拮抗或激活促肾上腺皮质激素释放因子受体对肠易激综合征大鼠内脏敏感性及结肠动力的影响

目的 探讨促肾上腺皮质激素释放因子(CRF)及其受体对肠易激综合征大鼠内脏敏感性及结肠动力的影响.方法 SD大鼠60只,随机平均分入空白组(不做处理)、模型组(特殊气味条件刺激和肢体束缚直肠刺激非条件刺激轮替致敏)、干预对照组(造模前侧脑室注射0.9%NaC1)、干预一组(造模前侧脑室注射CRF-R1拮抗剂)和干预二组(造模前侧腑室注射CRF-R2激动剂).采用腹部收缩反射(AWR)评分标准评估各组大鼠肠道敏感性,记录各组大鼠结肠快、慢波波动率、最大振幅、收缩波数及振幅指数等电生理活动改变.采用SPSS16.0统计软件分析,计量资料采用方差分析,等级资料采用秩和检验.结果 以AWR=3分时所需的直肠注水量作为评价指标,模型组大鼠[(0.90±0.11)ml]较空白组[(1.23±0.07)ml]内脏敏感性增高(F＝82.586,P＜0.01);结肠电生理活动增强,造模成功.干预对照组直肠注水量为(0.81±0.11)ml,与模型组[(0.90±0.11)ml]差异无统计学意义(F＝3.734,P＞0.05),干预一组[(1.28±0.07)ml,F＝161.878,P＜0.01]和干预二组[(1.22±0.05)ml,F＝121.564,P＜0.01]较干预对照组内脏敏感性降低.干预对照组大鼠结肠快、慢波波动率、最大振幅、收缩波数及振幅指数等电生理活动与模型组无明显差异(P均＞0.05);干预一组和干预二组大鼠结肠电生理活动均较干预对照组明显减弱(均P＜0.05).结论 CRF在IBS发病中起重要作用,抑制CRF-R1或激活CRF-R2可降低1BS大鼠内脏敏感性并抑制结肠运动.

Abstract：

Objective To explore the effect of corticotropin releasing factor (CRF) and its receptor on visceral sensitivity and colon motility of irritable bowel syndrome (IBS) rats. Methods sixty SD rats were divided randomly and equally into control group (without treatment),model group (sensitized in turn with camphor odor as conditional stimulation and physical restraint in combination with rectal distention pressure as non-conditional stimulation),treatment control group (injected physiological saline into lateral ventricles before treatment),treatment group 1 (injected CRF-R1antagonist into lateral ventricles before treatment),treatment group 2 (injected CRF-R2 agonist into lateral ventricles before treatment). Then the rats' visceral sensitivity were assessed by AWR,and colonic electricity activities such as volatility,maximum amplitude of fast wave and slow wave,interdigestive number of contraction wave and index of contraction were recorded. The data was analyzed with SPSS 16. 0 software. Results By the amount of ractal water injection to reach AWR=3 as the evaluation index,model group [(0. 90±0. 11) ml] showed higher visceral sensitivity than that of control group [(1. 23±0. 07) ml,F=82. 586,P＜0. 01],and colonic electricity activity increased (P＜0. 05),model was successfully set up. There was no significant difference of the amount of ractal water injection between model group [(0. 90±0. 11) ml] and treatment control group [(0. 81±0. 11) ml,F=3. 734,P＞0. 05]. Compared with treatment control group,the visceral sensitivity decreased in treatment group 1 [(1. 28±0. 07) ml,F=161. 878,P＜0. 01] and treatment group 2 [(1. 22±0.05) ml,F=121. 564,P＜0. 01]. There was no significant difference between treatment control group and model group in electricity activities such as volatility,maximum amplitude of fast wave and slow wave,interdigestive number of contraction wave and index of contraction (all P＞0. 05). While the electricity activities was weakened in treatment group 1 and 2 compared with the treatment control group (all P＜0. 05). Conclusions CRF plays an important role in the pathogenesis of IBS. Inhibition of CRF-R1 or activation of CRF-R2 may lower visceral hypersensitivity and decrease colon motility of rats.     

Effects of low-calorie diet on steatohepatitis in rats with obesity and hyperlipidemia

AIM: To evaluate the effects of low calorie diet (LCD) on nonalcoholic steatohepatitis (NASH) in rats with obesity and hyperlipidemia.METHODS: 29 Sprague-Dawley (SD) rats were randomly divided into three groups. The animals in control (n=9) and NASH group (n=10) were fed on standard rat diet and high fat diet respectively for 12 weeks, ten rats in LCD group were fed on high fat diet for 10 weeks and then low calorie diet for 2 weeks. At the end of the experiment, body weight, abdominal adipose content, liver function, and hepatopathological changes were examined to evaluate the effect of different feeding protocols on the experimental animals.RESULTS: There was no death of animal in the experimental period. All rats in the NASH group developed steatohepatitis according to liver histological findings. Compared with the control group, body weight (423.5±65.2 vs 351.1±43.0 g,P＜0.05), abdominal adipose content (14.25±1.86 vs9.54±1.43,P＜0.05), liver index (3.784-±0.533 vs2.957±±0.301%, P＜0.01),total serum cholesterol (1.60±0.41 vs 1.27±0.17 mmol/L, P＜0.05)and free fatty acids (728.2±178.5 vs 429.2±96.7 mmol/L,P＜0.01), serum alanine aminotransferase (1 257.51±671.34vs671.34±118.57 nkat/L, P＜0.05) and aspartic aminotransferse (2 760.51±998.66 vs 1 648.29±414.16 nkat/L, P＜0.01) were significantly increased in the NASH group. Whereas, when rats were fed on LCD protocol, their body weight (329.5±38.4 g,P＜0.01), abdominal adipose content (310.21±1.52 g, P＜0.05),liver index (3.199±0.552 %, P＜0.05), and serum alanine aminotransferase (683.03±245.49 nkat/L, P＜0.05) were significantly decreased, and the degree of hepatic steatosis (P＜0.05) was markedly improved compared with those in the NASH group. However, no significant difference was found in serum lipid variables and hepatic inflammatory changes between the two groups.CONCLUSION: LCD might play a role in the prevention and treatment of obesity and hepatic steatosis in SD rats,but it exerts no significant effects on both serum lipid disorders and hepatic inflammatory changes.     

Development of Wistar rat model of insulin resistance

AIM: To establish a simplified and reliable animal model of insulin resistance with low cost in Wistar rats. METHODS: Wistar rats were treated with a high fat emulsion by ig for 10 d. Changes of the diets, drinking and body weight were monitored every day and insulin resistance was evaluated by hyperinsulinemic-euglycemic clamp techniques and short insulin tolerance test using capillary blood glucose. Morphologic changes of liver, fat, skeletal muscles, and pancreatic islets were assessed under light microscope. mRNA expressions of GLUT2 and α-glucosidase in small intestine epithelium, GLUT4 in skeletal muscles and Kir6.2 in beta cell of islets were determined by in situ hybridization. RESULTS: KITT was smaller in treated animals (4.5±0.9) than in untreated control Wistar rats (6.8±1.5), and so was glucose injection rate. Both adipocyte hypertrophy and large pancreatic islets were seen in high fat fed rats, but no changes of skeletal muscles and livers were observed. mRNA levels of GLUT2, α-glucosidase in small intestinal epithelium and Kir6.2 mRNA in beta cells of islets increased, whereas that of GLUT4 in skeletal muscles decreased in high fat fed group compared with normal control group. CONCLUSION: An insulin resistance animal model in Wistar rats is established by ig special fat emulsion.     

Preventive effect of hydrotalcite on gastric mucosal injury in rats induced by taurocholate

AIM: To study the preventive effect of hydrotalcite on gastric mucosal injury in rat induced by taurocholate, and to investigate the relationship between the protective mechanism of hydrotalcite and the expression of trefoil factor family 2 (TFF2) mRNA and c-fos protein.METHODS: Forty five male Wistar rats were randomly divided into hydrotalcite group, ranitidine group and control group. Gastric mucosal injury was induced by introgastric acidified taurocholate. OD value of TFF2 mRNA expression in gastric mucous cells was determined by hybridization and computer image analysis system. OD value of c-fos protein expression in gastric mucous cells was measured by immunohistochemistry and computer image analysis system.RESULTS: The gross mucosal injury index in hydrotalcite group was significantly lower than that in ranitidine group and control group (8.60±2.20 vs 16.32±4.27, 29.53±5.39;P＜0.05, P＜0.01). The expression level of TFF2 mRNA in hydrotalcite group was markedly higher than that in ranitidine group and control group (0.56±0.09 vs 0.30±0.05, 0.28±0.03,P＜0.05). The OD value of c-fos protein in hydrotalcite group was higher than that in ranitidine group and control group (0.52±0.07 vs 0.31±0.04, 0.32±0.05, P＜0.05).CONCLUSION: Hydrotalcite can protect gastric mucosal injury in rats induced by taurocholate, which may be related to the increased expression of TFF2 and c-fos protein.     

慢性间歇低氧对大鼠颏舌肌细胞线粒体功能的影响及脂联素的干预作用

目的 探讨慢性间歇低氧(CIH)对大鼠颏舌肌细胞线粒体功能的影响及脂联素干预作用.方法 健康雄性Wistar大鼠39只,采用随机数字表法分成健康对照(NC)组、CIH组、CIH脂联素干预组(CIH+Ad组),每组13只.NC组大鼠呼吸正常空气,CIH组与CIH+Ad组均接受CIH环境(CIH 8 h/d,共5周),CIH+Ad组加用经静脉脂联素注射10 μg/次,2次/周,共5周.于实验终止(第35天)时测定并比较各组大鼠血清脂联素浓度、颏舌肌线粒体膜电位、线粒体复合物Ⅰ活性、线粒体复合物Ⅳ活性.结果 CIH组血清脂联素浓度明显低于NC组[(1108±112)ng/ml,(2241±121)ng/ml,P＜0.01];CIH+Ad组高于CIH组[(1889±119)ng/ml]但低于NC组[(2241±121)ng/ml,均P＜0.01].CIH组颏舌肌线粒体膜电位相对值(1.82±0.11)明显低于NC组(2.09±0.14,P＜0.01),CIH+Ad组(1.98±0.09)较CIH组略高但低于NC组,差异均有统计学差异(均P＜0.05).CIH组线粒体复合物Ⅰ、Ⅳ浓度[(35.68±1.73)μmol·min-1·mg-1,(2.37±0.11)nmol·min-1·mg-1]最低,CIH+Ad组[(37.18±1.95)μmol·min-1·mg-1,(2.49±0.09)nmol·min-1·mg-1]及NC组[(39.02±1.38)μmol·min-1·mg-1,(2.81±0.12)nmol·min-1·mg-1]依次增高.NC组与CIH组比较差异有统计学意义(P＜0.01),CIH+Ad组与CIH组和NC组比较差异有统计学意义(均P＜0.05).结论 CIH可致大鼠血清脂联素水平降低,并能显著损伤颏舌肌细胞线粒体功能,补充外源性脂联素能部分改善CIH对大鼠颏舌肌细胞线粒体功能的损伤,提示低脂联素血症可能参与CIH导致的颏舌肌能量代谢障碍.

Abstract：

Objective To investigate the effect of chronic intermittent hypoxia (CIH) on mitochondrial function in genioglossus cells of rats and intervention role of adiponectin (Ad). Methods Thirty-nine healthy male Wistar rats were randomly divided into 3 groups, normal control (NC) group, CIH group and CIH + Ad group with 13 rats in each. Rats in NC group were kept breathing normal air, while rats in both CIH and CIH + Ad groups experienced the same CIH environment ( CIH 8 h/day for successive 5 weeks). However, rats in CIH + Ad group was given intravenous Ad supplement at the dosage of 10 μg,twice a week for sucessive 5 weeks. At the end of experiment ( day 35 ), the levels of plasma adiponectin,mitochondrial membrane potential activities of respiratory chain complexes Ⅰ and Ⅳ in mitochondrion of genioglossus cells were compared among different groups. Results Serum Ad level was significantly lower in CIH group than that in NC group [(1108 ± 112) ng/ml vs (2241 ± 121) ng/ml, P＜0.01 ]. Serum Ad level in CIH + Ad group [ ( 1889 ± 119) ng/ml] was significantly higher than that in NC group but lower than that in CIH group ( all P ＜ 0. 01 ). Mitochondrial membrane potential was significantly lower in CIH group than that in NC group [ ( 1.82 ± 0. 11 ) vs (2. 09 ± 0. 14), P ＜ 0. 01 ]. Mitochondrial membrane potential in CIH + Ad group ( 1.98 ± 0. 09) was higher than that in CIH group but lower than that in NC group ( all P ＜ 0. 05 ). The concentrations of mitochondrial respiratory chain complexes Ⅰ and Ⅳ in CIH group ( 35.68 ± 1.73 ) μmol · min - 1 · mg- 1 and (2. 37 ± 0. 11 ) nmol · min - 1 ·mg - 1, respectively) were the lowest but became higher from CIH + Ad group [ (37. 18 ± 1.95) μ mol· min-1 · mg-1 and (2. 49 ±0.09) nmol · min-1 ·mg-1 ,respectively] to NC group (39.02 ± 1.38) μmol · min-1 · mg-1 and (2. 81±0. 12) nmol · min-1 ·mg-1 ,respectively), with a significant difference between NC and CIH groups ( P ＜ 0. 01 ), between CIH + Ad and CIH groups ( P ＜ 0. 05 ), as well as between CIH + Ad and NC groups (P ＜ 0. 05 ). Conclusion CIH could lead to hypoadiponectinemia and impaired mitochondrial function in genioglossus cells of rats. Since such changes could be partially improved by supplement of adiponectin, it was suggested that hypoadiponectinemia might be involved in CIH-induced impairment of genioglossus energy metabolism.     

Nucleus tractus solitarius mediates hyperalgesia induced by chronic pancreatitis in rats

BACKGROUND Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis(CP). We hypothesized that the nucleus tractus solitarius(NTS), a primary central site that integrates pancreatic afferents apart from the thoracic spinal dorsal horn, plays a key role in the pathogenesis of visceral hypersensitivity in a rat model of CP.AIM To investigate the role of the NTS in the visceral hypersensitivity induced by chronic pancreatitis.METHODS CP was induced by the intraductal injection of trinitrobenzene sulfonic acid(TNBS) in rats. Pancreatic hyperalgesia was assessed by referred somatic pain via von Frey filament assay. Neural activation of the NTS was indicated by immunohistochemical staining for Fos. Basic synaptic transmission within the NTS was assessed by electrophysiological recordings. Expression of vesicular glutamate transporters(VGlu Ts), N-methyl-D-aspartate receptor subtype 2 B (NR2 B), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subtype 1(Glu R1) was analyzed by immunoblotting. Membrane insertion of NR2 B and Glu R1 was evaluated by electron microscopy. The regulatory role of the NTS in visceral hypersensitivity was detected via pharmacological approach and chemogenetics in CP rats.RESULTS TNBS treatment significantly increased the number of Fos-expressing neurons within the caudal NTS. The excitatory synaptic transmission was substantially potentiated within the caudal NTS in CP rats(frequency: 5.87 ± 1.12 Hz in CP rats vs 2.55 ± 0.44 Hz in sham rats, P 0.01; amplitude: 19.60 ± 1.39 p A in CP rats vs14.71 ± 1.07 p A in sham rats; P 0.01). CP rats showed upregulated expression of VGlu T2, and increased phosphorylation and postsynaptic trafficking of NR2 B and Glu R1 within the caudal NTS. Blocking excitatory synaptic transmission via the AMPAR antagonist CNQX and the NMDAR antagonist AP-5 microinjection reversed visceral hypersensitivity in CP rats(abdominal withdraw threshold: 7.00± 1.02 g in CNQX group, 8.00 ± 0.81 g in AP-5 group and 1.10 ± 0.27 g in saline group, P 0.001). Inhibiting the excitability of NTS neurons via chemogenetics also significantly attenuated pancreatic hyperalgesia(abdominal withdraw threshold: 13.67 ± 2.55 g in Gi group, 2.00 ± 1.37 g in Gq group, and 2.36 ± 0.67 g in m Cherry group, P 0.01).CONCLUSION Our findings suggest that enhanced excitatory transmission within the caudal NTS contributes to pancreatic pain and emphasize the NTS as a pivotal hub for the processing of pancreatic afferents, which provide novel insights into the central sensitization of painful CP.     

Effect of retrograde colonic electrical stimulation on colonic transit and stress-induced visceral hypersensitivity in rats with irritable bowel syndrome

Objective: To evaluate the effects of retrograde colonic electrical stimulation(RCES) with trains of short pulses and RCES with long pulses on colonic transit in irritable bowel syndrome(IBS) rats and to investigate whether stress-induced visceral hypersensitivity could be alleviated by RCES so as to find a valuable new approach for IBS treatment. Methods:A total of 48 male rats were randomly divided into model group and control group. Visceral hypersensitivity model was induced by a 6-day HIS protocol composed of two stressors, restraint stress for 40 min and forced swimming stress for 20 min. The extent of visceral hypersensitivity was quantified by electromyography and abdominal withdrawal reflx scores(AWRs) of colorectal distension(use a balloon) at different pressures. After the modeling, all rats were equipped with electrodes in descending colon for retrograde electrical stimulation and a PE tube for perfusing phenol red saline solution in the ileocecus. After recovering from surgery, RCES with long pulses, RCES with trains of short pulses, and sham RCES were performed in colonic serosa of rats for 40 min in six groups of 8 each, including three groups of visceral hypersensitivity rats and three groups of health rats. Colonic transit was assessed by calculating the output of phenol red from the anus every 10 min for 90 min. Finally, the extent of visceral hypersensitivity will be quantified again in model group. Results: After the 6-day HIS protocol, the HIS rats displayed an increased sensitivity to colorectal distention, compared to control group at different distention pressures(P0.01). CRES with trains of short pulses and long pulses significantly attenuated the hypersensitive responses to colorectal distention in the HIS rats compared with sham RCES group(P0.01). The effects of RCES on rats colon transmission: In the IBS rats, the colonic emptying were(77.4 ± 3.4)%,(74.8 ± 2.4)% and(64.2 ± 1.6)% in the sham RCES group, long pulses group and trains of short pulses group at 90 min; In healthy rats, The colonic emptying was(65.2 ± 3.5)%,(63.5 ± 4.0)% and(54.0 ± 2.5)% in the sham RCES group, long pulses group and trains of short pulses group at 90 min. Conclusion: RCES with long pulses and RCES with trains of short pulses can significantly alleviate stress-induced visceral hypersensitivity. RCES with trains of short pulses has an inhibitory effect of colonic transit, both in visceral hypersensitivity rats and healthy rats.     

N-acetylcysteine attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis

AIM To evaluate attenuating properties of N-acetylcysteine (NAC) on oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis (NASH).METHODS Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 8) was free accessed to regular dry rat chow (RC) for 6 wk.Group 2 (NASH, n = 8) was fed with 100% fat diet for 6 wk. Group 3 (NASH NAC20, n = 9) was fed with 100% fat diet plus 20 mg/kg per day of NAC orally for 6 wk. All rats were sacrificed to collect blood and liver samples at the end of the study.RESULTS The levels of total glutathione (GSH)and hepatic malondialdehyde (MDA) were increased significantly in the NASH group as compared with the control group (GSH; 2066.7 ± 93.2 vs 1337.5 ± 31.5 μmol/L and MDA; 209.9± 43.9 vs 3.8 ±1.7 μmol/g protein, respectively, P ＜ 0.05). Liver histopathology from group 2 showed moderate to severe macrovesicular steatosis, hepatocyte ballooning, and necroinflammation.NAC treatment improved the level of GSH (1394.8 ± 81.2 μmol/L, P ＜ 0.05), it did not affect MDA (150.1 ± 27.0 μmol/g protein), but led to a decrease in fat deposition and necroinflammation.CONCLUSION NAC treatment could attenuate oxidative stress and improve liver histology in rats with NASH.     

Beneficial effects of antidepressant mirtazapine in functional dyspepsia patients with weight loss

AIM: To explore the effects and mechanism of action of antidepressant mirtazapine in functional dyspepsia(FD) patients with weight loss.METHODS: Sixty depressive FD patients with weight loss were randomly divided into a mirtazapine group(MG), a paroxetine group(PG) or a conventional therapy group(CG) for an 8-wk clinical trial. Adverse effects and treatment response were recorded. The Nepean Dyspepsia Index-symptom(NDSI) checklist and the 17-item Hamilton Rating Scale of Depression(HAMD-17) were used to evaluate dyspepsia and depressive symptoms, respectively. The body composition analyzer was used to measure body weight and fat. Serum hormone levels were measured by ELISA.RESULTS:(1) After 2 wk of treatment, NDSI scores were significantly lower for the MG than for the PG and CG;(2) After 4 or 8 wk of treatment, HAMD-17 scores were significantly lower for the MG and PG than for the CG;(3) After 8 wk of treatment, patients in the MG experienced a weight gain of 3.58 ± 1.57 kg, which was significantly higher than that observed for patients in the PG and CG. Body fat increased by 2.77 ± 0.14kg, the body fat ratio rose by 4%, and the visceral fat area increased by 7.56 ± 2.25 cm2; and(4) For the MG, serum hormone levels of ghrelin, neuropeptide Y(NPY), motilin(MTL) and gastrin(GAS) were significantly upregulated; in contrast, those of leptin, 5-hydroxytryptamine(5-HT) and cholecystokinin(CCK) were significantly downregulated. CONCLUSION: Mirtazapine not only alleviates symptoms associated with dyspepsia and depression linked to FD in patients with weight loss but also significantly increases body weight(mainly the visceral fat in body fat). The likely mechanism of mirtazapine action is regulation of brain-gut or gastrointestinal hormone levels.     

成年期追赶生长对大鼠胰岛素敏感性与应激水平的影响

目的 观察成年期追赶生长对大鼠胰岛素敏感性和应激水平的影响,并探讨其胰岛素抵抗形成的可能机制。方法 将7周龄雄性SD大鼠分为6组（共2个时间点）,即4周时间点2组：热卡限制4周组(R4),正常饮食4周组(NC4)作为R4组对照;8周时间点4组：正常饮食追赶生长组(RN4)、高脂饮食追赶生长组(RH4)、持续高脂饮食8周组（HF8）、持续正常饮食8周组(NC8)。通过先热卡限制后恢复饮食的方法建立追赶生长大鼠模型。检测大鼠高胰岛素-正糖钳夹试验过程中葡萄糖输注率和骨骼肌2-脱氧葡萄糖摄取、胰岛素刺激后的骨骼肌胰岛素信号通路、血皮质酮、骨骼肌11β-羟类固醇脱氢酶1(11β-HSD1)表达水平。结果 热卡限制4周时,R4组大鼠血皮质酮和骨骼肌11β-HSD1 mRNA表达水平明显高于NC4组(P＜0.05),骨骼肌蛋白激酶B( Akt) Ser473磷酸化和糖摄取与NC4组相比差异无统计学意义。热卡限制后恢复饮食4周时,血皮质酮和骨骼肌11β-HSD1表达水平RN4组明显高于NC8组,RH4组明显高于NC8和HF8组,而骨骼肌Akt磷酸化和糖摄取RN4组明显低于NC8组,RH4组明显低于NC8组、HF8组和RN4组（均P＜0.05）。结论正常饮食和高脂饮食追赶生长大鼠均可导致整体和骨骼肌应激水平上调及胰岛素抵抗,尤以高脂饮食追赶生长大鼠更为明显。应激和饮食状况的交互作用可能是追赶生长胰岛素抵抗形成的重要原因。     

大鼠追赶生长早期脂肪组织和骨骼肌葡萄糖代谢差异及其机制

目的 观察追赶生长过程中脂肪组织和骨骼肌葡萄糖利用状况,初步探讨脂肪组织追赶生长的发生机制.方法 将雄性Wistar大鼠随机分为正常对照组(NC)和追赶生长组(RN).通过限制饮食(NC组食量的50%)4周后给予自由开放普通饮食建立追赶生长大鼠模型,分别于实验的第5周末(NC1组和RN1组)、第6周末(NC2组和RN2组)进行检测.运用[ H]-脱氧葡萄糖测定骨骼肌、脂肪组织葡萄糖摄取率,RT-PCR、 Western印迹法检测葡萄糖转运子4(Glut4)的mRNA及膜蛋白水平.结果 与NC1组相比,RN1组大鼠脂肪组织葡萄糖摄取率升高了189.6%(P<0.01),而骨骼肌组织则下降了36.5%(P<0.05);开放饮食2周后,RN2组大鼠脂肪组织葡萄糖摄取率较NC2组增加了157.3%(P<0.01),骨骼肌组织则减少了41.5%(P<0.05).而RN与NC各组间骨骼肌和脂肪组织Glut4的mRNA表达水平的差异均无统计学意义.但RN1组和RN2组胰岛素刺激后骨骼肌细胞膜上Glut4蛋白水平分别较相应的NC1组和NC2组下降了46.5%(P<0.01)和32.1%(P<0.05);脂肪组织则分别升高了116.5%和89.9%(均P<0.01).结论 追赶生长的早期即出现了葡萄糖由骨骼肌向脂肪组织的转移,导致脂肪组织快速追赶生长.胰岛素刺激的Glut4向细胞膜的转位发生组织特异性改变可能是导致这一变化的机制之一.     

Physiological role of cholecystokinin B/gastrin receptor in leptin secretion.

In the present study, we investigated whether cholecystokinin (CCK) or its structurally related peptide gastrin participates in long term regulation of adipocyte leptin secretion. The levels of circulating leptin observed after 2 and 6 h of refeeding in 18-h fast rats were significantly lowered by injection of the specific gastrin/CCK-B receptor antagonist YM022 at doses that did not affect feeding behavior. Moreover, in normally fed animals, circulating leptin was markedly decreased by chronic injection of YM022 (from 4 +/- 0.6 to 2.1 +/- 0.5 ng/ml). Consistent with these observations, YM022 treatment decreased leptin messenger RNA (mRNA) levels and increased the leptin content in rat epididymal fat tissue. Rat adipocytes exclusively contain gastrin/CCK-B receptor mRNA, but not CCK-A receptor mRNA. Furthermore, adipocyte membranes bound [125I]CCK-8 in a saturable manner, with kinetics consistent with a single class of high affinity sites with a Kd of 0.2 nM. These data argue for a physiological role for the CCK-B/gastrin receptor in adipocyte leptin regulation. We therefore propose that gastrin is involved in long term regulation of leptin expression and secretion in rat fat tissues through activation of an adipocyte gastrin/CCK-B receptor.     

限食后追赶生长性肥胖与大鼠血浆葡萄糖依赖性胰岛素释放肽水平的关系

目的 探讨限食后追赶生长性肥胖与大鼠血浆葡萄糖依赖性胰岛素释放肽(GIP)水平的相关性.方法 6周龄健康雄性SD大鼠60只按随机数字表法随机分为普通饮食组(n=15,给予普通饮食)、普通饮食追赶生长组(n=15,给予同体重普通饮食组大鼠60%普通饲料限食喂养4周后饲以普通饲料)、高脂饮食组(n=15,给予高脂饮食)和高脂饮食追赶生长组(n=15,给予同体重普通饮食组大鼠60%普通饲料限食喂养4周后饲以高脂饲料),观察大鼠体重及进食量变化.分别于4、6、8周处死部分动物,检测体脂含量及血浆GIP浓度.采用配对t检验和单因素方差分析以及一元线性相关分析进行数据统计.结果 与普通饮食组相比,高脂饮食组、普通饮食追赶生长组和高脂饮食追赶生长组体脂含量[分别为(3.6±0.6)、(7.9±1.5)、(4.6±1.1)、(7.0±1.0)g;t值分别为-2.601、-2.305、-2.501,均P＜0.05]、血浆GIP水平升高[分别为(41±9)、(61±7)、(51±8)、(59±8)pmol/L;t值分别为-6.061、-3.452、-4.651,均P＜0.05].相关分析显示,体脂含量与血浆GIP水平显著相关(r2=0.9407).结论 限食后追赶生长性肥胖与大鼠血浆GIP水平高度相关,可能与追赶生长所引发的病理生理学变化有关.     

雌激素对去卵巢大鼠内脏脂肪细胞脂肪因子表达水平的影响

目的 观察雌激素对去卵巢大鼠内脏脂肪细胞瘦素、脂联素、抵抗素和肿瘤坏死因子-α(TNF-α)表达水平的影响,探讨雌激素对体脂分布的影响机制.方法 6周龄Sprauge-Dawley雌性大鼠30只,采用随机数字表法分成3组:假手术组、去卵巢组和去卵巢+戊酸雌二醇组(OVX+E2组),每组10只.术后1周,OVX+E2组大鼠每天按1 mg/kg体重灌胃戊酸雌二醇水溶液,其他组大鼠灌胃等体积蒸馏水.连续给药12周后,腹主动脉取血,迅速剥离内脏脂肪组织.采用全自动生化分析仪检测血脂、血糖.采用免疫组化染色、实时荧光定量RT-PCR和Western印迹检测脂肪细胞瘦素、脂联素、抵抗素和TNF-α的表达.结果 3组血清瘦素、脂联素和抵抗素水平差异无统计学意义(P均＞0.05),但去卵巢组TNF-α水平显著高于假手术组(F=4.785,P＜0.05).免疫组化显示,与假手术组相比,去卵巢组内脏脂肪组织中瘦素表达明显减弱,而脂联素、抵抗素和TNF-α表达明显增强;与去卵巢组相比,OVX+E2组内脏脂肪组织中瘦素表达明显增强,脂联素、抵抗素和TNF-α表达明显减弱(F =3.712 ～5.198,P均＜0.05).3组内脏脂肪细胞瘦素mRNA和蛋白表达水平差异无统计学意义(P均＞0.05);去卵巢组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著高于假手术组,而OVX+E2组内脏脂肪细胞脂联素、抵抗素和TNF-α的mRNA和蛋白表达水平显著低于去卵巢组(F=3.175～5.342,P均＜0.05).结论 雌激素可通过下调去卵巢大鼠内脏脂肪细胞脂联素、抵抗素和TNF-α的表达,进而影响去卵巢大鼠体脂再分布.     

肥胖大鼠肝脏、骨骼肌叉头状因子O1基因表达对胰岛素抵抗的影响

目的观察肥胖大鼠肝脏和骨骼肌叉头状因子O1（FoxO1）的表达,探讨Fox01在肥胖和胰岛素抵抗发生中的作用。方法30只SD大鼠随机分为对照组（NC,常规饲料）和高脂组（HF,高脂饲料）。饲养10周后测定有关指标。结果10周后与NC组相比,HF组体重、血糖、胰岛素、TG、TC、腹内脂肪／体重均升高,肝脏和骨骼肌FoxO1mRNA表达量分别增加84％和88％（P〈0．01）。TG、HOMA-IR及腹内脂肪／体重是肝脏FoxO1mRNA表达的独立相关因素,HOMA-IR及腹内脂肪／体重是骨骼肌FoxO1表达水平的主要影响因素。结论肥胖大鼠肝脏和骨骼肌FoxO1表达量升高,可能是肥胖状态下引发胰岛素抵抗的机制之一。     

热卡限制对高脂饲养大鼠肝脏糖异生相关基因表达的影响

目的 观察热卡限制对高脂饲养大鼠肝脏Forkhead转录因子O1(FoxO1)、磷酸烯醇丙酮酸羧激酶(PEPCK)和葡萄糖6磷酸酶(G-6-P)mRNA表达的影响,探讨其可能机制.方法 24只雄性Wistar大鼠随机分为正常对照组(7只)、高脂组(9只)和热卡限制组(8只),分别给予正常饮食、高脂饮食和60%热卡限制饮食,共饲养12周.实验终点时取空腹血测血糖、胰岛素、甘油三酯和总胆固醇;称内脏脂肪重量及体重,计算内脏脂肪重量占体重百分比;逆转录聚合酶链法检测肝脏FoxO1、PEPCK和G-6-P mRNA表达变化;光镜观察肝脏组织学改变.结果 高脂饮食大鼠出现明显腹型肥胖,空腹血糖、空腹胰岛素、甘油三酯和总胆固醇均升高,FoxO1、PEPCK和G-6-P mRNA表达较正常组分别增加18.9%、33.8%和24.6%(P值均＜0.01),且光镜下出现肝脏脂肪变性;热卡限制后大鼠体重、内脏脂肪含量显著下降,空腹血糖、空腹胰岛素、甘油三酯、总胆固醇均有所下降,FoxO1、PEPCK和G-6-P mRNA表达较正常组分别减少26.6%、35.0%和34.3%(P值均＜0.01),同时镜下脂肪变性有所好转.结论 热卡限制能有效降低FoxO1、PEPCK和G-6-P基因表达,增强胰岛素信号传导,抑制肝脏糖异生,调节糖代谢.     

Daily changes in hypothalamic gene expression of neuropeptide Y, galanin, proopiomelanocortin, and adipocyte leptin gene expression and secretion: effects of food restriction.

The participation of hypothalamic neuropeptide Y (NPY)-, galanin (GAL)-, and opioid-producing neurons in the restraint on food intake exerted by adipocyte leptin has recently been recognized. To further understand the interplay between the central appetite-stimulating- and peripheral appetite-inhibiting signals in the management of daily food intake, we have examined the daily patterns in expression of the hypothalamic neuropeptides and leptin receptor (R) and adipocyte leptin gene expression and secretion in freely feeding (FF) rats. These analyses were extended to determine the impact of food restriction (FR) to 4 h daily for 4 weeks. Groups of FF and FR rats were killed at 4-h intervals during a 24-h period, and hypothalamic NPY, GAL, POMC, and leptin-R gene expression and leptin gene expression were evaluated by RNase protection assays and serum leptin and corticosterone (CORT) levels were estimated by RIA. The following new findings emerged: 1) In FF rats, hypothalamic NPY messenger RNA (mRNA) levels fluctuated during the course of 24 h with high levels at 0700 h and 1100 h followed by a decrease at 1500 h during the lights-on phase that was sustained throughout the dark phase (1900 h-0500 h) of the light-dark cycle. Hypothalamic GAL and POMC mRNA also displayed daily patterns but with a different time course; GAL and POMC gene expression were elevated 4 h later than NPY mRNA at 1100 h and 1500 h. 2) Although FR to 4 h between 1100 h and 1500 h resulted in maintenance of body weight compared with a steady weight gain in FF rats, the daily patterns of fluctuations in hypothalamic neuropeptide gene expression were abolished. 3) In FF rats, hypothalamic leptin-R and adipocyte leptin gene expression and serum leptin levels displayed a daily pattern temporally different from that of hypothalamic neuropeptide gene expression. Adipocyte leptin mRNA remained low during the lights-on phase but increased at the onset of the lights-off phase (1900 h) and remained elevated through the dark phase. 4) Hypothalamic leptin-R gene expression, like that of adipocyte leptin gene expression, rose abruptly at the onset of nocturnal feeding behavior but receded progressively to low range thereafter. 5) On the other hand, a dichotomy in the daily rise in adipocyte leptin gene expression and leptin secretion was observed in FF rats. Unlike adipocyte leptin mRNA, serum leptin increased at 2300 h, 4 h after initiation of ingestive behavior. 6) In FR rats, adipocyte leptin gene expression fluctuated little over the 24-h period but, as in FF rats, leptin hypersecretion peaked 4 h after initiation of food intake. 7) In both FF and FR rats, increased serum CORT levels preceded serum leptin rise. Overall, these results show that in FF rats, gene expression of hypothalamic appetite stimulating peptides first rise and then fall to nadir during the lights-on phase when leptin levels are in low range; adipocyte leptin mRNA rises before impending ingestive behavior and increased leptin secretion reaching peak manifests itself during nocturnal feeding. The FR regimen, which curtailed the normal body weight gain, abolished these daily fluctuations in gene expression of hypothalamic orexigenic peptides and adipocyte leptin but permitted feeding-associated increased leptin secretion. Thus, it may be important to consider the daily patterns of gene expression and availability of hypothalamic orexigenic peptides in investigations aimed at elucidating the central mechanisms underlying the feedback action of the normal and altered leptin secretion patterns.     

胰岛素抵抗大鼠视黄醇结合蛋白4骨骼肌磷脂酰肌醇3激酶的表达及吡格列酮的干预作用

目的 观察胰岛素抵抗(IR)大鼠体内视黄醇结合蛋白4(RBP4)、骨骼肌磷脂酰肌醇3激酶( P13K)和晚期氧化蛋白产物(AOPP)的水平,以及给予吡格列酮干预后其活性的变化,探讨RBP4与IR的关系及其可能的机制。 方法 将SPF级雄性Wistar大鼠35只随机分为2组,正常对照组（对照组）11只,饲以普通饲料;模型组24只,饲以高糖高脂饲料。模型组造模成功后再随机分为2个亚组,IR组和IR+吡格列酮干预组（干预组）,每组12只;IR组和干预组继续饲以高糖高脂饲料,干预组大鼠同时给予吡格列酮20mg·kg 1·d-1灌胃,持续8周。第16周末处死大鼠取血检测三酰甘油(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)及空腹血糖(FBG)、空腹胰岛素(FINS),计算胰岛素抵抗指数(HOME-IR);酶联免疫吸附法(ELISA)检测血清RBP4水平,RT-PCR测定附睾脂肪组织RBP4表达;免疫组织化学染色检测骨骼肌PI3K水平;紫外分光光度计法测定AOPP水平;并取大鼠腹腔内肠系膜、附睾、腹膜反折处的脂肪组织称质量,计算腹部脂肪含量与体质量的比值。 结果 (1)IR组大鼠体质量16周后明显增加,TG、LDL-C、FINS以及脂体比较对照组均明显升高,而HDL-C则明显降低;吡格列酮干预后,干预组体质量、TG、LDL-C、FINS以及脂体比较IR组有明显下降,HDL-C明显升高;(2)IR组大鼠血清及附睾脂肪组织RBP4水平和血清AOPP水平明显高于对照组,干预组水平明显降低;(3)IR组大鼠骨骼肌组织PI3K表达水平明显低于对照组,吡格列酮干预其表达水平升高;(4)相关分析表明大鼠血清RBP4与FINS、脂体比、LDL-C呈正相关;与HDL-C、骨骼肌组织PI3K水平呈负相关。 结论 (1)IR大鼠RBP4、AOPP水平升高,RBP4是致IR的脂肪细胞因子,并可引起机体脂代谢紊乱及氧化应激增强;(2)RBP4降低大鼠胰岛素敏感性可能与其削弱胰岛素信号转导作用有关;(3)吡格列酮可降低IR大鼠RBP4及血清AOPP水平,增加骨骼肌PI3K表达,从而提高机体对胰岛素的敏感性。