| Purpose:This experiment is based on the theory of kidney dominating bone and marrow,study of Zuoguiwan and Yougui Wan osteogenic Oseogenic Differentiation of ovariectomized rat bone marrow,To further explore the relationship between "marrow" and "bone" of traditional Chinese medicine and the possible mechanism of PMOP.Material and method:65 SPF female rats at 2 months,according to the method of random weight,divided into blank group with 10 rats,Sham operation group with 10 rats,Model group with 45 rats.With10% chloral hydrate intraperitoneal anesthesia 0.3ml/100 g weight,except blank group,sham operation group,the other groups were from the back of ovariectomized,ovariectomized sham operation group only rmovaled of around the amount of fat tissue,not ovariectomized,and finally sutured incision,given intramuscular injection of penicillin to prevent infection.Seven days after the surgery,on the basis of the rat survival situation,were divided into blank group(KB)and Sham operation group(SHAM),Model group(OVX),Progynova group(BJL),Zuoguiwan group(ZGW),Youguiwan group(YGW),a total of six groups.After 12 weeks of medication,to remove due to other reasons such as surgery,lavage rats may only lead to 5 rats death,were to enter the experimental rats for 60,based on indicators for testing.All experimental rats were fed with 24 h before feeding,The first 2h was administered,weighed and anesthetized,abdominal aorta was taken from the rats,after static 2h at room temperature,centrifugation,serum separation,frozen in-80 ? refrigerator for blood lipid detection;Rats were second,third,and forth lumbar vertebrae with wet gauze and foil wrapped,freezing at-80 ? for refrigerator,dual energy X-ray absorptiometry;Take the side of rat femur,and will be attached to the muscle and fascia,peel removed,placed 4% poly Formaldehyde Solution fixed,then into about 21 days in decalcified decalcification,paraffin section,HE staining for histological observation of bone marrow fat cell number;Rat femur,femoral cut ends,exposing the bone marrow cavity,with PBS buffer repeatedly washed out of the medullary cavity,bone marrow,until the backbone of white transparent.Take out the bone marrow centrifuge,discard the upper PBS,will be equipped with centrifugal tube bone marrow frozen in-80 ? refrigerator,respectively by real-time fluorescent quantitative PCR and Western blot of bone marrow tissue of rat bone detection indexes of Runx2,Collagen?,ALP,OPN and expression of lipid index PPAR?,C/EBP?,C/EBP ?,FABP4 gene and protein.Results:1.Effects of Zuoguiwan and Yougui Wan on to second,third and forth of ovariectomized rats lumbar bone density The bone mineral density of second,third,and forth lumbar vertebrae in each group were as follows: After 12 weeks of treatment,compared with the blank group,the expression of BMD in sham operation group was fairly,no significant difference(P>0.05),the expression of BMD in model group decreased,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group,Yougui Wan group,the expression of BMD was increased,there was significant difference(P<0.01);compared with progynova group,Zuoguiwan group the expression of BMD was fairly,no significant difference(P>0.05),Yougui Pill group increased BMD than progynova group,there was significant difference(P<0.01);and compared with Zuoguiwan group,Yougui Wan group BMD increased less than Zuoguiwan group,there was significant difference(P<0.01).Zuoguiwan and Yougui Wan can improve BMD in ovariectomized rats,the effect of Zuogui wan group was better than Yougui Wan group.2.Effects of Zuoguiwan and Yougui Wan on blood lipid in ovariectomized rats The rats blood lipid test results are as follows: After 12 weeks of treatment,compared with the blank group,sham operation group serum TC,TG,LDL-C,HDL-C expression was fairly,no significant difference(P>0.05),the expression levels of serum TC,TG and LDL-C decreased,HDL-C expression increased significantly(P<0.01).Compared with the model group,progynova group,Zuoguiwan group,Yougui Wan group serum TC,TG,LDL-Cexpression decreased,HDL-C expression increased,there was significant difference(P<0.01 or P<0.05).Compared with progynova group,Zuoguiwan group and Yougui Wan group serum TC,TG,LDL-C,HDL-C expression was fairiy,no significant difference(P>0.05);Compared with Zuoguiwan group,Yougui Wan group serum TC,TG,LDL-C expression was fairly,no significant difference(P>0.05).3.Effects of Zuoguiwan and Yougui Wan on the expressions of Runx2,Collagen?,ALP,OPN in the bone marrow of ovariectomized rats Each rat bone marrow tissue osteogenesis m RNA test results are as follows: After 12 weeks of treatment,compared with the blank group,sham operation group Runx2,Collage?,ALP,OPNm RNA expression was fairly,no significant difference(P>0.05),model group Runx2,Collagen?,ALP,OPNm RNA expression was decreased,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group and Yougui Wan group Runx2,Collagen?,ALP,OPN m RNA expression was increased,there was significant difference(P<0.01 or P<0.05);compared with progynova group,Zuoguiwan group Runx2,Collagen?,ALP,OPNm RNA expression was fairly,no significant difference(P>0.05),Yougui Wan group Runx2,Collagen?m RNA increased less than progynova group,there was significant difference(P<0.05);compared with Zuoguiwan group,Yougui Wan Collagen?m RNA increased less than less than Zuoguiwan group,there was significant difference(P<0.05).Each rat bone marrow osteogenic protein detection results are as follows: 12 weeks of treatment,compared with the blank group,sham operation group Runx2,Collagen?,ALP,OPN protein expression was fairly,no significant difference(P>0.05),the model group Runx2,Collagen?,ALP and OPN protein expression decreased,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group,Yougui Wan group Runx2,Collagen?,ALP,OPN protein expression was increased,there was significant difference(P<0.01 or P<0.05);compared with progynova group,Zuoguiwan group Runx2,Collagen?,ALP,OPN protein expression was fairly,no significant difference(P>0.05),Yougui Wan Collagen?,ALP,OPN protein expression increased less thanprogynova group,there was significant difference(P<0.01 or P<0.05);compared with Zuoguiwan group,Yougui Wan Collagen?,ALP,OPN protein expression increased less than Zuoguiwan group,there was significant difference(P<0.01 or P<0.05).4.Effects of Zuoguiwan and Yougui Wan on proximal femur bone marrow fat cell number in ovariectomized rats The femur of rats proximal HE staining results are as follows: 12 weeks of treatment,compared with the blank group,sham operation group proximal femoral bone trabecula were compact and tidy,intact,there is a small amount of fat cells in the bone marrow,no significant difference(P>0.05),model group trabecular structure scattered sparse fine,the proximal femur bone marrow fat cells increased,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group,Yougui Wan group proximal femur bone marrow fat cell number was significantly reduced,there were significant differences(P<0.01);compared with progynova group,Zuoguiwan group of proximal femur bone marrow fat cell number was fairly,no significant difference(P>0.05),Yougui Wan group proximal femur bone marrow fat cells increased,there was significant difference(P<0.01);compared with Zuoguiwan group,Yougui wan group proximal femur bone marrow fat cells increased,there was significant difference(P< 0.01).5.Effects of Zuoguiwan and Yougui Wan on the expressions of PPAR?,C/EBP?,C/EBP ?,FABP4 in the bone marrow of ovariectomized rats Each rat bone marrow tissue into a fat m RNA test results are as follows: 12 weeks of treatment,compared with the blank group,sham operation group PPAR?,C/EBP?,C/EBP ?,FABP4 m RNA expression was fairly,no significant difference(P>0.05),model group PPAR?,C/EBP?,C/EBP ?,FABP4 m RNA expression was rised,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group,Yougui Wan group,PPAR?,C/EBP?,C/EBP?,FABP4 m RNA expression decreased,there was significant difference(P<0.01 or P<0.05);compared with progynova group,Zuogui wan group PPAR?,C/EBP?,C/EBP ?,FABP4 m RNA expression was fairly,no significant difference(P>0.05),Yougui Wan C/EBP?m RNA expression increased there was significant difference(P<0.01);compared with Zuoguiwan group,Youguiwan group C/EBP?m RNA expression increased,there was significant difference(P<0.01).Each rat bone marrow tissue lipoprotein results are as follows: 12 weeks of treatment,compared with the blank group,sham operation group PPAR?,C/EBP?,C/EBP ?,FABP4 protein expression was fairly,no significant difference(P>0.05),model group PPAR?,C/EBP?,C/EBP ?,FABP4 protein expression was rised,there was significant difference(P<0.01);compared with the model group,progynova group,Zuoguiwan group and Yougui Wan group PPAR?,C/EBP?,C/EBP ?,FABP4 protein expression was decreased,there was significant difference(P<0.01 or P<0.05);compared with progynova group,Zuoguiwan group PPAR?,C/EBP?,C/EBP?,FABP4 protein expression was fairly,no significant difference(P > 0.05),Yougui wan group PPAR?,C/EBP? protein expression increased,there was significant difference(P<0.01 or P<0.05);compared with Zuoguiwan group,Yougui wan group PPAR?,C/EBP? protein expression increased,there was significant difference(P< 0.01 or P < 0.05).Conclusion:1.The bone mineral density of lumbar spine was decreased after bilateral ovarectomy in experimental rats,indicating the success of rat model relication of postmenopausal osteoporosis.Zuoguiwan and Yougui Wan can effectively reduce bone density loss in rats with ovariectomized osteoporosis for the prevention and treatment of postmenopausal osteoporosis.2.Disordered metabolism of blood lipid and enhanced differentiation of bone marrow to adipocytes were found in experimental rats after bilateral ovariectomy.Zuoguiwan and Yougui Wan can effectively improve the lipid metabolism disorder in rats with ovariectomized osteoporosis,promote osteogenic differentiation,inhibit adipogenic differentiation,and regulate the balance between osteogenic and adipogenic differentiations of bone marrow.3.The unbalanced osteogenic and adipogenic differentiations is one of the pathogenesis of postmenopausal osteoporosis.Zuoguiwan and Yougui Wan can be effective in the preventionand treatment of postmenopausal osteoporosis,of which the mechanism is the regulation of the balance between osteogenic and adipogenic differentiations of bone marrow,which can provide an experimental basis for clinical application of Zuoguiwan and Yougui Wan in the treatment of postmenopausal osteoporosis. |
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