Extreme obesity reduces bone mineral density: complementary evidence from mice and women

Objective: To evaluate the effects of body adiposity on bone mineral density in the presence and absence of ovarian hormones in female mice and postmenopausal women. Research Methods and Procedures: We assessed percentage body fat, serum leptin levels, and bone mineral density in ovariectomized and non‐ovariectomized C57BL/6 female mice that had been fed various calorically dense diets to induce body weight profiles ranging from lean to very obese. Additionally, we assessed percentage body fat and whole body bone mineral density in 37 overweight and extremely obese postmenopausal women from the Women's Contraceptive and Reproductive Experiences study. Results: In mice, higher levels of body adiposity (>40% body fat) were associated with lower bone mineral density in ovariectomized C57BL/6 female mice. A similar trend was observed in a small sample of postmenopausal women. Discussion: The complementary studies in mice and women suggest that extreme obesity in postmenopausal women may be associated with reduced bone mineral density. Thus, extreme obesity (BMI > 40 kg/m²) may increase the risk for osteopenia and osteoporosis. Given the obesity epidemic in the U.S. and in many other countries, and, in particular, the rising number of extremely obese adult women, increased attention should be drawn to the significant and interrelated public health issues of obesity and osteoporosis.     

条件性缺氧诱导因子-1α RNAi转基因小鼠模型的建立

缺氧诱导因子1a (hypoxia inducible factor-1 a, HIF-1 a)是细胞在缺氧等条件下稳定表达的具有转录活性的蛋白,通过与多种靶基因调控区的缺氧反应元件(hypoxia response element, HRE)结合, 调控靶基因表达, 使机体对缺氧、缺血等病理生理过程产生适应性反应。为从整体动物水平研究HIF-1 a的作用, 需要建立HIF-1 a相关遗传修饰小鼠。分别针对HIF-1 a mRNA序列的两个靶位点合成两对互补的寡核苷酸链, 构建可诱导的RNA干扰真核表达载体HIF-AB和HIF-CD。分别将CRE重组酶真核表达载体CRE-ERT2与HIF-AB或HIF-CD转染入RAW264.7细胞, 筛选得到稳定表达CRE-ERT2与HIF-AB, 或CRE-ERT2与HIF-CD的稳定细胞系。在用4-HT诱导去除上述细胞系中HIF-AB或HIF-CD所含的Neo基因后, 用CoCl2诱导HIF-1 a表达, 采用半定量RT-PCR检测HIF-AB或HIF-CD对HIF-1 a 基因表达的影响。结果发现干扰载体(HIF-AB和HIF-CD)对HIF-1 a mRNA序列的沉默效果分别为85%和72%。选择干扰效率较高的表达载体HIF-AB经显微注射获得HIF-1 a基因敲低小鼠模型, 经PCR以及测序验证获得2个转基因阳性小鼠(Founders, G0代)。G0代雄鼠与FVB/N雌鼠交配后获得2只F1代(first filial generation)转基因阳性小鼠, 经与EIIA-Cre转基因小鼠交配, 得到EIIA-Cre; HIFRNAiflox/+小鼠, RT-PCR结果显示, EIIA-Cre; HIFRNAiflox/+小鼠肝、肺、肾等组织的HIF-1 a mRNA水平明显降低, 分别约为正常对照的44%、38.2%和23.5%。该小鼠模型的建立为进一步研究HIF-1 a的功能及作用机制提供了新的手段。     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

旋覆代赭汤对食管癌细胞干性的影响*

目的: 探究旋覆代赭汤对食管癌细胞干性的影响。方法: 将BALB/c裸鼠随机分为对照组与实验组,每组5只,分别连续给予生理盐水和旋覆代赭汤(9.89 g/kg)灌胃处理,在给予灌胃第8日时皮下接种5×10⁶细胞数量的人源食管癌ECA-109细胞,每周测量肿瘤大小,4周后处死小鼠。收取肿瘤组织和小鼠血清,采用RT-qPCR、Western blot和免疫组化方法检测肿瘤细胞干性相关转录因子NANOG、OCT4、SOX2的表达。分别用含10%胎牛血清和上述两组小鼠的血清处理48 h食管癌ECA-109细胞,每组设置三个复孔,用RT-qPCR和Western blot方法检测NANOG、OCT4、SOX2的mRNA和蛋白表达水平,以及AKT及其磷酸化(p-AKT)的蛋白表达水平;用流式细胞术检测肿瘤细胞中ALDH酶活性;用成球实验检测肿瘤细胞的成球数量。结果: 与对照组相比,旋覆代赭汤显著抑制食管癌小鼠肿瘤的生长和大小(P＜0.01或P＜0.05);旋覆代赭汤药物血清显著降低食管癌细胞的中NANOG、OCT4和SOX2 mRNA和蛋白的表达水平、ALDH酶活性和成球数量,以及AKT和AKT的磷酸化(p-AKT)水平(P＜0.01或P＜0.05)。结论: 旋覆代赭汤具有抑制食管癌细胞干性的作用,其可能是治疗食管癌的潜在有效药物,为食管癌治疗探寻新的有效药物提供理论依据。     

The CB2 cannabinoid agonist AM-1241 prolongs survival in a transgenic mouse model of amyotrophic lateral sclerosis when initiated at symptom onset

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, paralysis and death within 2-5 years of diagnosis. Currently, no effective pharmacological agents exist for the treatment of this devastating disease. Neuroinflammation may accelerate the progression of ALS. Cannabinoids produce anti-inflammatory actions via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), and delay the progression of neuroinflammatory diseases. Additionally, CB2 receptors, which normally exist primarily in the periphery, are dramatically up-regulated in inflamed neural tissues associated with CNS disorders. In G93A-SOD1 mutant mice, the most well-characterized animal model of ALS, endogenous cannabinoids are elevated in spinal cords of symptomatic mice. Furthermore, treatment with non-selective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. We demonstrate that mRNA, receptor binding and function of CB2, but not CB1, receptors are dramatically and selectively up-regulated in spinal cords of G93A-SOD1 mice in a temporal pattern paralleling disease progression. More importantly, daily injections of the selective CB2 agonist AM-1241, initiated at symptom onset, increase the survival interval after disease onset by 56%. Therefore, CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS.     

Mx—cre转基因小鼠品系的建立及其培育

将携带MX启动子调控的Cre基因真核表达载体pMx-cre线性化后,通过受卵显微注射途径制备转基因小鼠,共注射99个卵,产佴0只,利用PCR对小鼠进行筛选,以基因组Southern blot确证,最后得到一个阳性的小鼠品系,进而将其保护和扩大繁殖。     

Effects of captivity,diet, and relocation on the gut bacterial communities of white‐footed mice

Microbes can have important impacts on their host's survival. Captive breeding programs for endangered species include periods of captivity that can ultimately have an impact on reintroduction success. No study to date has investigated the impacts of captive diet on the gut microbiota during the relocation process of generalist species. This study simulated a captive breeding program with white‐footed mice (Peromyscus leucopus) to describe the variability in gut microbial community structure and composition during captivity and relocation in their natural habitat, and compared it to wild individuals. Mice born in captivity were fed two different diets, a control with dry standardized pellets and a treatment with nonprocessed components that reflect a version of their wild diet that could be provided in captivity. The mice from the two groups were then relocated to their natural habitat. Relocated mice that had the treatment diet had more phylotypes in common with the wild‐host microbiota than mice under the control diet or mice kept in captivity. These results have broad implications for our understanding of microbial community dynamics and the effects of captivity on reintroduced animals, including the potential impact on the survival of endangered species. This study demonstrates that ex situ conservation actions should consider a more holistic perspective of an animal's biology including its microbes.