the anti-osteoporosis effect of Gubao Pill;Through clinical research,the curative effect of Gubao pill on postmenopausal osteoporosis was confirmed.Through animal experiments,it has been confirmed that Gubao Pill plays an anti-osteoporosis role by regulating BMP pathway and promoting bone formation,so as to clarify its anti-osteoporosis mechanism.To clarify the mechanism of BMP pathway in osteoblast differentiation and bone formation,and to find out its possible action whip point,so as to provide experimental basis for the study of BMP pathway in Gubao Pill promoting bone formation and preventing and treating postmenopausal osteoporosis.Methods:Experiment 1: choose 80 patients with postmenopausal osteoporosis,randomly divided into two groups,control group(Allen phosphonic acid sodium)and treatment group(bone treasure bolus joint Allen phosphonic acid sodium)each 40 cases,treatment of 6 months in a row,to observe the effect of TCM syndrome score,and the two groups,bone mineral density test,serum ALP and osteocalcin and adverse reaction in the process of treatment and complications.Experiment 2: A total of 24 Balb/ C mice with SPF grade were randomly divided into two groups: normal control group,model group(cyclophosphamide model),normal control group and observation group(retinoic acid model).The two groups were observed for general conditions,thymus index and bone mineral density,so as to establish a method for establishing an osteoporosis mouse model.Experiment 3: Mice model of retinoic acid induced osteoporosis were used.Mice in model group and gubao pill each dose group were given retinoic acid(100 mg/kg)to establish an osteoporosis model.Mice in gubao pill each dose group were given drug solution for prevention and treatment for 30 consecutive days.After the end of the experiment,mice femurs were taken,their mass was weighed,and the diameter and length of femurs were measured.HE staining was used to observe the changes of femur tissues under an optical microscope.Micro-ct was used to detect bone density.The serum levels of OC and BALP were detected by ELISA kit.Experiment 4: The mouse embryonic osteoblastic precursor cells(Mc3t3-e1 cells)were selected as the research objects,and the effect of Gubao pill on the activity of Mc3t3-e1 cells was studied.The effect of Gubao pill on ALP activity of Mc3t3-e1 cells was detected by kit method.WB method was used to detect the effect of Gubao Pill on the expression of ALP and BMP-2 in Mc3t3-e1 cells,so as to further verify the mechanism of gubao pill in promoting osteogenesis.Results:Experiment 1: After 6 months,the TCM syndrome effect score of both groups was improved compared with that before treatment,and the treatment group was better than the control group(P<0.05).Bone mineral density in both groups was improved compared with that before treatment,and the treatment group was better than the control group(P<0.05).Serum ALP and osteocalcin in the two groups were continuously decreased,which was more obvious in the treatment group(P<0.05).Experiment 2: 1.The activity and food intake of mice in the cyclophosphamide model group decreased significantly,and the hair lost luster.After the end of the experiment,the body weight of mice in the model group was significantly lower than that in the normal group(P < 0.5).In most cases,there was no significant difference in activity and food intake between the retinoic acid model group and the normal group,but there was a decrease in activity and food intake one week before the end of the experiment(no significant difference).In addition,there was no significant difference in body mass between the model group and the control group after the end of the experiment(Figure 1B).2.Compared with before modeling,thymus index of cyclophosphamide modeling mice was significantly decreased(P < 0.01);There was no significant difference in thymus index in retinoic acid mice.3.Compared with the normal group,the BMD of mice in the cyclophosphamide model group showed a decreasing trend,but there was no statistical significance.The bone mineral density of the retinoic acid model group decreased compared with the normal group,and the difference was statistically significant(P < 0.05).Experiment3: 1.Gubao Pill can prevent and cure the decrease of the mass,diameter,bone density and bone marrow tissue of the femur of mice caused by retinoic acid.2.Gubao pill promoted the secretion of OC and P1 NP in the serum of mice induced by retinoic acid.Experiment4: 1.Gubao pill has almost no toxic side effect on Mc3t3-e1 cells,and promotes cell proliferation when the dose is 1-8 mg/ml.2.Gubao Pill significantly enhanced the ALP activity of Mc3t3-e1(P<0.05 or P<0.01),and presented a dose-dependent effect.3.Gubao pill significantly increased the levels of ALP protein and BM-2 protein in Mc3t3-e1 cells in a dose-dependent manner.Conclusion: 1.Gubao Pill has obvious clinical effect on primary osteoporosis,and can improve bone density and bone conversion rate in a short term.2.The mouse model of postmenopausal osteoporosis induced by retinoic acid is highly reliable.The follow-up study of this topic will use retinoic acid to make the model.3.Gubao Pill can prevent and cure retinoic acid-induced osteoporosis in mice,and its mechanism may be related to protecting b MP-2 secretion and promoting bone formation.4.Gubao Pill can promote the proliferation and differentiation of mouse embryonic osteoblastic precursor cells(Mc3t3-e1 cells),and its mechanism may be related to the protection of BMP-2 secretion and the promotion of bone formation.