Oral administration of egg white ovotransferrin prevents osteoporosis in ovariectomized rats

Ovotransferrin,an iron-binding glycoprotein,accounting for approximately 12%of egg white protein,is a member of transferrin fam ily.Our previous studies showed that ovotransferrin stimulates the proliferation and differentiation of osteoblasts,while inhibits osteoclastogenesis and resorption activity.The work aims to study the efficacy of orally administered ovotransferrin on the prevention of osteoporosis using ovariectomized(OVX)Sprague-Dawley rats.Oral administration of ovotransferrin showed no negative effect on body weight,food intake and organ weight.After 12-week treatment,feeding ovotransferrin at a dose of 1%(1 g ovotransferrin/100 g diet)prevented OVX-induced bone loss and maintained relatively high bone mineral density and integrated bone microarchitecture.The serum concentration of biomarkers indicating bone formation was increased in ovotransferrin administration groups,while the bone resorption biomarkers were decreased.Ovotransferrin feeding also decreased the production of serum cytokine TNF-αand IL-6,which are two stimulators for osteoclast differentiation.In addition to its direct regulatory role on bone turnover,ovotransferrin supplementation might benefit osteoporosis prevention by inhibiting adipogenesis,and regulating immune response.Our results suggested the potential application of ovotransferrin as a functional food ingredient on the prevention of osteoporosis.

Effect of Mushroom Extract Hericium erinaceus on Spatial Memory and Morphology of Neurons in the CA1 and CA3 Regions of the Hippocampus in Ovariectomized Rats

Various studies indicate that low levels of estradiol negatively impact cognitive abilities. Extracts from the fungus Hericium erinaceus (HE) contain bioactive components that promote the proper functioning of the nervous system and potential effects on protection against neurodegenerative diseases, including dementia and motor dysfunctions. The objective was to evaluate the effects of the administration of the HE mushroom extract on visuospatial memory and morphology of neurons in the CA1 and CA3 regions of the hippocampus in ovariectomized rats. 40 young Wistar rats weighing 90 ± 10 g BW were used, which were distributed into four groups of 10 animals;Control Group, non-ovariectomized and untreated rats;Group E2, ovariectomized rats treated with estradiol (2 μg/kg/body weight);Group HE, ovariectomized rats treated with the extract of the fungus Hericium erinaceus (0.5 mg/kg body weight) and Group Ovx/ST, ovariectomized rats, without treatment. The animals were tested in the Barnes and Open Field maze, then they were sacrificed, and their brains were obtained to perform a histological analysis of neuronal morphology in the CA1 and CA3 areas of the hippocampus. The most outstanding results showed that the Ovx/ST group recorded the longest time to arrive at the escape box and stay in the Barnes maze. A correlation was observed between neuronal damage and function;in the groups that did not present satisfactory performance in the maze tests, morphological alterations were identified such as the presence of some neuronal somata with degeneration characteristics such as pyknosis, nuclear basophilia and shrinkage of the cells. Its soma, as well as a decrease in the nuclear area of CA1 and CA3 neurons. It is concluded that the fungus Hericium erinaceus exerted a neuroprotective effect on the neuronal bodies of the hippocampus, associated with better performance in the visuospatial recognition memory test.

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 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.

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.

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.

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.

青娥方对去卵巢骨质疏松大鼠骨组织PI3K/Akt信号通路的影响

目的 探讨青娥方对去卵巢骨质疏松大鼠骨组织磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路的影响。方法 采用随机数字表法将72只3月龄SPF级雌性SD大鼠分为假手术组、模型组和青娥方组,每组24只。大鼠均按40 mg/kg腹腔注射2%戊巴比妥钠溶液麻醉,于下腹部正中作切口,模型组和青娥方组均切除双侧卵巢,假手术组切除卵巢周围少量脂肪组织后立即关腹,缝合伤口。造模1周后,假手术组和模型组予2 mL0.9%氯化钠溶液灌胃,青娥方组按浸膏量0.42 g/(kg·d)予青娥方药液灌胃,每日1次,连续灌胃12周。3组分别于干预4、8、12周后各处死8只并取材。Masson染色法观察股骨颈形态;检测股骨骨密度和骨生物力学指标最大载荷;ELISA法检测血清PI3K、Akt含量;qPCR和Western blot检测腰椎PI3K、Akt和糖原合成酶激酶3β(GSK3β)mRNA相对表达水平和蛋白表达量。结果 与假手术组比较,模型组干预第4周腰椎Akt mRNA相对表达水平明显升高(P<0.05);第12周股骨最大载荷明显降低(P<0.05);第8、12周股骨骨密度明显降低(P<0.05),腰椎PI3K mRNA相对表达水平均明显升高(P<0.05),PI3K蛋白表达量明显升高(P<0.05);第4、8、12周血清PI3K、Akt含量均明显升高(P<0.05),腰椎Akt、GSK3β蛋白表达量均明显升高(P<0.05),GSK3β mRNA相对表达水平明显升高(P<0.05)。与模型组比较,青娥方组干预第4周腰椎Akt mRNA相对表达水平和蛋白表达量明显降低(P<0.05);第12周股骨最大载荷明显升高(P<0.05),血清Akt含量均明显降低(P<0.05),腰椎Akt蛋白表达量明显降低(P<0.05);第8、12周股骨骨密度明显升高(P<0.05),腰椎PI3K、GSK3β mRNA相对表达水平均明显降低(P<0.05),PI3K蛋白表达量明显降低(P<0.05);第4、8、12周血清PI3K含量明显降低(P<0.05)。结论青娥方可改善去卵巢骨质疏松模型大鼠骨密度及骨生物力学性能,其机制可能与抑制PI3K/Akt信号通路有关。

基于代谢组学探究电针干预去势大鼠骨质疏松的起效机制研究

目的:利用LC-MS技术探究电针对去势骨质疏松大鼠粪便代谢物的影响,解析其起效机制。方法:SD大鼠随机分为假手术组(Con)、模型组(Mod)、电针A组(MA)与电针B组(MB)。切除双侧卵巢建立去势大鼠骨质疏松模型。MA、MB接受电针治疗,1次/d,连续治疗6 d后休息1 d。MA治疗4周,MB治疗8周。治疗结束后,检测胫骨和椎骨骨密度(BMD),靶向代谢组学定量血浆E2和HIF-1α,非靶向代谢组学呈现粪便代谢物轮廓及特征。结果:与Con相比,Mod大鼠BMD、E2水平下降,HIF-1α水平升高(P<0.01)。与Mod相比,MA、MB的BMD和E2均升高(P<0.01),MA和MBHIF-1α水平下降(P<0.05或P<0.01);且MB在BMD、E2和HIF-1α水平改善方面均优于MA,差异均具有统计学意义(p<0.05或P<0.01)。PCA和OPLS-DA结果显示各组间代谢物差异有统计学意义;治疗组排名前10的差异代谢物中,MA中6个回调,MB全部回调;KEGG分析发现,Con和Mod差异代谢物的显著富集通路主要为Vitamin digestion and absorption,电针干预后为Bile secretion通路。结论:电针可显著改变去势骨质疏松大鼠粪便代谢物,而且可能通过升高E2水平来抑制HIF-1α活性进而减轻体内氧化炎症反应,从而改善骨质,粪便代谢物主动或被动参与其中。

电针干预去势骨质疏松大鼠“骨微生态”的作用机制研究

目的:利用16S rRNA测序技术研究电针对去势骨质疏松大鼠肠道菌群的影响,探讨电针改善去势大鼠骨质疏松的作用机制。方法:SD大鼠随机分为假手术组(Con)、模型组(Mod)、电针A组(MA)和电针B组(MB)。切除双侧卵巢建立去势大鼠骨质疏松模型。MA、MB大鼠接受电针治疗,1次/d,连续治疗6 d后休息1 d。MA治疗4周,MB治疗8周。治疗结束后,进行胫骨三点抗弯实验、椎骨压缩实验和粪便16S rRNA测序。结果:与Con比较,Mod大鼠胫骨和椎骨的最大载荷、弹性模量均下降,差异具有统计学意义(P<0.01);与模型组比较,MA和MB大鼠胫骨和椎骨的最大载荷、弹性模量均升高,差异具有统计学意义(P<0.05或P<0.01);且MB的椎骨最大载荷、椎骨弹性模量、胫骨最大载荷和胫骨弹性模量均优于MA,差异均具有统计学意义(P<0.05或P<0.01)。厚壁菌门、拟杆菌门和放线菌门为各组排名前3的菌门。与Con比较,Mod厚壁菌门、放线菌门丰度增加,拟杆菌门减少,电针干预后出现不同程度的回调。综合LEfSe和DESeq2结果分析各组具有显著统计学差异的优势菌属,Con为考拉杆菌属、耐盐咸海鲜球菌属,MA为螺杆菌属、颤螺菌属,MB为假单胞菌属、Dysgonomonas属与紫色杆菌属。Beta多样性分析示微生物组间丰度有差异。结论:电针可能通过调节肠道菌群丰度和组成结构来发挥抗骨质疏松作用。

杜仲汤对去卵巢大鼠骨质疏松症的影响及机制研究

目的探讨杜仲汤对去卵巢大鼠骨质疏松症的影响及机制。方法将42只雌性SD大鼠随机分为假手术组、模型组、阳性药组(戊酸雌二醇,0.09 mg·kg^(-1))、谷胱甘肽组(36 mg·kg^(-1))以及杜仲汤低、中、高剂量组(0.54、1.08、2.16 g·kg^(-1))。去卵巢模型大鼠复制成功后,按照设定剂量灌胃给药,灌胃体积为10 mL·kg^(-1),每日1次,连续给药200 d。采用生化试剂盒检测大鼠血清中谷胱甘肽过氧化物酶(GSH)含量;ELISA法检测大鼠血清中抗酒石酸酸性磷酸酶(TRACP)、骨保护素(OPG)含量;骨密度仪检测大鼠股骨骨密度;HE染色法观察大鼠胫骨组织病理变化;Western Blot法检测骨组织中骨形态发生蛋白2(BMP-2)、OPG、一型胶原(COL1)蛋白表达水平;RT-PCR法检测骨组织中BMP-2、Runt相关转录因子2(RUNX2)mRNA表达水平。结果与假手术组比较,模型组大鼠体质量增长值显著升高(P<0.01);血清GSH、OPG的水平显著降低(P<0.05,P<0.01),TRACP水平显著升高(P<0.01);骨密度明显降低(P<0.05);骨小梁结构不完整,出现明显断裂,小梁数稀疏且明显变薄;骨组织中BMP-2、OPG、COL1蛋白表达水平均显著下降(P<0.01),BMP-2、RUNX2mRNA表达水平明显降低(P<0.05)。与模型组比较,杜仲汤低、中、高剂量组大鼠体质量增长值均显著降低(P<0.05,P<0.01),血清GSH水平显著升高(P<0.01),骨小梁结构明显改善,骨小梁变粗,数量增加,骨组织中BMP-2、RUNX2 mRNA表达水平均显著升高(P<0.01);杜仲汤中剂量组大鼠的血清OPG水平显著升高(P<0.01);杜仲汤低剂量组大鼠的血清TRACP水平显著降低(P<0.01),骨组织中COL1蛋白表达水平明显升高(P<0.05);杜仲汤低、中剂量组大鼠的骨密度显著升高(P<0.01),骨组织中OPG蛋白表达水平明显升高(P<0.01);杜仲汤中、高剂量组大鼠骨组织中BMP-2蛋白表达水平明显升高(P<0.05)。结论杜仲汤能提高去卵巢大鼠的骨密度,保护骨小梁完整性,对去卵巢大鼠骨质疏松症有一定防治作用,其机制可能与提高血清GSH、OPG水平,降低TRACP水平,上调BMP-2、OPG、COL1蛋白以及BMP-2、RUNX2mRNA表达,从而促进骨形成有关。

益骨汤对去势大鼠骨密度及经典BMP2信号通路的影响

目的观察益骨汤对去势大鼠骨组织中经典骨形态发生蛋白(bone morphogenetic protein,BMP)2信号通路的影响,旨在探讨益骨汤防治骨质疏松症(osteoporosis,OP)的作用机制。方法将44只12周龄雌性Wistar大鼠随机分为四组,每组11只。正常组不进行任何处理,模型组、戊酸雌二醇组和益骨汤组大鼠进行去势手术以模拟骨质疏松。造模成功后,分别以10ml/kg双蒸水灌胃(正常组、模型组)、0.36mg/kg戊酸雌二醇水溶液灌胃及10ml/kg益骨汤灌胃,1次/d。12周后,双能X射线骨密度仪测定股骨骨密度(bone mineral density,BMD),实时聚合酶链反应(polymerase chain reaction,PCR)检测BMP2经典信号通路BMP2、Smad1、Runx2、OSX基因的表达,免疫组织化学法、Western blot法检测BMP2经典信号通路BMP2、Smad1、Runx2、OSX蛋白的表达量。结果①双能X射线骨密度仪测定股骨骨密度,结果显示,与模型组比较,益骨汤组大鼠的BMD升高(P<0.01)。②实时荧光定量PCR检测,与模型组比较,益骨汤组大鼠的BMP2、Smad1、Runx2、Noggin基因表达量升高(P<0.05)。③免疫组织化学法检测:与模型组比较,益骨汤组大鼠的BMP2、Smad1、Runx2、OSX蛋白表达量升高(P<0.05)。④Western blot法检测:与模型组比较,益骨汤组大鼠的Smad1、Runx2、OSX蛋白含量升高(P<0.05)。结论益骨汤可能通过激活BMP2经典信号通路及抑制Noggin的高表达,促进骨形成和防治骨质疏松。

胡椒碱逆转氟西汀所致骨破坏作用的实验研究

目的针对氟西汀的骨破坏作用,研究胡椒碱与其联用的骨保护作用并探讨作用机制。方法建立SD大鼠去卵巢模型,分别灌胃给予雌二醇、氟西汀、胡椒碱及氟西汀与胡椒碱联用处理共8周。双能X射线吸收法测量骨密度;microCT检测骨微观结构;三点弯曲检测骨生物力学性能;ELISA法检测血清骨转换生化指标。培养大鼠原代成骨细胞,检测药物处理对细胞成骨分化及矿化的影响。结果与去卵巢组比,雌二醇及胡椒碱明显提高骨密度和生物力学性能,并改善骨小梁微观结构;给予氟西汀后,骨密度、骨生物力学性能和骨小梁微观结构进一步恶化,并加速骨转换;胡椒碱与氟西汀联用后,能明显减少氟西汀对骨的破坏作用。氟西汀明显降低原代成骨细胞的分化和矿化能力;胡椒碱明显提高其成骨分化。结论氟西汀引起去卵巢大鼠骨质进一步流失,其机制可能与抑制成骨细胞分化及矿化相关,氟西汀与胡椒碱联用可逆转氟西汀对骨的破坏作用。

灵芝孢子粉对去卵巢大鼠骨量、骨生物力学和骨代谢的影响

目的研究灵芝孢子粉对去卵巢骨质疏松大鼠的治疗作用。方法随机取39只SD雌性大鼠进行去卵巢手术建立骨质疏松模型,剩余20只进行假手术。饲养16周建模成功后随机从假手术大鼠中取10只设为空白组(C组),随机取30只去势大鼠分为模型组(M组)、灵芝孢子粉低浓度组(BL组,400 mg/kg)和灵芝孢子粉高浓度组(BH组,1600 mg/kg),每日1次灌胃治疗,持续10周。比较各组大鼠的体重变化、脏器指数、股骨骨密度、骨小梁微观结构变化、骨小梁参数、骨生物力学性能、血清中骨代谢物水平。结果各组大鼠体重变化差异无统计学意义(P>0.05),肝、脾和胸腺的脏器指数差异无统计学意义(P>0.05),与C组相比,M组大鼠的子宫系数显著降低(P<0.01),而BL组与BH组大鼠的子宫系数显著高于M组(P<0.05)。与M组相比,BL组与BH组大鼠的股骨骨密度(BMD)、骨体积分数(BV/TV)、骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、最大载荷、弯曲强度、弹性模量和血清中雌二醇水平(E2)均显著提升(P<0.05);而骨小梁分离度(Tb.Sp)、血清中骨钙素(OC)、骨碱性磷酸酶(BALP)和抗酒石酸酸性磷酸酶5b(TRACP⁃5b)水平均显著降低(P<0.01)。Micro⁃CT骨微结构图像显示M组大鼠骨小梁减少而稀疏,而BL组与BH组大鼠骨小梁增多而致密。结论灵芝孢子粉对去卵巢大鼠的骨质疏松有一定治疗作用,推测其通过改善内分泌功能提升雌激素水平,以此平衡骨吸收与骨形成,从而改善骨质量、提升骨强度、提高骨密度。