| Osteoporosis(OP) is a systemic metabolic bone disease, which is marked by the bone micro structural damage, a loss of bone mass, fragile and more prone to fracturing. Osteoporosis has become a public health problem which is impact in old people’s physical and mental health. Currently, it has nearly 100 million osteoporosis patients in China. In the middle of the 21 st century, Chinese patients will up to 200 million. In our country, aged 50 to 59 male with osteoporosis prevalence was 14%, and the female prevalence was 20%. The risk of the death form the female was 2.8%, and which is fairly with the risk of death from breast cancer. In our country, aged 60 to 69 male with osteoporosis prevalence was 30%, and the female prevalence was 50%-57%. With the aggravation of the aging society, the number of patients with osteoporosis is increasing, and the serious consequences of brittle fracture have greatly increased the elderly morbidity and mortality.At present, the western medicine treatment of osteoporosis mainly includes bone mineral drugs, estrogen and bone formation stimulating agents(calcium preparation, estrogen, parathyroid hormone, statins, etc.). These drugs have a certain effect on the treatment of osteoporosis in the short term, but treatment effect is not satisfactory in the long-term, and it has lots of toxic effects. For example, estrogen therapy can significantly increased the incidence of breast cancer.Traditional Chinese medicine has great advantages in the treatment of osteoporosis, and traditional Chinese medicine can significantly improved the physical and mental health of patients. DYGP derived from clinical prescription, and it is curative effect. The experiment is to establish the osteoporosis rats model by retinoic acid. Preliminary experiments showed that, no calcium preparations by DYGP could significantly increased the content of bone calcium and bone density in ovariectomized rats of osteoporosis. So that, DYGP how to increase the content of bone calcium and bone mineral density? This study would explore OPG/RANK /RANKL system, and initially revealed the mechanism for DYGP in the treatment of OP.Objective:This experiment is to investigate DYGP on osteoporosis rats induced by retinoic acid, through the influence of OPG, RANKL, TGF-β1 gene expression and bone tissue morphology, initially revealed the mechanism of DYGP in the treatment of osteoporosis.Methods:1 Group, Modeling, and Administration: Totally sixty six months old female SD rats were randomly divided into: the control group(N), the model group(MO), the positive control group(XC), the low-dose group of DYGP(L), the middle-dose group of DYGP(M), the high-dose group of DYGP(H), 10 rats in each group. In the first 14 days, except the N group, each group of the rats were given retinoic acid(7 mg·m L-1) by gavage in the morning. In the afternoon, three groups of DYGP were given DYGP(25 mg·m L-1, 50 mg·m L-1, 100 mg·m L-1) by gavage, the positive control group was given Xianling Gubao capsule(31.2 mg·m L-1) by gavage. In the second 14 days, the model group was given distilled water by gavage, The different treatment groups were given the corresponding drugs. In the whole process, the normal group was only given distilled water by gavage, and each solution volume was in accordance with 10 m L·kg-1.2 Body Weight Monitoring: Weighed the rats body weight in the modeling and drug intervention in each week.3 Materials and Pretreatment: In the 29 th day, the rats were injected with intraperitoneal of 0.5 m L/100 g 4% chloral hydrate, then took abdominal aortic blood 5 m L quickly, 4℃ stood for 20 minutes, 4000 rpm centrifuged for 15 minutes, and then stored in-80℃ refrigerator. Rats were killed, and took a segment of bone tissue quickly, removed the ligaments, cleaned with physiological saline, absorbed surface water with dry filter paper, the femoral were placed in-80℃ refrigerator, the tibia were placed in 4% paraformaldehyde.4 Indexes Detection: The content of OPG, TGF-β1 and osteocalcin(OC) were detected by enzyme linked immune sorbent assay(ELISA). The expressions of OPG m RNA, RANKL m RNA, TGF-β1 m RNA in the bone were detected by using reverse transcription polymerase chain reaction(RT-PCR),the right were weighed and then to chloroform: methanol mixture(2:1) skim for 72 hours, set the oven drying at 120℃ for 6 hours, the cooling constant weight for the dry weight, calculated the ratio of dry weight/wet weight. The right of tibia were removed the ligaments, cleaned with physiological saline, absorbed surface water with dry filter paper, then fixed in 4% paraformaldehyde for 24 hours, 10% EDTA decalcified for 42 days, then under the epiphyseal line 0.5 cm was taken, which was for the morphological changes of bone tissue.Results:1 effect of DYGP on the weight of rats with OPBefore modeling, among six groups, there were no significant difference(P>0.05); After modeling, compared with the control group, the body weight in model group was sharply decreased(P<0.05). Compared with model group, the body weight of high dose group of DYGP was significantly increased(P<0.05).2 Bone tissue morphology change in rats with OPThe control group of bone was dense, uniform distribution; The model group of bone became thinner, sparse fracture connection, some turned into “buttons” shape, mesh structure destroyed, the number of osteoclasts increased significantly, the number of osteoblasts were decreased, marrow cavity significantly larger and irregular arrangement, calcium salt deposition reduced sharply. Compared with the model group, two kinds of dose-group(M, H) of DYGP and the positive control group of bone became wider, significant increased in the number of mesh structure and parts of it was recovered, the number of osteoblasts increased significantly, the number of osteoclasts decreased, bone marrow cavity size distribution and rules, calcium salt deposition significantly increased.3 Effect of DYGP on the ratio of dry weight/wet weight in the femur of rats with OPCompared with the control group, the ratio of dry weight/wet weight in femur of rats was sharply decreased in model group(P< 0.01). Compared with the model group, two kinds of dose-group(M, H) of DYGP and the positive control group could significantly increased the ratio of dry weight/wet weight(P< 0.05).4 Effects of DYGP on the content of OPG, TGF-β1 and OC in serum of rats with OPCompared with the control group, the content of OPG and TGF-β1 in serum of rats was decreased sharply in model group(P< 0.01), the content of OC in serum of rats in model group was significantly increased(P< 0.05). Compared with the model group, two kinds of dose-group(M, H) of DYGP and the positive control group could significantly increased the content of OPG in serum of rats(P< 0.05, P< 0.01), high dose group of DYGP and the positive control group could significantly increased the content of TGF-β1in serum of rats(P< 0.05), two kinds of dose-group(M, H) of DYGP and the positive control group could increased the content of OPG in serum of rats(P< 0.05).5 Effects of DYGP on the expression of OPG, RANKL and TGF-β1 m RNA in the femur of rats with OPCompared with the control group, the expression of OPG m RNA and TGF-β1 m RNA was significantly decreased in femur of rats in model group(P< 0.01), the expression of RANKL m RNA and the ratio of RANKL/OPG was significantly increased in femur of rats in model group(P< 0.01). Compared with the model group, two kinds of dose-group(M, H) of DYGP and the positive control group could increased the expression of OPG m RNA and TGF-β1 m RNA in femur of rats(P< 0.01), two kinds of dose-group(M, H) of DYGP and the positive control group could reduced the ratio of RANKL/OPG in femur of rats(P< 0.01), three kinds of dose-group(L, M, H) of DYGP and the positive control group could reduced the expression of RANKL m RNA in femur of rats(P< 0.01).Conclusion:DYGP improved the bone microstructure degeneration of OP rats. The mechanisms of DYGP in the prevention and treatment of OP rats may be related to increased osteoblast or stromal cells expressed the femoral of OPG and TGF-β1, inhibited the expression of RANKL, and increased OPG and RANKL competitive combination. Consequently, the effect inhibited osteoclast differentiation, activation and maturation, antaonized bone absorption. |
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