Study On The Effects Of Donkey-hide Glue Reinforcing Bone Oral Solution On Osteoporosis In Ovariectomized Rats.

Objective: To investigate the effects of donkey-hide glue reinforcing bone oralsolution (DGRBOS) on osteoporosis to explore the mechanism of treatingosteoporosis with DGRBOS.Methods: (1) 16 female SD rats aged 6 months, one week after the feedingadaptation, were randomly divided into two groups: sham-operated group and theovariectomized group, 8 in each group. 2ï¼…pentobarbital sodium (40mg/kg)intraperitoneal anesthesia, the ovariectomized group were completely removal ofligation and ovariectomy, sham-operated group equivalent adipose tissue wereremoved, testing after three months on both sides of femur, conventional HE staining,light microscope observation of bone tissue structure; to use of dual-energy X-rayabsorptiometry of the femoral head BMD measured in rats; Biomechanical detector todetermine the maximum load (ML) and maximum compressive strain (MS); (2) 12rats were randomly selected from 36 female SD rats aged 6 months which wereequally divided into group A of sham operation with normal physiological saline.theleft 24 rats were randomly divided into 2 groups after ovariectomy:B(group ofosteoporotic animal model with normal physiological saline) ,C(treated group ofosteoporotic animal model with DGRBOS). each group included 12 rats. Theconcentration of 25-OH-VD₃,1,25-(OH)2-VD₃ were detected by ELISA, theexpression of VDR mRNA in kidney tissue was detected by real time fluorescencequantitative PCR(FQ - PCR) method; (3) 12 rats were randomly selected from 36female SD rat s aged 6 months which were equally divided into group A of shamoperation with normal physiological saline. The left 24 rats were randomly dividedinto 3 groups after ovariectomy:A(sham group),B(group of osteoporotic animal modelwith normal physiological saline) ,C(treated group of osteoporotic animal model withDGRBOS). Each group included 12 rats. the expression of typeâ… collagen gene inbone tissue was detected by real time fluorescence quantitative PCR(FQ - PCR)method and Western blot on the use of typeâ… collagen protein analysis; (4) 16 female SD rats aged 6 months, 12 of which are prepared only for oral administrationcontaining serum and control serum, 4 of which were for cell extraction. These cellswill be divided into two groups, namely, drug-containing serum group (M group) andcontrol serum group (C group). M group with the cell culture medium containingserum and C group of cells cultured with control serum-free medium, the two groupswere added 25ng/ul M-CSF and RANKL whose size were 1 ul respectively, TRAPstaining cells after interventing a week.To measure proliferation using thiazole salt(MTT) colorimetric. To detect apoptosis rate using flow cytometry by Annexin Vjointing PI.Results: (1) 1. Sham-operated rats femur bone were rich, continuous, dense,however, ovariectomized rats were sparse trabeculae and the average width oftrabecular were smaller and trabecular spacing increased. Mean trabecular width (μm):sham-operated group was 71.23±10.62, ovariectomized group was 52.65±12.17,there was statistically significant difference (p＜0.01); mean trabecular spacing (μm):sham-operated group was 83.68±3.69, ovariectomized group was 105.32±3.55,there was statistically significant difference (p＜0.01); 2.biomechanics results of thefemoral head in sham-operated group and ovariectomized group: the largestcompressive stress (N): sham-operated group was 345.87±32.16, ovariectomizedgroup was 266.13±30.05, there was statistically significant difference (p＜0.01);maximum load (N/mm2): sham-operated group was 39.13±4.28, ovariectomizedgroup was 32.39±4.37, there was statistically significant difference (p＜0.01); 3.femoral bone mineral density (gms/cm2): sham-operated group was 0.316±0.015,ovariectomized group was 0.281±0.017, there was statistically significant difference(p＜0.01); (2) The concentration of 25-OH-VD₃,1,25-(OH)₂-VD₃ in C group washigher than B ( P＜0. 01) ,which was closed to group of A(sham operation), theexpression of VDR mRNA on gene level in kidney tissue of C group was higher thanB ( P＜0. 01); (3) typeâ… collagen protein of Group C (OVX + DGRBOS) wassignificantly higher than B group (ovariectomized + saline group), p = 0.002 (p＜0.01 ), there are obvious differences in statistical significance. Typeâ… collagen gene fluorescence quantitative PCR (FQ-PCR) results of the B group and C group, p =0.004 (p＜0.01), that amplified the differences in the efficiency of a clear statisticalsignificance; (4) 1.Osteoclast cell morphology: osteoclast cytoplasm containing manyvacuoles structure, nuclear were large and circular, nucleolus were clear, a smallnumber of cells containing several nuclei,cell body and abundant cytoplasm; 2.TRAP staining showed that osteoclast cytoplasm were positive, showing red wine,nuclear were negative; 3. The impact of DGRBOS on osteoclast proliferation rate:OD (490nm) values were 0.1825±0.03641 (M group) and 0.1819±0.03532 (Cgroup), there was no significant difference statistically (p＞0.05); 4. The impact ofDGRBOS on osteoclast apoptosis rates were 89.51±1.62ï¼…(M group) and 63.79±0.87ï¼…(C group), there was statistically significant difference (p＜0.01).Conclusion. (1) There was significantly difference in the histological structure ofbone, BMD, biomechanics after removal of both ovaries between sham-operatedgroup and ovariectomized rats, as a result , to build a animal model of osteoporosis tomeet the requirements of this experiment successfully; (2) DGRBOS can not onlyimprove the concentration of 25-OH-VD₃,1,25-(OH)₂-VD₃,but also increasingregulate the VDR mRNA; (3) DGRBOS can increase typeâ… collagen mRNAexpression level and at the same time significantly increase the typeâ… collagencontent of expression, which was one of the mechanisms that DGRBOS can be treatedfor osteoporosis; (4) The use of RANKL and M-CSF in vitro differentiate bonemarrow mesenchymal stem cells to become osteoclasts successfully, DGRBOS groupand control group on the proliferation rate of osteoclasts was no significant difference,and of osteoclast apoptosis rate were significantly different. DGRBOS group canincrease osteoclast apoptosis rate, which was one of the mechanisms that DGRBOScan be treated for Osteoporosis. Chapterâ… To establish animal models for osteoporosisObjective: To observe the ovariectomized rats and sham-operated group the structureof bone tissue in rats, BMD, the difference between biomechanics in order to build tomeet the requirements of the experimental animal model of osteoporosis.Methods: 16 female SD rats aged 6 months, one week after the feeding adaptation,were randomly divided into two groups: sham-operated group and the ovariectomizedgroup, 8 in each group. 2ï¼…pentobarbital sodium (40mg/kg) intraperitoneal anesthesia,the ovariectomized group were completely removal of ligation and ovariectomy,sham-operated group equivalent adipose tissue were removed, testing after threemonths on both sides of femur, conventional HE staining, light microscopeobservation of bone tissue structure; to use of dual-energy X-ray absorptiometry ofthe femoral head BMD measured in rats; Biomechanical detector to determine themaximum load (ML) and maximum compressive strain (MS).Results: 1. Sham-operated rats femur bone were rich, continuous, dense, however,ovariectomized rats were sparse trabeculae and the average width of trabecular weresmaller and trabecular spacing increased. Mean trabecular width (μm): sham-operatedgroup was 71.23±10.62, ovariectomized group was 52.65±12.17, there wasstatistically significant difference (p＜0.01); mean trabecular spacing (μm):sham-operated group was 83.68±3.69, ovariectomized group was 105.32±3.55,there was statistically significant difference (p＜0.01); 2.biomechanics results of thefemoral head in sham-operated group and ovariectomized group: the largestcompressive stress (N): sham-operated group was 345.87±32.16, ovariectomizedgroup was 266.13±30.05, there was statistically significant difference (p＜0.01);maximum load (N/mm2): sham-operated group was 39.13±4.28, ovariectomizedgroup was 32.39±4.37, there was statistically significant difference (p＜0.01); 3.femoral bone mineral density (grns/cm2): sham-operated group was 0.316±0.015,ovariectomized group was 0.281±0.017, there was statistically significant difference (p＜0.01).Conclusion: There was significantly difference in the histological structure of bone,BMD, biomechanics after removal of both ovaries between sham-operated group andovariectomized rats, as a result , to build a animal model of osteoporosis to meet therequirements of this experiment successfully. Chapterâ…¡Effects of donkey-hide glue reinforcing boneoral solution on expression of 25-OH-VD3,1,25-(OH)2-VD3and VDR mRNA in ovariectomized rats.Objective: To investigate the effects of donkey-hide glue reinforcing bone oralsolution (DGRBOS) on expression of 25-OH-VD₃,1,25-(OH)2-VD₃ and VDR inrats,and explore the mechanism of treating osteoporosis with DGRBOS.Methods: 12 rats were randomly selected from 36 female SD rats aged 6 monthswhich were equally divided into group A of sham operation with normal physiologicalsaline.the left 24 rats were randomly divided into 2 groups after ovariectomy:B(groupof osteoporotic animal model with normal physiological saline) ,C(treated group ofosteoporotic animal model with DGRBOS). each group included 12 rats. Theconcentration of 25-OH-VD₃,1,25-(OH)2-VD₃ were detected by ELISA, theexpression of VDR mRNA in kidney tissue was detected by real time fluorescencequantitative PCR( FQ - PCR) method.Results: The concentration of 25-OH-VD₃,1,25-(OH)₂-VD₃ in C group was higherthan B ( P＜0.01) ,which was closed to group of A(sham operation), the expression ofVDR mRNA on gene level in kidney tissue of C group was higher than B ( P＜0.01)Conclusion: DGRBOS can not only improve the concentration of 25-OH-VD₃,1,25-(OH)₂-VD₃,but also increasing regulate the VDR mRNA. Chapterâ…¢Effects of donkey-hide glue reinforcing boneoral solution on expression of typeâ… collagen gene andprotein expression in ovariectomized rats.Objective: To investigate the effects of donkey-hide glue reinforcing bone oralsolution (DGRBOS) on expression of typeâ… collagen gene and protein expression inrats, and to explore the mechanism of treating osteoporosis with DGRBOS.Methods: 12 rats were randomly selected from 36 female SD rat s aged 6 monthswhich were equally divided into group A of sham operation with normal physiologicalsaline. The left 24 rats were randomly divided into 3 groups afterovariectomy:A(sham group),B(group of osteoporotic animal model with normalphysiological saline) ,C(treated group of osteoporotic animal model with DGRBOS).Each group included 12 rats. the expression of typeâ… collagen gene in bone tissue wasdetected by real time fluorescence quantitative PCR( FQ - PCR) method and Westernblot on the use of typeâ… collagen protein analysis;Results: typeâ… collagen protein of Group C (OVX + DGRBOS) was significantlyhigher than B group (ovariectomized + saline group), p = 0.002 (p＜0.01 ), there areobvious differences in statistical significance. Typeâ… collagen gene fluorescencequantitative PCR (FQ-PCR) results of the B group and C group, p = 0.004 (p＜0.01),that amplified the differences in the efficiency of a clear statistical significance.Conclusion: DGRBOS can increase typeâ… collagen mRNA expression level and atthe same time significantly increase the typeâ… collagen content of expression,which was one of the mechanisms that DGRBOS can be treated for osteoporosis. Chapterâ…£Effects of donkey-hide glue reinforcing boneoral solution on osteoclasts cultured in vitroObjective: To study the effects of donkey-hide glue reinforcing bone oral solution onosteoclasts cultured in vitro when serum containing DGRBOS, and to studyosteoclasts proliferation and apoptosis to explore the mechanism of DGRBOSefficacy in treatment of osteoporosis.Methods: 16 female SD rats aged 6 months, 12 of which are prepared only for oraladministration containing serum and control serum, 4 of which were for cellextraction. These cells will be divided into two groups, namely, drug-containingserum group (M group) and control serum group (C group). M group with the cellculture medium containing serum and C group of cells cultured with controlserum-free medium, the two groups were added 25ng/ul M-CSF and RANKL whosesize were 1 ul respectively, TRAP staining cells after interventing a week.To measureproliferation using thiazole salt (MTT) colorimetric. To detect apoptosis rate usingflow cytometry by Annexin V jointing PI.Results: 1.Osteoclast cell morphology: osteoclast cytoplasm containing manyvacuoles structure, nuclear were large and circular, nucleolus were clear, a smallnumber of cells containing several nuclei,cell body and abundant cytoplasm; 2.TRAP staining showed that osteoclast cytoplasm were positive, showing red wine,nuclear were negative; 3. The impact of DGRBOS on osteoclast proliferation rate:OD (490nm) values were 0.1825±0.03641 (M group) and 0.1819±0.03532 (Cgroup), there was no significant difference statistically (p＞0.05); 4. The impact ofDGRBOS on osteoclast apoptosis rates were 89.51±1.62ï¼…(M group) and 63.79±0.87ï¼…(C group), there was statistically significant difference (p＜0.01).Conclusion: The use of RANKL and M-CSF in vitro differentiate bone marrowmesenchymal stem cells to become osteoclasts successfully, DGRBOS group andcontrol group on the proliferation rate of osteoclasts was no significant difference, and of osteoclast apoptosis rate were significantly different. DGRBOS group can increaseosteoclast apoptosis rate, which was one of the mechanisms that DGRBOS can betreated for Osteoporosis.