Fasting produces antidepressant-like effects via activating mammalian target of rapamycin complex 1 signaling pathway in ovariectomized mice

Recent studies have shown that a 9-hour fast in mice reduces the amount of time spent immobile in the forced swimming test.Howeve r,whether 9-hour fasting has therapeutic effects in female mice with depressive symptoms has not been established.Therefore,in this study,we simulated perimenopausal depression via an ovariectomy in mice,and subjected them to a single 9-hour fasting 7 days later.We found that the ovariectomy increased the time spent immobile in the forced swimming test,inhibited expression of the mammalian target of rapamycin complex 1 signaling pathway in the hippocampus and prefro ntal cortex,and decreased the density of dendritic spines in the hippocampus.The 9-hour acute fasting alleviated the above-mentioned phenomena.Furthermore,all of the antidepressant-like effects of 9-hour fasting were reve rsed by an inhibitor of the mammalian to rget of rapamycin complex 1.Electrophysiology data showed a remarkable increase in long-term potentiation in the hippocampal CA1 of the ovariectomized mice subjected to fasting compared with the findings in the ovariectomized mice not subjected to fasting.These findings show that the antidepressant-like effects of 9-hour fasting may be related to the activation of the mammalian target of the rapamycin complex 1 signaling pathway and synaptic plasticity in the mammalian hippocampus.Thus,fasting may be a potential treatment for depression.

Effects of genistein on neuronal apoptosis,and expression of Bcl-2 and Bax proteins in the hippocampus of ovariectomized rats

Genistein is one of several isoflavones that has a structure similar to 17β-estradiol, has a strong antioxidant effect, and a high affinity to estrogen receptors. At 15 weeks after ovariectomy, the expression of Bcl-2 in the hippocampus of rats decreased and Bax expression increased, with an obvious upregulation of apoptosis. However, intraperitoneal injection of genistein or 17β-estradiol for 15 consecutive weeks from the second day after operation upregulated Bcl-2 protein expression downregulated Bax protein expression, and attenuated hippocampal neuron apoptosis. Our experimental findings indicate that long-term intervention with genistein can lead to a decrease in apoptosis in hippocampal neurons following ovadectomy, upregulate the expression of Bcl-2, and downregulate the expression of Bax. In addition, genistein and 17β-estradiol play equal anti-apoptotic and neuroprotective roles.

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.

Bone loss prevention in ovariectomized rats using stable amorphous calcium carbonate

In assessing the relationship between calcium supplementation and maintaining bone mass or reducing the risk of fracture, the effectiveness of calcium supplementation has never been decisive. Freshwater crayfish rely on amorphous calcium carbonate (ACC), an instable polymorph of calcium carbonate, as the main mineral in the exoskeleton and in the temporary storage organ, the gastrolith. Inspired by the crayfish model, we have previously shown an increase in calcium bioavailability in rats administered with synthetic stable ACC vs. crystalline calcium carbonate (CCC). The current study compared the effects of amorphous calcium derived from either gastrolith or synthetic ACC with those of crystalline calcium, found in commercial CCC or calciumcitrate supplements, in a bone loss prevention model. Rats were subjected to either sham or ovariectomy (OVX) operation (n^20/ group) followed by administration of food pellets supplemented with 0.5% calcium from either source over 12 weeks. Micro-computed tomography (μCT) and histomorphometric analyses revealed bone loss prevention by both gastrolith and ACC treatments, manifested by an increase in morphometric bone parameters, compared to both CCC-?and calcium citrate-treated groups. Both gastrolith and ACC treatments resulted in bone formation in the tibia cancellous bone, indicated by dynamic histomorphometry parameters, compared to either the CCC or calcium citrate treatments. Levels of urine deoxypyridinoline (DPD), suggested an anti-resorptive effect of ACC, which was also the only treatment that led to a significant increase in vertebral mechanical strength, as supported by μCT analysis of topology and orientation parameters of the vertebral trabeculae. To our knowledge, such levels of bone loss prevention by calcium supplements have never been reported. These findings thus suggest the potential of both natural (crayfish gastrolith) and, to a greater extent, synthetic ACC sources for the prevention of metabolic bone disorders and possibly of osteoporotic processes.

Dynamic changes of beta-amyloid protein deposition in hippocampus of female ovariectomized rats

BACKGROUND： To evaluate and summarize the effects of cerebral perfusion and vascular reserve on the treatment of SICAS. Recently, research on β-amyloid protein has focused on the regulatory effects of estrogen or phytoestrogen on its deposition. However, there have been only a few reports on dynamic changes of β -amyloid protein deposition in hippocampus of ovariectomized rats. OBJECTIVE： To measure β -amyloid protein deposition in the hippocampal formation of ovariectomized rats by using immunohistochemistry; to observe time-dependent dynamic changes. DESIGN： Randomized controlled animal study. SETTING： Third Xiangya Hospital of Central South University. MATERIALS： The experiment was carried out in the Central Laboratory of the Third Xiangya Hospital of Central South University from November 2005 to December 2006. Fifty healthy female Sprague Dawley （SD） rats, weighing （293 ± 10） g, were provided by the Animal Laboratory of Xiangya Medical College, Central South University. All rats had neither a childbearing history nor hepatic or renal disease, or skeletal deformity. β-amyloid protein immunohistochemical kit was provided by Wuhan Boster Company. The experiment was in accordance with animal ethics standards. METHODS： All rats were randomly divided into five groups, including normal control group （n = 10）, sham operation group （n = 10）, and ovariectomized group （n = 30）. After anesthesia in the ovariectomized group, the bilateral ovaries were separated and resected. The same volume of fat was resected in the sham operation group. Rats from the normal control group, however, did not receive any surgical treatments. Rats in the normal control group and sham operation group were sacrificed by anesthesia 7 weeks after surgery. Every ten rats from the ovariectomized group was respectively sacrificed at 7, 15, and 30 weeks after surgery. lmmunohistochemistry was used to detect β-amyloid protein deposition in hippocampal sections. Cell counting and gray value measurements served to record the dynamic changes in β-amyloid protein deposition. MAIN OUTCOME MEASURES： （1） Morphological changes. （2） Positive cell counts from β -amyloid protein stainings and gray value measurements. RESULTS： All 50 rats were involved in the final analysis. （1） Morphological changes. β -amyloid-positive cells were detected in the hippocampus of all rats. Biebrich scarlet stained neurites with a swollen cytoplasm. A few β -amyloid-positive cells were observed in all groups 7 weeks after surgery, and plasma and neurites were slightly stained. By 15 weeks after surgery, a number of β -amyloid-positive cells were observed in the ovariectomized group, and plasma and neurites were also slightly stained. By 30 weeks after surgery, however, many β-amyloid-positive cells were observed in the ovariectomized group. These cells were partially aggregated and darkly stained. （2） Positive cell counts and gray value of β-amyloid protein in hippocampus. At 7 weeks after surgery, cell counts and gray value measurements were not significantly different in the ovariectomized group compared to the sham operation group and normal control group （P 〉 0.05）. Cell counts and gray value measurements were higher in the ovariectomized group by 15 weeks compared to those by 7 weeks in the normal control group, sham operation group and ovariectomized group （P 〈 0.05）. At 30 weeks after surgery, cell counts and gray value measurements were higher in the ovariectomized group compared to the normal control group. In addition, there were significant differences between sham operation group and ovariectomized group, at 7 and 15 weeks after operation （P 〈 0.05-0.01 ）. Cell counts and gray value measurements increased in all groups over time. CONCLUSION： Extended estrogen deficiency in rats can increase β-amyloid protein deposition in the hippocampus and the deposition increases over time.

Effects of genistein and 17 beta-estradiol on hippocampal synaptophysin expression in ovariectomized rats

BACKGROUND： Phytoestrogen, derived from plants, is an estrogen-like element, and is effective and safe for estrogen replacement. OBJECTIVE： To compare the interventional effects of genistein and 17 S-estradiol on learning and memory and synaptophysin （SYN） expression in the hippocampus of ovariectomized rats.DESIGN： Randomized controlled animal study.SETTING： Department of Neurology, the Third Affiliated Hospital, Xiangya Medical College, Central South University. MATERIALS： 130 healthy female Sprague Dawley （SD） rats, 6 months old and weighing （293.1 ± 10.2） g, were provided by the Second Xiangya Hospital of Central South University. This animal experiment received confirmed consent from the local ethics committee. All rats were randomly divided into 5 groups, including baseline group （n= 10）, sham operation group （n = 30）, ovariectomized group （n = 30）, genistein group （n = 30）, and 17 β -estradiol group （n = 30）. Rats in the latter four groups were observed for 3 weeks （n = 10） and for 15 weeks （n = 20） after model establishment. METHODS： This study was performed at the Department of Endocrinology, the Second Affiliated Hospital, Xiangya Medical College, Central South University from August 2005 to January 2006. Animals were not submitted to any treatment in the baseline group, but anesthetized and sacrificed at the 7 months of age. After anesthesia in the ovariectomized, genistein, and 17 S-estradiol groups, both ovaries were separated and resected to establish an ovariectomized model. The same volume of fat was resected in the sham operation group. After surgery, rats were intraperitoneally injected with 5 mg/kg genistein in the genistein group,10μg/kg 17 β -estradiol in the 17 β-estradiol group, and 0.1 mL/100 g dimethyl sulfoxide （DMSO）/polyethylene glycol （PEG）-200 stock solution in the sham operation and ovariectomized groups once a day until one day before sacrifice. MAIN OUTCOME MEASURES：① Learning and memory changes of SD rats were detected using water maze behavioral testing 3 and 15 weeks after surgery. ② SYN expression in the hippocampus was measured using immunohistochemistry. RESULTS： A total of 16 out of 130 rats died due to infection, and 114 rats were included in the final analysis. ① Comparison of water maze results from the five groups： by 3 and 15 weeks after surgery, escape latency was prolonged and platform-crossing times decreased in the ovariectomized group compared to the baseline, genistein, 17 β-estradiol, and sham operation groups （t = 4.17 14.64, P 〈 0.05）. However, there were no significant differences in escape latency and platform-crossing times among the sham operation, genistein, and 17 S-estradiol groups （P 〉 0.05）. ② Distribution and quantity of SYN immunoreactive products in hippocampus： SYN-immunoreactive cells stained darkly in the baseline and sham operation groups, but were lightly stained in the genistein, 17 S -estradiol, and ovariectomized groups. In particular, SYN-immunoreactive cells stained lightly in the ovariectomized group 15 weeks after surgery. SYN correction gray values in hippocampal sub-regions, especially in the mossy fiber layer of the CA3 region, of the ovariectomized group was lower compared to the baseline, sham operation, 17 β -estradiol, and genistein groups （t = 12.57 23.92, P 〈 0.05） 15 weeks after surgery. However, there were no significant differences in SYN correction gray values among the baseline, sham operation, 17 β -estradiol and genistein groups （P 〉 0.05）. CONCLUSION： Genistein or 17 β -estradiol supplemental therapy antagonizes memory deterioration, due to endogenous estrogen deficiency and blocks the decrease of SYN expression in the hippocampus. The effect of genistein is similar to 17 β -estradiol.

Estrogenic activity of hydroalcoholic extract of Clitoria ternatea Linn. leaves on rats

Objective:To assess the estrogenic activity of hydroalcoholic extract of Clitoria(C.)ternatea leaves in female Wistar rats.Methods:Hydroalcoholic extract of C.ternatea leaves prepared by using cold maceration method was tested for estrogenic activity.An acute toxicity study was conducted to estimate the safe dose using OECD 423 guidelines.For estrogenic activity,ovariectomized female rats were divided into four groups,with 6 rats in each group.The control and standard groups were administered with 1%carboxymethyl cellulose orally and estradiol valerate at 1μg/rat/day subcutaneously,respectively.The third group was administered with hydroalcoholic extract of C.ternatea at the dose 500 mg/kg body weight orally and the fourth group was administered with hydroalcoholic extract of C.ternatea at the dose 500 mg/kg body weight orally along with estradiol valerate at dose 1μg/rat/day subcutaneously.All treatments lasted for 7 consecutive days and estrogenic activity was assessed by observing vaginal cornfication.On day 8,all animals were sacrificed and uterine horns were dissected out.Utrine weight was measured and blood serum was further processed for the estimation of biochemical parameters like cholesterol,total proteins,alkaline phosphatase and estrogen by autoanylser.Histological studies of uterus were also carried out.Results:Acute toxicity studies indicated the hydroalcoholic extract of C.ternatea leave was found to be safe up to the dose level of 2000 mg/kg.Oral administration of C.ternatea extract at the dose 500 mg/kg body weight and and estradiol valerate(1μg/rat/day)caused morphological changes i.e.increase in uterine weight,vaginal opening and cornification of cells;biochemical changes i.e.increase in cholesterol,total protein,alkaline phosphatase and estrogen contents;histological changes i.e.increase in uterine diameter,thickness and height of endometrium.Simultaneous administration of C.ternatea extract with estradiol valerate showed a synergistic effect.Histological investigations further confirmed the strong estrogenic nature of C.ternatea extract.Conclusions:C.ternatea extract(500 mg/kg)showed a significant estrogenic activity which is also supported by biochemical and histological studies.So,on the basis of these findings,it can be concluded that C.ternatea can be used as an alternative to synthetic oral contraceptives.

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.

Effects of Lepidium sativum supplementation on growth and gonadotropins secretion in ovariectomized, estrogen-implanted rabbits

Objective: To test the effects of dietary supplementation of Lepidium sativum (LS) seed powder on growth performance and gonadotropins secretion in ovariectomized, estradiol-implanted rabbits. Methods: Ovariectomized, estradiol-implanted Chinchilla rabbits were assigned into four experimental groups: LS seed powder was included into normal rabbit chow at 0% (control), 5% (low), 7% (mid) and 10% (high) w/w. Experimental feed and water were given ad-libitum for 3 weeks. Weekly body weights and daily feed intake of rabbits were recorded. Twenty-one days post-feeding, blood samples were collected at 15-minute interval for 3 h (PeriodⅠ) after which 2.5 μg gonadotropin-releasing hormone (GnRH) was injected intravenously and the sampling continued for another hour (PeriodⅡ). Plasma was harvested and analyzed for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by radioimmunoassay. Results: Feed intake was significantly (P<0.05) increased in LS-supplemented rabbits. However, the increase in feed intake did not result in significant body weight gain. LS seed supplementation significantly (P<0.001) increased mean plasma LH dose-dependently from the low- to the mid-LS level and then decreased LH at the high-LS level. LS supplementation increased (P<0.001) plasma FSH secretion. Injection of GnRH had no effect on plasma LH, however significantly (P<0.05) decreased overall plasma FSH secretion. Conclusions: LS seed supplementation stimulates feed intake and gonadotropins secretion in rabbits. Gonadotropins effect may be mediated through LS seeds phytosterols through the activation of estrogen receptors thereby producing agonistic effects resulting in LH and FSH secretion. The differential responses of gonadotropins to GnRH in LS-supplemented rabbits suggest differential regulation of the synthesis and secretion of these gonadotropins.

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.

Agnuside Reduces Visceral Adipocyte Size,Improves Insulin Sensitivity and Reverses Hepatic Steatosis in Ovariectomized Rats

This study aimed to investigate the effects of agnuside(AGN),a phytoestrogen in Vitex agnus-castus,on cellular and metabolic alterations induced by ovariectomy in rats.Ovariectomized(OVX)Wistar rats were used as an animal model of postmenopausal metabolic syndrome.The visceral and subcutaneous adipocyte sizes,the peripheral insulin sensitivity and the occurrence of nonalcoholic fatty liver disease(NAFLD)and liver oxidative damage were evaluated.The results were compared with those obtained from control rats(sham-operated)and OVX rats treated daily with AGN.Obese OVX rats exhibited visceral adipocyte hypertrophy and developed glucose intolerance,hyperinsulinemia,NAFLD and dyslipidemia.AGN exerted beneficial effects on several metabolic parameters in OVX rats,including the improvement in glucose intolerance,hyperinsulinemia and NAFLD.Besides,AGN promoted a general improvement in the liver redox state.Although the reduction of the mesenteric adipocyte size is very likely to be an important mode of action of AGN,some actions of AGN could also involve the interaction with estrogen receptors.

S1-5 Effects of Osthole on Learning and Memory and the Estrogen Pathway in Ovariectomized Rats

Objective:To investigate the effects of osthole,a natural coumarin first derived from the Cnidium plant,on learning and memory,physiological and pathological changes,and expression of estrogen receptor(ER)αandβin the brain of ovariectomized(OVX)rats of Alzheimer’s disease(AD)models.Methods:Female rats were randomly divided into six groups:①sham operation,and OVX plus:②saline,③Estradiol(0.1 mg·kg-1;positive control),④osthole at 12.5 mg·kg-1,⑤osthole at 25 mg·kg-1,and⑥osthole at 50 mg·kg-1;intragastric administration for 30 days.The Morris water-maze test was used to evaluate the learning and memory ability of rats,ELISA to measure the levels of estradiol in the serum,Western blotting to detect the expression of ERαand ERβin the hippocampus,and HE staining to determine the histopathological changes in the brain.Results:①Effects on learning and memory:compared to the OVX alone,osthole at 25 or 50 mg·kg-1 signifi cantly increased the number of entries and the duration in the target quadrant in the water-maze probe trial test(P<0.05).②Effects on the estrogen pathway in the brain:the level of estradiol in the serum and expression of ERβin the hippocampus in the OVX alone were signifi cantly lower,while the expression of ERαwas higher,relative to the sham operation control(P<0.01);osthole at 25 mg·kg-1 reversed the OVX-induced changes in expression of ERαand ERβ(P<0.01).③Effects on histopathological change in the brain:in comparison with the sham operation group,the OVX rats treated with saline displayed increases in the number of apoptotic cells in the hippocampus,which was reversed by osthole at 25 or 50 mg·kg-1(P<0.05),but not the lower dose of 12.5 mg·kg-1.Conclusion:Osthole produced enhancement of learning and memory in the ovariectomized dementia model,which was mediated,at least in part,by regulating neuronal apoptosis and the estrogen pathway.Therefore,osthole is potent in delaying the development of female neurodegenerative diseases,which provides a potential,new approach to treatment of female AD.

NP-15 Effects of Biochanin A on Learning and Memory in Ovariectomized Rats

Objective:To observe the effects of biochanin A on learning and memory of ovariectomized rats and the histopathological changes in uterus and breast tissues,so as to look for a new drug instead of estradiol to prevent or treat neuro-degenerative diseases after menopause.Method:A total of 48 six-month-old female rats were randomly divided into six groups:sham group,model group,17β-estrodiol,Biochanin A high-dose group,middle-dose group and low-dose group.The changes of the spatial learning and memory function were observed through Morris water maze.After the treatment,morphous of the hippocampus were observed after HE stained by microscope.Internal structure of hippocampal neurons was observed under transmission electron microscope.Colorimetry method was used to test SOD and GSH-PX activity and MDA content in the left cerebral homogenate.Western Blot was used to examine the expression of PPAR gamma and MMP-2 in right hippocampus of rats.After the treatment,the histopathological changes in uterus and breast tissues were observed to probe its safety for therapy.Result:Biochanin A could significantly antagonize the decrease of spatial learning and memory induced by OVX.Under the optical microscope and Electron microscopy observation,The number of necrosis nerve cells in Biochanin A treatment groups were significantly reduced.Biochanin A groups could decrease the content of MDA,and increase the activity of GSH-PX,SOD.The expression of PPAR gamma was down regulated and the expression of MMP-2 was up regulated in Biochanin A groups.The number of acini decreased,tissue atrophy in model group,low-dose group and middle-dose group,but the situatiion reserved in high-dose group.Conclusion:Biochanin A could significantly improve memory dysfunction due to lower estrogen levels in rats,the mechanism of which may be related to antioxidative stress.

NP-21 Effects and Mechanisms of Estrogen on Endogenous Growth Hormone in Brain of Ovariectomized Rats

Objective:To observe the effects of estrogen/plant estrogen(Biochanin A,BCA)on learning and memory of ovariectomized rats,so as to explore the possible mechanisms of neuroprotective effect on endogenous growth hormone.Method:A total of 50 six-month-old female rats were randomly divided into five groups:sham group,model group,17β-estrodiol,Biochanin A high-dose group,and low-dose group.The changes of the spatial learning and memory function were observed through Morris water maze and shuttle box.After the treatment,morphological structures of rat neurons were observed by HE staining and electron microscopy.The contents of E2 and GH in serum were determined by ELISA and associated proteins of growth hormone JAK/STAT signaling pathway were examined by western blot.Result:Estrogen/BCA could enhance learning and memory ability of the aging rats,improve the structures of the nerve cells,increase E2,GH and IGF1 levels,improve STAT5 and p-STAT5 protein expression and reduce SOCS2 protein expression.Conclusion:Estrogen/BCA could significantly improve memory dysfunction,the mechanisms may be related to increase secretion of endogenous growth hormone,regulate growth hormone JAK/STAT signaling pathway.

Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro

The present study aimed at investigating the effects of Puerarin(PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized(OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR(2, 4, and 8 mg·d^(-1), respectively) or 17β-estradiol(E_2, 0.03 μg·d^(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density(BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase(TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand(RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin(OPG) and RANKL m RNA expression in osteoblasts. The expression of OPG m RNA and RANKL m RNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG m RNA and reduced the expression of RANKL m RNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts.

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.

Different effects of L-arginine on morphine tolerance in sham and ovariectomized female mice

Objective:The roles of gonadal hormones and nitric oxide(NO) on the analgesic effects of morphine,tolerance to morphine,and their interactions have been widely investigated.In the present study,the effect of L-arginine(an NO precursor) on morphine tolerance in sham and ovariectomized(OVX) female mice was investigated.Methods:Forty mice were divided into sham and OVX groups.On the first day,a hot plate test((55±0.2) °C;cut-off 30 s) was carried out as a base record 15 min before injection of morphine(10 mg/kg,subcutaneously(s.c.)) and was repeated every 15 min after injection.The sham group was then divided into two subgroups:sham-tolerance-L-arginine(Sham-Tol-LA) and sham-tolerance-saline(Sham-Tol-Sal) which received either L-arginine 50 mg/kg(intraperitoneally(i.p.)) or saline 10 ml/kg(i.p.),respectively,three times in a day for three consecutive days.Morphine tolerance was induced in animals by injecting 30 mg/kg morphine(s.c.) three times/day for three days.This treatment was also used for OVX subgroups.On the fifth day,the hot plate test was repeated.The analgesic effect of morphine was calculated as the maximal percent effect(MPE).The results were compared using repeated measure analysis of variance(ANOVA).Results:There was no significant difference in MPE between the OVX and sham groups.The MPEs in both the Sham-Tol-Sal and OVX-Tol-Sal groups were lower than those in both the sham and OVX groups(P<0.01).The MPE in the OVX-Tol-Sal group was greater than that in the Sham-Tol-Sal group(P<0.01).The MPE in the Sham-Tol-LA group was higher than that in the Sham-Tol-Sal group(P<0.01).However,there was no significant difference between the Sham-Tol-LA and sham groups or between the OVX-Tol-LA and OVX-Tol-Sal groups.Conclusions:The results of the present study showed that repeated administration of morphine causes tolerance to the analgesic effect of morphine.L-Arginine could prevent tolerance to morphine but its effect was different in the presence of ovarian hormones.

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.