叶酸对老年脑梗死伴高同型半胱氨酸血症患者血管内皮功能的影响

目的探讨叶酸对老年脑梗死伴高同型半胱氨酸(Hcy)血症患者血浆Hcy水平和血管内皮功能的影响。方法前瞻性纳入2012年11月—2013年10月南京市市级机关医院神经内科住院腔隙性脑梗死伴高Hcy血症老年患者112例,按随机数字法分成两组,两组患者同时接受缺血性卒中常规药物治疗。A组56例,口服叶酸20mg,1次/d;B组56例,未口服叶酸治疗。检测两组患者入院时、治疗后24周血浆Hcy水平。采用高分辨率超声检测肱动脉介导内皮依赖性舒张功能(FMD),并同时检测一氧化氮(NO)、一氧化氮合酶(NOS)、内皮素(ET)水平。结果治疗后24周A组血浆Hcy水平较治疗前明显降低[分别为(12.2±4.2)和(22.6±3.7)μmol/L,P0.05],同时低于B组同期水平[(21.8±4.0)μmol/L,P0.05)];A组FMD水平明显高于治疗前及B组同期水平[分别为(11.2±3.6)、(9.2±3.3)、(9.3±3.3)%,P0.05]。A组治疗后24周NO、NOS水平明显高于治疗前及B组同期水平[分别为(76.2±9.4)、(49.2±9.0)、(50.0±9.1)μmol/L;(47.6±9.5)、(38.4±7.5)、(37.8±8.8)kU/L,P0.05)],A组ET水平较治疗前及B组降低[(81.3±20.5)、(105.6±25.2)、(105.3±21.8)ng/L,P0.05)]。结论较大剂量叶酸能够明显降低老年脑梗死伴高Hcy血症患者血浆Hcy水平,降低FMD水平,改善血管内皮功能。     

B-vitamin deficiency causes hyperhomocysteinemia and vascular cognitive impairment in mice

In older adults, mildly elevated plasma total homocysteine (hyperhomocysteinemia) is associated with increased risk of cognitive impairment, cerebrovascular disease, and Alzheimer's disease, but it is uncertain whether this is due to underlying metabolic, neurotoxic, or vascular processes. We report here that feeding male C57BL6/J mice a B-vitamin-deficient diet for 10 weeks induced hyperhomocysteinemia, significantly impaired spatial learning and memory, and caused a significant rarefaction of hippocampal microvasculature without concomitant gliosis and neurodegeneration. Total hippocampal capillary length was inversely correlated with Morris water maze escape latencies (r = −0.757, P < 0.001), and with plasma total homocysteine (r = −0.631, P = 0.007). Feeding mice a methionine-rich diet produced similar but less pronounced effects. Our findings suggest that cerebral microvascular rarefaction can cause cognitive dysfunction in the absence of or preceding neurodegeneration. Similar microvascular changes may mediate the association of hyperhomocysteinemia with human age-related cognitive decline.     

Relationship between carbamoyl-phosphate synthetase genotype and systemic vascular function

Endothelial cells can convert l-citrulline to l-arginine, the precursor of nitric oxide. The present study tests the hypothesis that a C-to-A nucleotide transversion (T1405N) in the gene-encoding carbamoyl-phosphate synthetase 1, the enzyme catalyzing the rate-limiting step in l-citrulline formation, influences nitric oxide metabolite concentrations or nitric oxide-mediated vasodilation in humans. Bradykinin (100, 200, and 400 ng/min) was infused via brachial artery in 106 (CC:AC:AA=40:54:12) healthy subjects. Sodium nitroprusside (1.6, 3.2, and 6.4 microg/min) was also infused in 87 (CC:AC:AA=31:46:10) subjects. Forearm blood flow was measured by plethysmography and blood samples were collected for tissue-type plasminogen activator antigen, nitric oxide metabolites, and cyclic GMP. There was a significant relationship between carbamoyl-phosphate synthetase 1 genotype and nitric oxide metabolites, such that nitric oxide metabolite concentrations were highest in individuals homozygous for the C allele (mean+/-SD, 14.0+/-8.5 micromol/L), lowest in individuals homozygous for the A allele (9.1+/-3.1 micromol/L), and intermediate (11.8+/-6.6 micromol/L) in heterozygotes (P=0.036). There was a significant effect of carbamoyl-phosphate synthetase 1 genotype on forearm blood flow during bradykinin (P=0.028), such that the vasodilator response was greatest in C allele homozygotes (22.2+/-9.1 mL/min/100 mL at 400 ng/min), least in A allele homozygotes (13.6+/-6.2 mL/min/100 mL), and intermediate (19.4+/-10.7 mL/min/100 mL) in heterozygotes. Similarly, carbamoyl-phosphate synthetase 1 genotype influenced forearm blood flow during nitroprusside (maximal flow 19.2+/-8.3, 18.1+/-8.3, and 11.5+/-4.9 mL/min/100 mL in the CC:AC:AA groups, respectively; P=0.022). In contrast, there was no effect of carbamoyl-phosphate synthetase 1 genotype on the nitric oxide-independent tissue-type plasminogen activator response to bradykinin (P=0.943). These data indicate that a polymorphism in the gene encoding carbamoyl-phosphate synthetase 1 influences nitric oxide production as well as vascular smooth muscle reactivity.     

Analysis of the effect of leptin on immune function in vivo using diet-induced obese mice

Leptin can regulate several immune functions. However, the role of leptin on lymphocyte function has not been recognized in vivo. Accordingly, we have investigated the effect of leptin on starvation-induced immune dysfunction using diet-induced obese mice. To induce obesity, C57BL/6J mice were fed a high-fat diet for 14 weeks and control mice were fed a standard diet for the same period. The obese and control groups of mice were then starved for 48 h, and received intraperitoneal injections of recombinant leptin or phosphate-buffered saline four times during starvation. Other control mice in both diet groups were free fed without being starved. Although starvation of the control mice dramatically reduced the weights of the immune organs, cytokine production and increased proliferation of cultured splenocytes, these levels returned to those of the free-feeding groups with exogenous leptin administration. However, these effects of leptin were not observed in obese mice. These findings provide some evidence that leptin can regulate the immune function in vivo. It is also suggested that the action of leptin might not appear in obesity.     

Murine models of hyperhomocysteinemia and their vascular phenotypes

Hyperhomocysteinemia is an established risk factor for arterial as well as venous thromboembolism. Individuals with severe hyperhomocysteinemia caused by inherited genetic defects in homocysteine metabolism have an extremely high incidence of vascular thrombosis unless they are treated aggressively with homocysteine-lowering therapy. The clinical value of homocysteine-lowering therapy in individuals with moderate hyperhomocysteinemia, which is very common in populations at risk for vascular disease, is more controversial. Considerable progress in our understanding of the molecular mechanisms underlying the association between hyperhomocysteinemia and vascular thrombotic events has been provided by the development of a variety of murine models. Because levels of homocysteine are regulated by both the methionine and folate cycles, hyperhomocysteinemia can be induced in mice through both genetic and dietary manipulations. Mice deficient in the cystathionine beta-synthase (CBS) gene have been exploited widely in many studies investigating the vascular pathophysiology of hyperhomocysteinemia. In this article, we review the established murine models, including the CBS-deficient mouse as well as several newer murine models available for the study of hyperhomocysteinemia. We also summarize the major vascular phenotypes observed in these murine models.     

Diet-induced mild hyperhomocysteinemia and increased salt intake diminish vascular endothelial function in a synergistic manner.

OBJECTIVES: We investigated the influence of hyperhomocysteinemia and high salt intake on sodium handling, oxidative state, vascular endothelial function and blood pressure in a rat model. METHODS: Eight-week-old male Sprague-Dawley rats were divided into subgroups and maintained for 4 weeks prior to experimentation on either control chow containing 0.36% methionine and 0.5% NaCl; or one of the following modified diets containing either 0.7% methionine, 8% NaCl or 0.7% methionine + 8% NaCl. Sodium handling, homocysteine metabolism, lipid profile, NO synthesis, oxidative state, blood pressure and relaxation to acetylcholine of carotid rings were evaluated and compared. RESULTS: Diet-induced mild hyperhomocysteinemia (plasma homocysteine levels 1.4-fold higher than control), by itself, had no significant influence on sodium excretion, vascular endothelial function and blood pressure. Increased salt intake had no influence on homocysteine metabolism, vascular endothelial function and blood pressure. The coexistence of mild hyperhomocysteinemia and high salt intake significantly diminished vascular endothelial function (rmax to acetylcholine; control chow 83.2 +/- 6.2%, 0.7% methionine diet 74.7 +/- 3.9%, 8% NaCl diet 85.1 +/- 4.6%, 0.7% methionine + 8% NaCl diet 57.9 +/- 6.6%) but manifested no rise in blood pressure. No significant difference in oxidative state was observed in this analysis. CONCLUSIONS: Diet-induced mild hyperhomocysteinemia, the extent of which is comparable with the levels that are associated with a predisposition to common atherosclerotic diseases, was found to induce vascular endothelial dysfunction only when accompanied by high salt intake.     

Effect of rAd-p53 on glucose metabolism in diet-induced and genetic T2DM mice

正Objective To explore the possible mechanism of rAd-p53 involved in glucose metabolism by observing the different effects of rAd-p53 on high-fat diet(HFD)fed with low-dose streptozotocin(STZ)treated(HFD/STZ)mice(β-cell dysfunction model)and db/db mice(genetic insulin resistance model).Methods The established HFD/STZ and db/db diabetic mice were respectively and     

盐酸多奈哌齐对血管性痴呆小鼠认知功能及P300电位的影响

目的探讨具有认知功能改变的血管性痴呆小鼠P300电位的变化特征及盐酸多奈哌齐对其影响。方法48只小鼠随机分为假手术组、血管性痴呆模型组(模型组)和多奈哌齐组,每组16只。应用双侧颈总动脉线结反复缺血再灌注法制备血管性痴呆小鼠模型,采用盐酸多奈哌齐连续治疗4周,利用跳台试验和水迷宫试验观测其行为学改变,测定P300电位。结果与多奈哌齐组和假手术组比较,模型组小鼠学习成绩为反应时间长,游全程时间长,错误次数多,记忆成绩为跳台试验潜伏时间短,游全程时间长,错误次数多(P<0.05)。模型组小鼠P300电位的测定,N2潜伏期、P3潜伏期均长于多奈哌齐组和假手术组(P<0.01);P3波幅低于多奈哌齐组和假手术组(P<0.05)。结论盐酸多奈哌齐对血管性痴呆认知功能损伤有肯定的干预和治疗作用。     

Effects of vitamin supplementation and hyperhomocysteinemia on atherosclerosis in apoE-deficient mice

It has been demonstrated that hyperhomocysteinemia (HHcy) accelerates atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. In this study, vitamin-defined chow diets were used to induce HHcy in apoE(-/-) mice in an attempt to identify possible pathogenic pathways. Six-week-old female apoE(-/-) mice were divided into seven groups: vitamin-defined purified chow diet alone (control), or same diet supplemented with either D,L-homocysteine (upward arrow Hcy) or L-homocystine (upward arrow Hcy-Hcy), or diet high in L-methionine (upward arrow Met), or diet high in B-vitamins (upward arrow vitamin), or diets deficient in folate (downward arrow folate) or vitamin B(6) ( downward arrow B(6)). Eighteen weeks later, plasma total homocysteine (tHcy), lipids and atherosclerotic plaque burden (aortic root, aortic arch, and brachiocephalic trunk) were measured. tHcy levels were similar in the upward arrow vitamin, downward arrow folate, downward arrow B(6) and control groups (9.2-10.1 micromol/l, NS), but elevated mildly in the upward arrow Hcy-Hcy group (16.1 micromol/l) and moderately in the upward arrow Met and upward arrow Hcy groups (53.6 and 51.5 micromol/l, respectively). Mice in the latter two groups had significantly more atherosclerosis in the aortic root. Although B vitamin-supplementation failed to lower tHcy levels, mice had less atherosclerosis in the aortic arch. In summary, dietary methionine and homocysteine, but not homocystine, enhanced the development of atherosclerosis. Supplementation with B vitamins appeared to confer homocysteine-independent protection against atherosclerosis. These results suggest that (1) there may be a threshold level below which homocysteine is not atherogenic; (2) the atherogenic effect of HHcy may be mediated via an intracellular pathway; and/or (3) the anti-atherogenic effect of B vitamins in normohomocysteinemic mice is independent of tHcy levels.     

Characterization of high-salt and high-fat diets on cardiac and vascular function in mice

This study compared two established dietary formulations, high salt and high fat-high carbohydrate, separately or in combination on the induction cardiovascular dysfunction. One-month-old C57BL/6J mice were fed one of the following diets for 3 mo: (1) control diet consisting of a high fat-high simple carbohydrate (HFHSC); (2) 8% NaCl diet (HS); or (3) HFHSC diet supplemented with 1% NaCl (HFHS). After 3 mo, the HFHSC mice demonstrated significantly increased end-diastolic volume and end-systolic volume, specifically increases of 35% and 78%, respectively (p<0.01) and a reduction of ventricular stiffness by 27% (p=0.015). The HS mice exhibited arterial hypertension with an increase of 33% in maximum end-systolic pressure (p=.024) and a decrease of 44% in arterial elastance (p=0.020), corroborated by an increase in the heart weight to body weight ratios (p=0.002) and vascular types I and III collagen (p=0.03 and p=0.0008, respectively). The HFHS group revealed a striking response of 230% to the α₁-adrenergic challenge (p=0.00034). These data suggest that the HFHSC diet causes dilaeed cardiomyopathy, whereas the HS diet produces arterial hypertension and the HFHS diet causes a vascular dysfunctional state that was highly responsive to α-adrenergic stimulation.     

Dysfunction of nitric oxide mediation in isolated rat arterioles with methionine diet-induced hyperhomocysteinemia.

In humans, increased plasma homocysteine (Hcy) has been shown to be correlated with occlusive arterial diseases and atherosclerosis. Studies of isolated conductance vessels of experimental animals suggest that Hcy may interfere with local vasoregulatory mechanisms, yet the effect of hyperhomocysteinemia (HHcy) on the function of microvessels, such as skeletal muscle arterioles, has not been investigated. Male Wistar rats were divided into 2 groups: control rats (C; plasma Hcy, 7.1+/-0.3 micromol/L; n=25), and rats made HHcy by 1 g/kg body weight daily intake of methionine in the drinking water for 4 weeks (plasma Hcy, 23.6+/-2.9 micromol/L; P<0.01 versus C; n=25). First-order arterioles ( approximately 130 micrometer in diameter) were isolated from gracilis muscle, cannulated, and pressurized (80 mm Hg, no-flow conditions). Changes in diameter were observed by videomicroscopy. Arteriolar constrictions to norepinephrine (NE; 3x10(-7) mol/L) were significantly (P<0.01) greater in HHcy compared with C rats (C, 37.7+/-4.9%; HHcy, 59.5+/-5. 2%). Removal of the endothelium (-E) augmented NE-induced constrictions only in arterioles from C rats, whereas it had no effect on responses of arterioles from HHcy rats (C-E, 55.9+/-6.9%; HHcy-E, 56.5+/-7.0%). Dilations to cumulative doses of acetylcholine (ACh; 10(-8) mol/L) were significantly reduced in arterioles from HHcy rats (C, 64.0+/-5.2%; HHcy, 24.1+/-6.8%). Inhibition of nitric oxide (NO) synthesis with N(omega)-nitro-L-arginine (L-NNA; 10(-4) mol/L) significantly decreased ACh-induced dilations of C arterioles, whereas it did not affect HHcy arterioles. Similar alterations were found in arteriolar dilations to histamine, another known NO-dependent agonist. Endothelium-independent dilations to the NO donor sodium nitroprusside were not different in arterioles from C and HHcy rats, either in the presence or absence of L-NNA. Presence of superoxide dismutase and catalase (scavenger of reactive oxygen metabolites) did not affect HHcy-induced alterations in the ACh response. We conclude that hyperhomocysteinemia reduces rat skeletal muscle arteriolar dilations in response to ACh and histamine, and enhances constrictions to NE, alterations that are likely to be caused by the reduced mediation of these responses by NO. The reduced activity of NO in arterioles may contribute to the microvascular impairment described in HHcy.     

葡萄籽原花青素提取物对膳食诱导肥胖小鼠氧化应激的影响

目的观察强抗氧化剂葡萄籽原花青素提取物（grape seed procyandins extract,GSPE,低聚物〉85％）对肥胖伴氧化应激小鼠抗氧化能力的影响。方法选用C57BL／6J小鼠,观察高、中、低剂量GSPE（400mg/kg·d^-1、200mg／kg·d^-1和100mg／kg·d^-1）对膳食诱导肥胖（diet-induced obesity,DIO）小鼠成模过程中体重、体脂、血清游离脂肪酸、血清肿瘤坏死因子α、肝谷胱甘肽过氧化物酶（GSH-Px）、肝过氧化物歧化酶（SOD）、肝总抗氧化能力（T-AOC）和丙二醛（MDA）的影响。结果GSPE能显著降低DIO小鼠的体重、体脂比,降低血清游离脂肪酸、血清肿瘤坏死因子α的水平,提高肝GSH-Px、SOD、T-AOC活性。结论GSPE有良好的预防DIO小鼠肥胖及其相伴的氧化应激的作用。     

三氧化二砷对小鼠免疫功能的影响

为研究砷在蓄积中毒过程中对免疫功能的影响，用Ａｓ２Ｏ３染毒昆明种小鼠，测定小鼠脏体系数，外周血Ｔ淋巴细胞α－醋酸萘酯酶的阳性率（ＡＮＡＥ），迟发型变态反应（ＤＴＨ），血清溶血素（ＨＣ５０），碳粒廓清吞噬指数（α）五个指标。结果显示小鼠的脾脏系数和血清溶血素明显低于对照组（Ｐ＜０．０１）。ＡＮＡＥ阳性率、吞噬指数的中、高剂量组和迟发型变态反应的高剂量组较对照组均降低（Ｐ＜０．０５），这说明Ａｓ２Ｏ３对免疫器官、体液免疫功能、细胞免疫功能以及单核巨噬细胞系统均有不同程度的损伤。     

Altered hypothalamic function in diet-induced obesity

Energy homeostasis involves a complex network of hypothalamic and extra-hypothalamic neurons that transduce hormonal, nutrient and neuronal signals into responses that ultimately match caloric intake to energy expenditure and thereby promote stability of body fat stores. Growing evidence suggests that rather than reflecting a failure to regulate caloric intake, common forms of obesity involve fundamental changes to this homeostatic system that favor the defense of an elevated level of body adiposity. This article reviews emerging evidence that during high-fat feeding, obesity pathogenesis involves fundamental alteration of hypothalamic systems that regulate food intake and energy expenditure.     

高同型半胱氨酸对高血压大鼠血管平滑肌细胞GRP78和CHOP的表达的影响与血管重构的关系

目的：观察同型半胱氨酸（Homocysteine,Hcy）对高血压大鼠内质网应激因子GRP78和CHOP表达及血管中层厚度变化,探讨Hcy对血管重构的影响。方法：采用腹主动脉缩窄术建立高血压大鼠模型,术后两周采用无创尾套法测量大鼠血压,选取24只成年雄性大鼠[平均动脉压（MAP）〉150mmHg]随机等分为2组,即对照组和蛋氨酸组,每组12只（n=12）。对照组给予普通饮食＋双蒸水灌胃,蛋氨酸组给于30g/L蛋氨酸饮食＋双蒸水灌胃;根据实验结束的时间,又将各组分为4W和8W两个亚组,各亚组6只（n=6）。用同型半胱氨酸检测仪检测血清同型半胱氨酸浓度,用颈动脉插管法测定各组大鼠的MAP,并利用图像分析软件测量血管中层的厚度;HE染色观察大鼠主动脉血管形态学变化;通过免疫组化及Western blot检测大鼠主动脉血管平滑肌细胞（VSMCs）组织中GRP78及CHOP表达水平。结果：①对照组大鼠血清Hcy浓度在正常值范围（〈10μmol／L）;蛋氨酸组大鼠血清Hcy浓度明显高于对照组（P〈0．01）。②两组大鼠MAP均显著增高,4周时两者之间差异不明显,8周时两者差异显著（P〈0．05）;③蛋氨酸组大鼠血管平滑肌中层厚度显著大于对照组（P〈0．05）;④HE染色显示,与对照组相比,蛋氨酸组大鼠主动脉VSMCs显著肥大,管壁显著增厚;⑤两组CRP78和CHOP蛋白表达量增高,与对照组相比蛋氨酸组表达更明显。结论：高同型半胱氨酸可能通过促使高血压大鼠动脉VSMCs内质网应激反应,导致CRP78及CHOP的平衡失调加剧,从而引起血管损伤加重,而发生血管重构加重。     

Signaling mechanisms involved in altered function of macrophages from diet-induced obese mice affect immune responses

Recent research links diet-induced obesity (DIO) with impaired immunity, although the underlying mechanisms remain unclear. We find that the induction of inducible NO synthase (iNOS) and cytokines is suppressed in mice with DIO and in bone marrow macrophages (BMMΦ) from mice with DIO exposed to an oral pathogen, Porphyromonas gingivalis. BMMΦ from lean mice pre-treated with free fatty acids (FFAs) and exposed to P. gingivalis also exhibit a diminished induction of iNOS and cytokines. BMMΦ from lean and obese mice exposed to P. gingivalis and analyzed by a phosphorylation protein array show a reduction of Akt only in BMMΦ from mice with DIO. This reduction is responsible for diminished NF-κB activation and diminished induction of iNOS and cytokines. We next observed that Toll-like receptor 2 (TLR2) is suppressed in BMMΦ from DIO mice whereas carboxy-terminal modulator protein (CTMP), a known suppressor of Akt phosphorylation, is elevated. This elevation stems from defective TLR2 signaling. In BMMΦ from lean mice, both FFAs and TNF-α—via separate pathways—induce an increase in CMTP. However, in BMMΦ from DIO mice, TLR2 can no longer inhibit the TNF-α-induced increase in CTMP caused by P. gingivalis challenge. This defect can then be restored by transfecting WT TLR2 into BMMΦ from DIO mice. Thus, feeding mice a high-fat diet over time elevates the CTMP intracellular pool, initially via FFAs activating TLR2 and later when the defective TLR2 is unable to inhibit TNF-α-induced CTMP. These findings unveil a link between obesity and innate immunity.     

Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice

A rodent model of diet-induced obesity revealed that obesity significantly altered hematopoietic and lymphopoietic functions in the bone marrow and thymus. C57BL/6 mice were fed a mixed high-fat diet (HFD) of 45% fat or 10% fat diet (lean controls) for 180 d. A sustained increase in the numbers of cells found in bone marrow and thymus of HFD mice occurred from day 90 to day 180. However, with the exception of a 10-18% increase in the proportion of lymphocytes, the composition of monocytes, granulocytes, erythrocytes, and mixed progenitor lineages remained normal in the marrow. Likewise, thymuses of HFD mice increased 30-50% in size compared with controls, with analogous increases in thymocyte numbers. The overall thymus cellular composition remained normal. Although increased blood and lymphatic volume in obese mice would play a role in increased hematopoiesis, there were large and disproportionate increases in blood leukocytes of HFD mice, indicating that homeostasis was not maintained. Leptin, which promotes lymphopoiesis and myelopoiesis, reached 100 ng/mL in sera from HFD mice. Moreover, a three- to sixfold increase in adipocytes in marrow resulted in spiked leptin mRNA expression in bones of HFD mice compared with lean controls. Other cytokines and growth factors did not show any increases in obese marrow. The substantial increase in lymphopoietic and hematopoietic processes in HFD mice indicates that the primary tissues are another facet of the immune system dysregulated by obesity, which was perhaps fostered by higher amounts of leptin in marrow and serum.