奥美沙坦减轻高脂饮食诱导的非酒精性脂肪肝病的机制研究

目的探讨奥美沙坦对于高脂诱导的非酒精性脂肪肝病（NAFLD）的影响及可能机制。方法健康雄性8周龄C57BL／6小鼠24只随机分为高脂组（n=16）和正常饮食组（n=8）,高脂组小鼠高脂饮食（60％的脂肪）12w后再随机分为高脂饮食对照组（n=8）、高脂饮食治疗组（n=8）。高脂饮食治疗组小鼠给予0．75mg／kg／d的奥美沙坦灌胃8w,灌胃结束后处理小鼠,留取空腹血样本检测AST和ALT。肝组织冰冻切片行油红O染色观察脂肪变;石蜡切片行HE和F4／80免疫组化染色观察肝脏炎症变化;实时荧光定量PCR检测肝脏TNF-α和IL-6mRNA的表达水平;WesternBlot检测肝组织中IκB-α、p-IκBa、NF—κB信号通路的活化。结果奥美沙坦显著抑制了高脂诱导的NAFLD脂肪变性,并明显改善肝功能。实时荧光定量PCR结果表明奥美沙坦能显著降低肝脏组织中TNF-α和IL-6mRNA表达水平（P〈0．05）;Western Blot结果显示奥美沙坦显著抑制肝脏NF-κB信号通路活化。结论奥美沙坦显著抑制NAFLD小鼠肝脏炎性病变而保护肝功能,其机制与抑制NF-κB信号通路活化以及降低肝脏TNF-α和IL-6mRNA水平有关。     

雌激素在中枢肾素-血管紧张素系统功能异常诱导的高血压中的保护作用

雌激素主要由卵巢分泌,是一种类固醇激素。流行病学研究显示,绝经前女性的高血压发病率明显低于同龄男性。研究表明,雌激素可通过影响中枢肾素-血管紧张素系统(RAS)组分的功能发挥其在中枢血压调控中的保护作用。雌激素作用于雌激素受体可抑制RAS增压信号通路或/和激活其减压信号通路,衰减中枢核团内自主神经元兴奋性进而延缓外界刺激诱导的高血压的发生。本文通过对雌激素在中枢RAS活动增强诱导的高血压发生中心血管保护作用及作用机制等方面进行综述,为临床开展性别差异性的高血压防治提供新思路。     

Effect of age on high-fat diet-induced hypertension

Aging and obesity both have a significant impact on central blood pressure (BP) regulation, and previous studies indicated that changes in central redox signaling with age may affect high-fat (HF) diet-induced cardiovascular responses. Therefore, we investigated the effects of 60% HF feeding on BP regulation in young adult (5 mo) and old (26 mo) Fischer-344 × Brown-Norway rats. Radiotelemetric transmitters were implanted to measure BP, heart rate (HR), locomotor activity, and spontaneous baroreflex sensitivity. Expression and activity of NADPH oxidase and ANG II type 1 receptor were assessed in the hypothalamus and in the nucleus tractus solitarii. Old animals gained more weight on HF diet compared with young, whereas central NADPH oxidase expression and activity elevated similarly in the two age groups. After an initial hypotensive and tachycardic response during the first week of HF feeding, BP in young animals increased and became significantly elevated after 6 wk of HF feeding. In contrast, BP in old animals remained depressed. Nighttime HR and locomotor activity decreased in both young and old rats fed with HF diet, but these changes were more significant in young rats. As a result, amplitudes of circadian variation of BP, HR, and activity that were originally higher in young rats declined significantly and became similar in the two age groups. In conclusion, our experiments led to the surprising finding that HF diet has a more serious impact on cardiovascular regulation in young animals compared with old.     

Profiling of gender-specific rat plasma proteins associated with susceptibility or resistance to diet-induced obesity

Obesity-prone (OP) and obesity-resistant (OR) rats with different responses to development of obesity in spite of the same genetic background are useful animal models for searching for markers during the development of obesity. Here, we investigated whether plasma proteins of OP and OR rats may behave in a different way in males and females. We performed a comparative proteomic analysis using 2-DE combined with MALDI-TOF/MS on proteins from OP and OR male and female rats to discover gender-specific rat plasma proteins associated with susceptibility or resistance to diet-induced obesity. A total of 29 proteins showing differential expression between the groups were identified by MALDI-TOF/MS and database searches. These proteins were classified into 4 groups according to their regulation patterns in response to diet and gender. 22 proteins showed significant differences between OP and OR rats in males and/or females (Group I, II, and III) and 7 proteins exhibited only a high fat diet (HFD)-responsive difference in male or female rats (Group IV). In conclusion, the proteins negatively (ITIH3, FGG, TUBB5, and ZAG) or positively (Hp, ITIH4, and RBP) correlated with obesity found in this study could be used for selection of new targets for gender specific-medical treatment of obesity.     

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

Protective effect of Morus rubra L. leaf extract on diet-induced atherosclerosis in diabetic rats

The antiatherosclerotic effect of aqueous leaves extract of Morus rubra was studied in streptozotocin-induced diabetic rats fed with atherosclerotic (Ath) diet [1.5 ml olive oil containing 8 mg (3, 20,000 IU) vitamin D2 and 40 mg cholesterol] for 5 consecutive days. A short-term toxicity assessment was also conducted in healthy rats to examine toxic effects of the extract. Oral administration of extract to diabetic rats (100, 200 and 400 mg/kg body weight per day for a period of 30 days) produced significant (p<0.001) fall in fasting blood glucose (FBG) in a dose-dependent manner. Treatment with the extract (400 mg/kg) showed significant (p<0.001) improvement in body weight and serum lipid profile i.e., total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol and VLDL-cholesterol, when compared with diabetic control. Endothelial dysfunction parameters (sVCAM-1, Fibrinogen, total NO levels and oxidized LDL), apolipoprotein A and apolipoprotein B were significantly (p<0.001) reversed to near normal, following treatment with the extract. Thus, our study shows that aqueous leaf extract of Morus rubra (400 mg/kg) significantly improves the homeostasis of glucose and fat and possesses significant anti-atherosclerotic activity.     

Protective effects of hydrogen-rich saline on monocrotaline-induced pulmonary hypertension in a rat model

Background

Hydrogen-rich saline has been reported to have antioxidant and anti-inflammatory effects and effectively protect against organ damage. Oxidative stress and inflammation contribute to the pathogenesis and/or development of pulmonary hypertension. In this study, we investigated the effects of hydrogen-rich saline on the prevention of pulmonary hypertension induced by monocrotaline in a rat model.

Methods

In male Sprague-Dawley rats, pulmonary hypertension was induced by subcutaneous administration of monocrotaline at a concentration of 6 mg/100 g body weight. Hydrogen-rich saline (5 ml/kg) or saline was administred intraperitoneally once daily for 2 or 3 weeks. Severity of pulmonary hypertension was assessed by hemodynamic index and histologic analysis. Malondialdehyde and 8-hydroxy-desoxyguanosine level, and superoxide dismutase activity were measured in the lung tissue and serum. Levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6) in serum were determined with enzyme-linked immunosorbent assay.

Results

Hydrogen-rich saline treatment improved hemodynamics and reversed right ventricular hypertrophy. It also decreased malondialdehyde and 8-hydroxy-desoxyguanosine levels, and increased superoxide dismutase activity in the lung tissue and serum, accompanied by a decrease in pro-inflammatory cytokines.

Conclusions

These results suggest that hydrogen-rich saline ameliorates the progression of pulmonary hypertension induced by monocrotaline in rats, which may be associated with its antioxidant and anti-inflammatory effects.     

Protective effect of digitoxin in adrenal-compression hypertension.

Bilateral compression of the adrenal glands combined in mononephrectomy and followed by the imposition of a high NaC1 intake resulted in severe hypertension in all rats so treated. It was accompanied by enlargement of the heart, kidneys, and adrenal glands, atrophy of the thymus, and the occurrence of severe nephrosclerosis. Digitoxin treatment delayed the onset, reduced the incidence, and ameliorated the magnitude of the hypertensive response in such animals; it also reduced the degree of cardiac hypertrophy and the severity of nephrosclerosis and completely prevented enlargement of the adrenals and kidneys and atrophy of the thymus.     

Comparative assessment of the effects of gender-specific heparan sulfates on mesenchymal stem cells

We compare here the structural and functional properties of heparan sulfate (HS) chains from both male or female adult mouse liver through a combination of molecular sieving, enzymatic cleavage, and strong anion exchange-HPLC. The results demonstrated that male and female HS chains are significantly different by a number of parameters; size determination showed that HS chain lengths were ～100 and ～22 kDa, comprising 30-40 and 6-8 disaccharide repeats, respectively. Enzymatic depolymerization and disaccharide composition analyses also demonstrated significant differences in domain organization and fine structure. N-Unsubstituted glucosamine (ΔHexA-GlcNH(3)(+), ΔHexA-GlcNH(3)(+)(6S), ΔHexA(2S)-GlcNH(3)(+), and N-acetylglucosamine (ΔHexA-GlcNAc) are the predominant disaccharides in male mouse liver HS. However, N-sulfated glucosamine (ΔHexA-GlcNSO(3)) is the predominant disaccharide found in female liver. These structurally different male and female liver HS forms exert differential effects on human mesenchymal cell proliferation and subsequent osteogenic differentiation. The present study demonstrates the potential usefulness of gender-specific liver HS for the manipulation of human mesenchymal cell properties, including expansion, multipotentiality, and subsequent matrix mineralization. Our results suggest that HS chains show both tissue- and gender-specific differences in biochemical composition that directly reflect their biological activity.     

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.     

Hypoglycemic effects of brassinosteroid in diet-induced obese mice

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.     

Intermittent hypoxia exacerbates metabolic effects of diet-induced obesity

Obesity causes insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD), but the relative contribution of sleep apnea is debatable. The main aim of this study is to evaluate the effects of chronic intermittent hypoxia (CIH), a hallmark of sleep apnea, on IR and NAFLD in lean mice and mice with diet-induced obesity (DIO). Mice (C57BL/6J), 6-8 weeks of age were fed a high fat (n = 18) or regular (n = 16) diet for 12 weeks and then exposed to CIH or control conditions (room air) for 4 weeks. At the end of the exposure, fasting (5 h) blood glucose, insulin, homeostasis model assessment (HOMA) index, liver enzymes, and intraperitoneal glucose tolerance test (1 g/kg) were measured. In DIO mice, body weight remained stable during CIH and did not differ from control conditions. Lean mice under CIH were significantly lighter than control mice by day 28 (P = 0.002). Compared to lean mice, DIO mice had higher fasting levels of blood glucose, plasma insulin, the HOMA index, and had glucose intolerance and hepatic steatosis at baseline. In lean mice, CIH slightly increased HOMA index (from 1.79 ± 0.13 in control to 2.41 ± 0.26 in CIH; P = 0.05), whereas glucose tolerance was not affected. In contrast, in DIO mice, CIH doubled HOMA index (from 10.1 ± 2.1 in control to 22.5 ± 3.6 in CIH; P < 0.01), and induced severe glucose intolerance. In DIO mice, CIH induced NAFLD, inflammation, and oxidative stress, which was not observed in lean mice. In conclusion, CIH exacerbates IR and induces steatohepatitis in DIO mice, suggesting that CIH may account for metabolic dysfunction in obesity.     

Resveratrol inhibits the deleterious effects of diet-induced obesity on thymic function

Obesity is associated with an increased risk of infectious diseases. It has been shown to have deleterious effects on cell-mediated immunity, including reducing thymocyte numbers and altering responses of thymocytes to pathogens. In the current study, we examined the efficacy of the antiobesity phytochemical resveratrol in preventing the deleterious effects of a high-fat diet on thymic anatomy and function. Compared to C57Bl/6 male mice fed a low-fat diet, mice on a high-fat diet had a significant increase in thymic weight and lipid content, and a disrupted anatomy, including a reduction of the medullary compartment and absence of a corticomedullary junction. There were a decrease in thymic cellularity and mature T-cell output, and a disrupted T-cell maturation, as evidenced by increased double-negative and decreased single- and double-positive thymocytes. Mice that had been fed resveratrol along with a high-fat diet had a dose-dependent reversal in all these parameters. Western blots from thymi showed that obese mice had lower levels of the key stimulators of lipid metabolism, phospho-5′ adenosine monophosphate-activated protein kinase and its downstream target, carnitine palmitoyl transferase-1; this was restored to normal levels in resveratrol-fed mice. Resveratrol also reversed an increase in glycerol-3-phosphate acyltransferase-1, the enzyme that catalyzes the first step in triglycerol synthesis. Taken together, these results indicate that resveratrol is a potent inhibitor of the deleterious effects of diet-induced obesity on thymic anatomy and function, and this may hold promise in preventing obesity-related deficits in cell-mediated immunity.     

Morphological and immunocytochemical analyses on the effects of diet-induced hypocalcemia on enamel maturation in the rat incisor.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff- and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.     

Differential effects of two estrogen antagonists on the development of masculine and feminine sexual behavior in hamsters

To determine whether nonsteroidal antiestrogens can be used to investigate the role of aromatization in behavioral masculinization and defeminization, newborn male and female hamsters were administered 0.5 or 5.0 μg of the antiestrogens nafoxidine or tamoxifen on postnatal Days 1 and 2. Other females received 1.0 μg of the synthetic estrogen RU-2858 (RU) alone or in combination with 0.5 or 5.0 μg of nafoxidine or tamoxifen. All animals were tested for the display of masculine and feminine sexual behaviors in adulthood. Nafoxidine, tamoxifen, and RU all reduced lordosis behavior in adult females, indicating that the antiestrogens probably have some estrogenic properties. Nafoxidine had no effect on male mating behavior in female hamsters when given alone; when given to male hamsters or female hamsters receiving a partially masculinizing dose of RU, this compound effectively reduced the frequencies of masculine sexual behaviors (mounts and intromissions) displayed by the treated animals. However, nafoxidine-treated males had the same mating efficiency (ME = intromissions/ mounts) as control males. Tamoxifen, in contrast, facilitated the display of mounting behavior in females when given alone or in combination with RU. Male hamsters receiving 5.0 μg of tamoxifen had high mount frequencies and slightly reduced intromission frequencies, but their ME scores were only half of control levels. Thus nafoxidine itself simultaneously promoted defeminization and antagonized masculinization while tamoxifen appeared to facilitate both processes. The data support the hypothesis that estrogens derived via aromatization from androgens play an important role in both masculinization and defeminization, at least in hamsters. The differential effects of tamoxifen and nafoxidine suggest that these and other antiestrogens might serve as useful tools for dissociating and independently examining these two components of the sexual differentiation process.