Activities of the Deriviatives of Chitin on the Osbeoblast Proliferation and the Effect on Bone Strength in Ovariectomized Rats

This study intends to examine the effects of different concentrations of four kinds of degradations of chitin： glucosamine （GLC）, N-acetyl-D-glucosamine （NAG）, chitooligosaccharide（COS）, CM-chitooligosaccharide （CM-COS）—on the proliferation of MC3T3-E1 cell line cultured in vitro. Results suggest that all of the glucoses mentioned above promoted the proliferation of osteoblast, and various concentrations have different effects： the proliferation was remarkable when the concentration of GLC, NAG, COS, CM-COS was 100 , 100, 500, 500 μg/mL ,respectively. Furthermore we choose the glucosamine as the material and study the effect on the bone strength in ovariectomized rats. The results showed that the middle does of glucosamine can significantly increase the hone strength of femur in ovariectomized rats.

The Effect of Estrogen on the Restoration of Bone Mass and Bone Quality in Ovariectomized Rats

To evaluate the effect of estrogen on its ability to restore the bone mass and bone quality in ovariectomized rats by examining the changes of bone morphology and histomorphometry, 3- month-old rats were divided randomly into 4 groups: normal control, ovariectomized (OVX), sham- operated (Sham-O) and OVX plus estrogen (OVX+E2). Treatment initiated from the day 8 weeks after operation and continued for 12 weeks. Bone morphology and histomorphometry were examined afterwards. Results showed that comparing to control group, the trabecular bone in OVX appeared thinner and reduced in the amount. The connectivity between trabecula was decreased and the struc- ture disordered. The free-end of trabecula was increased. The cavity of bone marrow enlarged. After treatment with estrogen, above changes improved remarkably by different degree, although did not reach the normal face. The bone histomorphometry results damonstrated that estrogen treatment in- creased bone mass and the amount of trabecula by 129% and 132% respectively (P<0. 05). The activity of bone resorption decreased significantly and the rate of bone formation increased to 203%. These results suggest that treatment of ovariectomized rats with estrogen can not only increase bone mass, also improve the bone structure and enhance the property of bone mechanics.

Effect of Soy Isoflavone Crude Extract Supplementation on High Fat Diet-induced Insulin Resistance in Ovariectomized Rats

Female Wister rats aged 8 weeks were randomly divided into sham operation group, ovariectomized （OVX） control group, and 20VX groups fed with soy isoflavone crude extract supplementation. The rats had free access to high fat diet and water for 9 weeks. No significant difference was found in body weight （BW）, total abdominal fat, food intake and food utilization rate between OVX control group and 20VX groups. However, the fasting blood glucose and blood lipid levels were significantly higher in 20VX groups than in OVX control group （P〈0.05）. Intraperitoneal glucose tolerance test （IGTI＂） showed that the area under AUC was smaller in 20VX groups than in OVX control group （P〈0.05）. These findings showed that soy isoflavone crude extract supplementation can improve glucose tolerance and prevent high fat diet-induced insulin resistance in ovariectomized rats.

The effect of Wujiadan Recipe on serum sex hormone and expression of Kiss-1, GPR54 and GnRH in arcuate nucleus of hypothalamus in ovariectomized rats

Objective:To explore the effect of wujiadan Recipe on the expression of FSH,E2 in serum and KiSS-1,GPR54,GnRH mRNA in hypothalamic arcuate nucleus tissue homogenate of ovariectomized rats,and to further explore the mechanism of wujiadan Recipe on menopausal syndrome.Methods:Sixty-four adult female rats were randomly divided into three groups:blank group,model group,Chinese medicine control group,western medicine control group,and wujiadan high,medium and low dose groups.Except for the blank group,all the other groups were ovariectomized.The rats in the normal group and the model group were given the same dose of saline once a day.One rat in the blank group died during the experiment.After 4 weeks of continuous administration,blood was collected from abdominal aorta and brain stereotaxic localization was performed to preserve the arcuate nucleus of hypothalamus.The levels of FSH and E2 in serum were detected by enzyme-linked immunosorbent assay(ELISA),and the expression of Kiss-1,GPR54 and GnRH mRNA in arcuate nucleus of hypothalamus were detected by PCR.Results:1.Compared with the blank group,FSH level in the model group was significantly higher(P<0.01),and E2 level was significantly lower(P<0.01).Compared with the model group,the level of serum E2 in the western medicine control group increased significantly(P.The serum FSH content in each group decreased,especially in the high dose group and the western medicine control group(P<0.01),and in the Chinese medicine control group,the middle dose group and the low dose group(P<0.05).Compared with the blank group,the expression of KiSS-1 mRNA,GPR54 mRNA and GnRH mRNA in the model group increased significantly(P<0.01);compared with the model group,the expression of KiSS-1 mRNA and GPR54 mRNA in the high dose group,middle dose group and Western medicine control group of wujiadan prescription decreased significantly(P<0.05).Compared with the model group,the expression of GnRH mRNA in the high dose group and the western medicine control group decreased significantly(P<0.01);the middle dose group of wujiadan decreased significantly(P<0.05).Conclusion:Wujiadan recipe can improve the serum sex hormone level of castrated rats,and reduce the over expression of KiSS-1,GPR54 and GnRH mRNA in hypothalamic arcuate nucleus of castrated rats,which may be the reproductive endocrine mechanism of wujiadan recipe in the treatment of menopausal syndrome.

Effects of a Herbal Formula on Biochemical Markers of Bone Turnover in Ovariectomized Rats

Objective: To investigate the effect and mechanism of a Chinese herbal formula MG in ameliorating estrogen deficiency bone loss. Methods: 10 month old female rats were randomly divided into three groups: ovariectomized (OVX), OVX treated with MG decoction (OVX M) and OVX treated with estrogen injection (OVX E). The urinary pyridinoline creatinine ratio (Pyd/Cr), deoxypyridinoline creatinine ratio (Dpd/Cr), and plasma alkaline phosphatase (ALP) were determined at the baseline, 1, 4, 8 and 12 postoperative weeks. Results: Our data showed an average increase of 93% in Pyd/Cr ( P <0.001), 180% in Dpd/Cr ( P <0.005) and 59% in plasma ALP ( P <0.001) respectively in the OVX group after ovariectomy. Compared with OVX rats, the increase of the OVX M group were prevented 39% in Pyd/Cr, 102% in Dpd/Cr and 19% in ALP respectively ( P <0.05). Conclusion: The herbal formula MG possesses a therapeutic effect on estrogen dependent bone loss by inhibiting the bone turnover as estrogen replacement.

Effects of long-term estrogen replacement therapy on beta-amyloid precursor protein and mRNA expression in ovariectomized rat hippocampus

BACKGROUND： In vitro cultures of neural stem cells have shown that estrogen can regulate beta-amyloid precursor protein （β-APP） metabolism and reduce amyloid-beta production. OBJECTIVE： To investigate the effects of long-term oral administration of compound nylestriol or low-dose 17beta-estradiol on β-APP and mRNA expression in the hippocampus of ovariectomized （OVX） rats. DESIGN, TIME AND SETTING： This randomized and controlled experiment was performed at the Animal Laboratory and Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University between April 2003 and May 2004. MATERIALS： According to body mass, 50 six-month-old female Sprague-Dawley rats were randomly divided into five groups （n = 10 per group）： normal control, sham operation, OVX model, 17beta-estradiol （Sigma, USA）, and compound nylestriol tablet （Laboratory of Endocrine and Metabolic Disease, Xiangya Second Hospital of Central South University） groups. METHODS： Rats in OVX plus 17beta-estradiol and OVX plus compound nylestriol tablet groups underwent ovariectomy. On the second day after surgery, rats were intragastrically given 17beta-estradiol （100 μg/kg）, once per day or compound nylestriol tablet （0.5 mg/kg） and levonorgestrel （0.15 mg/kg） every 2 days. MAIN OUTCOME MEASURES： β-APP expression in the hippocampus of OVX rats was determined using immunohistochemistry （SABC method） and β-APP mRNA expression was analyzed by in situ hybridization. The results were quantitatively analyzed using cell counting and average optical density. RESULTS： The number and optical density of β-APP-positive neurons in every subregion of the hippocampus of OVX rats was dramatically increased compared with normal and sham operation groups following 35 weeks of administration （P 〈 0.05）. Levels of β-APP were decreased following oral administration of compound nylestriol or 17beta-estradiol. In situ hybridization showed that long-term estrogen deficiency and oral administration of compound nylestriol or 17beta-estradiol did not alter the number of β-APP mRNA-positive neurons. CONCLUSION： The results show that long-term estrogen deficiency results in an increase of expression of β-APP though no changes in the expression of β-APP mRNA are detected. Replacement of estrogen with low-dose 17 beta-estradiol or compound nylestriol tablet inhibits the expression of β-APP in the hippocampus to the same extent.

A Preliminary Study of The Short Circuit Current(I_(sc)) Responses of Endometria from Estrum and Bilateral Ovariectomy Female Rats

Objective To investigate the property of the endometrium ion transport in the normal estruation and ovariectomize rats. Methods The basic electrophysiological property of the endometrium was carried out by mean of the short circuit current （Isc） recording with the rats. Results The baseline Lsc and the transepithelial resistance （Rte） were different between the estruation and ovariectomized groups. Apical application of Na＋ channel blocker, amiloride （10 μmol/L）, or CFTR Cl- channel agonist, forskolin （10 μmol/L） did not significantly affect the Lc in the two groups. However, the 1sc in the model group was more sensitive to the CFTR Cl- channel blocker （glibenclamide, 1 mmol/L, apical） decreased about 27.0% （P〈0.05, n=6）, no remarkably changes in the estruation rats. While apical application CA CC CI-channel blocker, DIDS （4, 4 ＇-diisothiocyanostilbene- 2,2 ＇-disulfonic, 100 μmol/L）, the Isc was much more sensitive, percentage almost to 36.52% （P〈0.01） in the model group than that in the control Furthermore, basolateral application of bumetanide （100μmol/L）, a blocker of Na＋-K＋-2Cl- cotransport, didn＇t significantly reduce the Lc in the two groups. Conclusion The baseline Lsc and membrane resistance in the ovariectomize group is much higher than that in the normal estruation group, and the endometrial epithelium CI- secretion may be mediated by predominantly calcium-dependent CI- channels （CACC） and activating adenylate cyclase and apical cAMP-dependent Cl- channels （CFTR） in the model group with minor contributions in the control. Furthermore, the endometrial epithelium responses to different stimulants exists difference in the control and model group a likely mechanism for the ovary hormone.

Effect of Oviductus Ranae and Oviductus Ranae Eggs on Bone Metabolism and Osteoporosis

To evaluate the roles or effects of oviductus ranae （OR） or oviductus ranae eggs （ORE） in preventing and treating postmenopausal osteoporosis. Methods： In vivo experiment： Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized （OVX）, with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens （CE）, OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein （BGP）, serum calcium, phosphorus, and alkaline phosphatase （ALP） were assayed; bone mineral densities （BMD） were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment： Eighty 10-month old SD rats, with equal numbers of mates and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase （TRAP）. Results： In vivo experiment： The body weight of the four OVX groups increased significantly （P〈0.01）. Uterine weight of the CE group was the highest （P〈0.01）; Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly （P〈0.05, P〈0.01）; femoral volume in the ORE group increased significantly （P〈0.05）; and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly （P〈0.05, P〈0.01）. In vitro experiment： Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts （P〈0.05 and P〈0.01, repsectively） and stimulated the formation of calcium nodes （P〈0.05, P〈0,01）, while the enhancement of ALP activity in osteoblasts was significant （P〈0.05, P〈0.01）. The number of TRAP-positive cells was significantly reduced as well （P〈0.01）. Conclusions： OR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.