Study On Osteogenesis And Adipose Balance In Ovariectomized Osteoporosis Rats By Left, Right Gui Pill And Its Decomposition

Purpose: To research the effects of Zuogui pill, Yougui pill and prevention treatment and action mechanism of their disassembled prescriptions for ovariectomized osteoporosis rats, and explore the relation of bone metabolism and lipid metabolism from Yinyang point of view. All those practice have been made on basis of the theory called kidney storing the body essence and dominating bone and manufacturing marrow.Material and method: Dividing 90 SPF female rats at 2 months into 3 groups randomly: Normal group with 10 rats, Sham group with 10 rats, for the rest 70 rats were eccoped ovaries. Eccoped ovaries group established PMOP models, and sham group only eccoped the fat tissue around the ovaries. 1 week later, dividing the rats into 9 groups randomly: Control group, Sham group, OVX group, BJL group, ZGW group, YGW group, GTY group, ZYY group, BYY group. After 12 weeks drug treatment rats were removed, excluding the death caused by the mold gavage, including 76 rats. No eating but giving water for 24 hours before operation, 2h after the last administration weighed and gave anesthesia, taking the blood from abdominal aortic blood, serum centrifugation aliquots was stored-80 ℃, which was used for the detection of bone metabolism ELISA, enzyme-linked immunosorbent serum alkaline phosphatase enzymes ALP, osteocalcin OC, Collagen Ⅰ procollagen propeptide hydroxyl end PICP; removal from rat 3rd, 4th and 5th lumbar section, which was wrapped in wet gauze placed-80 ℃ frozen for dual-energy X-ray absorptiometry detection; taking out of one third the distal femur in rats, carefully peeling adhesion of muscle fascia and connective tissue, after frozen in liquid which was wrapped by nitrogenthe foil, and stored in-80 ℃ frozen for real-time quantitative PCR was used to detect bone tissue gene expression RUNX2, CollagenⅠ, ALP, BGP, PPARγ, C/EBPβ, and Western blot assay of bone tissue the protein expression of RUNX2, CollagenⅠ, ALP, BGP, PPARγ, C/EBPβ; taking out of one third from rat proximal tibia part, making paraffin section after the fixed decalcified for a few weeks, in order to prepare for morphology HE detection and RUNX2, CollagenⅠ, ALP, BGP, PPARγ, C/EBPβ indicators for immunohistochemistry.Results: 1. The impact for PMOP on bone mineral density for Zuogui pill, Yougui pill and their disassembled prescriptions Compared with the Control group, except for the Sham group, BMD each group has a downward trend, rat lumbar spine BMD was significantly decreased in the OVX group(P<0.01), BJL group and ZGW group was not significant downward, which was no statistically significant. Duplicating PMOP model successfully; compared with the OVX group, each treatment group for bone density was improved indifferent degrees(P<0.05 or P<0.01), which the effects of BJL group and ZGW group were better(P<0.01), basically reaching Sham group level. Comparision results between the other groups were no significant difference. 2. The morphological effects on bone tissue PMOP for Zuogui pill, Yougui pill and their disassembled prescriptions Control group and the Sham group showed trabecular bone tightly packed, which morphological structure was complete and trabecular bone was connected by reticular, trabecular bone surface was more visible and a single row of osteoblasts, marrow cavity was relatively small. While the total number of trabecular bone in the OVX group was significantly reduced, the structure was more sparse, connectivity was poor and irregular, showing a large number of blind side in trabecular bone, trabecular thickness of the wall was inconsistency, a number of bone cells were turned into trabecular bone surface, which was significantly decreased, and seen bulky, irregular and multinucleated osteoclasts, and the bone marrow cavity increased. Each treatment group after treatment for 12 weeks, the number of trabecular bone have been increased and widened in different degrees, structural arrangement and shape gradually approaching the OVX group, and it was shown that trabecular bone was characterized by reticular connection and marrow cavity which was narrowing. In all groups, effects of BJL group, ZGW group and ZYY group were marked. 3. The impact of indicators on bone metabolism in rats PMOP for Zuogui pill, Yougui pill and their disassembled prescriptions Compared with the Control group, the content in OVX group were significantly higher(P<0.01); compared with the OVX group, ALP content in each treatment group had decreased(P<0.05 or P<0.01), ZGW group decreased significantly. Compared with the Control group, OC content in OVX group were significantly increased(P<0.05); compared with the OVX group, OC content BJL group, ZGW group, YGW group, ZYY group and BYY group decreased in different degrees(P<0.05 or P<0.01), the effects for ZGW group was significantly marked, there are differences compared with the other treatment groups. Compared with the Control group, PICP content in OVX group increased, and the difference was significant(P<0.01); compared with the OVX group, each treatment group made PICP values downward and the difference was significant,(P<0.05 or P<0.01), ZGW group in treatment group is better. 4. The impact on PMOP rat osteoblast differentiation for Zuogui pill, Yougui pill and their disassembled prescriptions The results of Immunohistochemical expression: RUNX2, CollagenⅠ, ALP, BGP is mainly expressed in the cytoplasm and into the nucleus of the bone, trabecular bone structural was complete in Control group and expression of osteoblasts was normal. The number of visible osteoblasts was significantly reduced in model group, rarely showing brown coloring. After each treatment after administration the number of osteogenic cells positively stained cells increased significantly, the cell membrane and cytoplasmic were shown brown granules, so was the small amount of brown osteogenic nucleus. Gene expression results: Compared with the Control group, OVX group RUNX2, CollagenⅠ, ALP, BGP gene expression were decreased, there was a significant difference(P<0.05); compared with the OVX group, the gene expression of each treatment group RUNX2, CollagenⅠ, ALP, BGP increased in different degrees, while the content in BJL group and ZGW group RUNX2, CollagenⅠ, ALP, BGP increased significantly(P<0.01), up to the level of the Control group. Zuogui pill was superior to Yougui pill and better than all the disassembled prescriptions groups. Protein expression results: Compared with the Control group, protein expression in OVX group RUNX2, CollagenⅠ, ALP, BGP has decreased, which was a significant difference(P<0.05); compared with the OVX group, the treatment group RUNX2, CollagenⅠ, ALP, BGP protein expression were increased in different degrees, which the content of BJL group and ZGW group RUNX2, CollagenⅠ, ALP, BGP were increased significantly(P<0.01). 5. The impact on PMOP adipogenic differentiation of rat for Zuogui pill, Yougui pill and their disassembled prescriptions Immunohistochemical expression of the results: PPARγ, C/EBPβ was mainly expressed in the cytoplasm, and the Control group and Sham group were barely PPARγ, C/EBPβexpression of a small number of cells. Trabecular bone fracture in OVX group, then a small number of bone cells was reducing, which was visibly shown other intracellular membranes and cytoplasm brown coloring, the number of bone cells in each treatment after given administration increased, but the number of positive stained cells reduced, showing rarely brown coloring. Gene expression results: Compared with the Control group, gene expression in OVX group PPARγ, C/EBPβ are increased, which was significantly different(P<0.05); compared with the OVX group, PPARγ, C/EBPβ gene expression in the treatment was decreased in different degrees, which the content of BJL group and ZGW group PPARγ, C/EBPβ increased significantly(P<0.01), basically reaching the level of the Control group. Zuogui pill was superior to Yougui pill and better than all the disassembled prescriptions groups. Protein expression results: Compared with the Control group, protein expression in OVX group PPARγ, C/EBPβ was increased significantly(P<0.05); compared with the OVX group, PPARγ and C/EBPβ protein expression in each treatment group were decreased in different degrees, which the protein content in the BJL group and ZGW group PPARγ, C/EBPβ decreased significantly(P<0.01).Conclusion: 1. After eccoping the both ovaries of rats can duplicate successfully the PMOP models. 2. Zuogui pill and Yougui pill can effectively prevent and treat osteoporosis, both of them can restrict bone resorption and promote bone formation to some degree; which Zuogui pill was characterized by nourishing yin and invigorating the kidney and nourishing marrow, its effects was more significant. Zuogui pill was superior to Yougui pill and better than all the disassembled prescriptions groups. 3. Bone metabolism has relative relationship with lipid metabolism, Zuogui pill prevention osteoporosis mechanism, which can promote bone formation and bone cell proliferation, regulate fat metabolism and inhibit the formation of fat cells, has significant efficacy in preventing postmenopausal osteoporosis and regulating the symptoms of lipid metabolism disorder in the body. 4. Zuogui pill and Yougui pill all from the theory of Yin Yang Hu JI, regulate the balance between bone and fat, which achieve the prevention and treatment of postmenopausal osteoporosis, especially therapeutic effects of Zuogui pill based on the theory from obtaining Yin from Yang is the most effective.