Oral Insulin Enhances Intestinal Regrowth Following Massive Small Bowel Resection in Rat

Experimental studies have suggested that insulin (INS) plays an important role in small intestinal growth and development. In the present study we investigated the effect of oral INS on structural intestinal adaptation and enterocyte proliferation and loss via apoptosis in a rat model of short bowel syndrome (SBS). Male Sprague–Dawley rats were divided into three experimental groups: sham rats underwent bowel transection, SBS rats underwent 75% small bowel resection, and SBS-INS rats underwent bowel resection and were treated with oral INS given in the drinking water from the 3rd to the 15th postoperative day. Parameters of intestinal adaptation (bowel and mucosal weight, mucosal DNA and protein, villous height, and crypt depth), enterocyte proliferation, and apoptosis were determined on day 15. SBS-INS rats demonstrated a significant increase (vs SBS rats) in jejunal and ileal overall bowel and mucosal weight, ileal mucosal DNA and protein, ileal villous height, and crypt depth. SBS-INS rats also showed an increased cell proliferation index in jejunum and ileum and decreased apoptotic index in jejunum compared to SBS animals. In conclusion, in a rat model of SBS, oral INS strongly enhances intestinal adaptation.Possible mechanisms may include increased cell proliferation and decreased enterocyte loss via apoptosis. Contributed equally to the preparation of this article.     

Dietary Glutamine Supplementation Prevents Mucosal Injury and Modulates Intestinal Epithelial Restitution Following Ischemia-Reperfusion Injury in the Rat

The aim of the present study was to evaluate the preventive effect of a 2-day oral glutamine supplementation against intestinal ischemia-reperfusion (IR) injury in a rat. Male Sprague-Dawley rats were divided into four experimental groups: sham rats underwent laparotomy, sham-GLU rats underwent laparotomy and were treaded with enteral glutamine (GLU) given in drinking water (2%) 48 hr before and following operation, IR rats underwent occlusion of both the superior mesenteric artery and the portal vein for 30 min followed by 24 hr of reperfusion, and IR-GLU rats were treated with enteral glutamine 48 hr before and following IR. Intestinal mucosal damage (Park’s injury score), mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were determined 24 hr following IR. Sham-GLU rats demonstrated a lower rate of cell apoptosis in jejunum and ileum compared to sham animals. IR-GLU animals demonstrated a greater jejunal and ileal bowel and mucosal weight, mucosal DNA, villous height and crypt depth, and enterocyte proliferation index in ileum and a lower injury score grade in jejunum compared to IR-nontreated rats. In conclusion, pretreatment with oral glutamine prevents mucosal injury and improves intestinal recovery following IR injury in the rat.     

黄芩苷对脂多糖诱导的巨噬细胞Toll样受体4表达的影响

目的 研究黄芩苷对脂多糖诱导的小鼠巨噬细胞Toll样受体4和其下游信号分子抑制蛋白kB以及效应分子肿瘤坏死因子α表达的影响.方法 分别用脂多糖(终浓度1 mg/L)或脂多糖+黄芩苷(终浓度10、50和100 μmol/L)处理生长良好的小鼠巨噬细胞RAW264.7,逆转录聚合酶链反应和Western Blot检测细胞Toll样受体4表达情况和抑制蛋白kB含量变化,酶联免疫吸附法检测细胞上清液中肿瘤坏死因子α的浓度.结果 脂多糖刺激RAW264.7细胞可导致Toll样受体4表达增高,促进抑制蛋白kB降解,上调肿瘤坏死因子α表达;黄芩苷预处理能降低脂多糖诱导的Toll样受体4表达增高,降低抑制蛋白kB降解,下调肿瘤坏死因子α分泌.结论 黄芩苷可通过抑制Toll样受体4表达和降低抑制蛋白kB降解,影响Toll样受体4/抑制蛋白kB-核因子kB炎症信号途径,阻碍炎症因子肿瘤坏死因子α的生成,发挥抗炎作用,这可能是其抗动脉粥样硬化的作用机制之一.     

Topical and Oral Anti-inflammatory Activity of Budesonide Compared with Oral Prednisolone in an Animal Model Using Allergen-induced Gut Mucosal Exudation of Plasma as a Marker

Background: Development of topically active glucocorticosteroids with minimal systemic effects is paramount in improving therapy in inflammatory bowel disease. Our experimental model in the rat has proved useful for assessing topical versus systemic anti-inflammatory potency of glucocorticosteroids on the inflamed gut. Methods: Experiments were performed on allergen-sensitized perfused rat ileum in vivo. Mucosal exudation of plasma, induced by local allergen perfusion, was measured as the appearance of circulating ¹²⁵     

Toll样受体4在CCl_4诱导的大鼠慢性肝损伤过程中的表达

背景:Toll样受体4(TLR4)在内毒素的信号转导过程中具有重要作用。目的:动态观察在CCl4诱导的大鼠慢性肝损伤过程中,肝组织和Kupffer细胞TLR4基因表达的变化,探讨TLR4在肝损伤中的作用。方法:以CCl4诱导慢性肝损伤纤维化大鼠模型,分离肝Kupffer细胞,以逆转录聚合酶链反应(RT-PCR)检测肝组织和Kupffer细胞TLR4 mRNA的表达;将Kupffer细胞分别与不同浓度的脂多糖(LPS)孵育,以酶联免疫吸附测定(ELISA)检测细胞培养上清的肿瘤坏死因子(TNF)-α水平。以基质显色法测定大鼠血浆内毒素水平。结果:正常大鼠肝组织TLR4 mRNA表达水平较低,Kupffer细胞未检测到TLR4 mRNA表达;CCl4处理2-6周大鼠的肝组织和Kupffer细胞TLR4 mRNA表达水平显著增高(P<0．05)。CCl4处理4周和6周大鼠Kupffer细胞的TNF-α基础分泌水平显著高于正常大鼠(P<0．05); 在LPS的刺激下,TNF-α的分泌水平较基础值进一步增高(P<0．05),呈浓度依赖性。大鼠血浆内毒素水平在肝损伤过程中逐渐增高．相关分析显示在慢性肝损伤的早、中期,肝组织和Kupffer细胞TLR4 mRNA的表达与血浆内毒素水平呈正相关。结论:在CCl4诱导的慢性肝损伤过程中,大鼠肝脏TLR4基因表达上调,与Kupffer细胞活化和肝脏的炎症损伤有关。     

Dobutamine Enhances Alveolar Fluid Clearance in a Rat Model of Acute Lung Injury

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), remain leading factors for morbidity and mortality in critically ill patients. A significant aspect of ALI and ARDS is impaired alveolar fluid clearance (AFC). Improvements in therapies for these types of respiratory illnesses will require an understanding of the mechanisms that control AFC. The present study was designed to determine whether the administration of dobutamine decreases pulmonary edema and stimulates AFC in a rat model of lipopolysaccharide-induced lung injury. Adult male Sprague–Dawley rats were randomly divided into three groups: control, lipopolysaccharide, and lipopolysaccharide + dobutamine. The effect of dobutamine on AFC and the expression of aquaporin-1 and aquaporin-5 were examined. Lipopolysaccharide administration results in significant lung injury with impaired AFC, while dobutamine improves alveolar fluid reabsorption with elevation of aquaporin-1 and aquaporin-5. Our study indicates that dobutamine may enhance alveolar fluid reabsorption by increasing the expression of aquaporin-1 and aquaporin-5.     

Intestinal and Systemic Effects of Oral Insulin Supplementation in Rats After Weaning

Oral insulin has intestinal trophic effects in suckling animals. In mice, lower glucose and lipid levels may be seen when oral insulin is given after the weaning period. The purpose of the present study is to examine local and systemic effects of oral insulin supplementation in rats in the postweaning period. Immediately after weaning, Sprague–Dawley rats received either drinking water (controls) or oral insulin in their drinking water (1 U/ml) for either 1 week or 6 weeks. Intestinal mucosal parameters (bowel and mucosal weight, mucosal DNA and protein) and histological changes were examined in all study groups. Glucose levels were monitored weekly, and at the end of the study, blood levels of glucose, lipids, and insulin were measured in the fasting state. After 1 week of insulin supplementation, mucosal weight in duodenum and jejunum as well as jejunal DNA content were significantly higher in insulin-supplemented rats compared to controls. Duodenal circumference and villus height in jejunum were significantly higher in insulin-supplemented rats compared to controls on both day 7 and day 42. Total cholesterol levels were significantly lower in the study group compared with the controls. We conclude that oral insulin supplementation exerts intestinal trophic effects, as well as systemic effects in the postweaning period in rats.     

Inhibition of HA Synthase 3 mRNA Expression, with a Phosphodiesterase 3 Inhibitor, Blocks Lung Injury in a Septic Ventilated Rat Model

Low-molecular-weight hyaluronan produced by hyaluronan synthase 3 (HAS3) has been shown to play a role in acute lung injury secondary to high-tidal-volume ventilation. Phosphodiesterase 3 inhibitors have been shown to decrease HAS3 expression. We hypothesized that low-molecular-weight hyaluronan (LMW HA) produced by HAS3 mediates LPS-induced lung injury in the mechanically ventilated rat and that milrinone (MIL), by blocking HAS3 mRNA expression, would prevent the injury. Rats were randomized to four groups: controls with mechanical ventilation at 7 cc/kg MV, MV+LPS, MV+MIL, and MV+LPS+MIL. Rats were intubated and ventilated without PEEP for 4 h. Lipopolysaccharide (LPS) (1 mg/kg) was infused into the arterial line 1 h prior to MV. MIL 10 μg/kg/min (or an equivalent volume of saline) was infused through the venous line at the beginning of MV. Bronchoalveolar lavage fluid (BAL) was collected after 4 h of ventilation and lungs were saved for histopathology. LPS significantly increased neutrophil infiltration and protein concentration in the BAL and augmented lung injury score on histology. MIL significantly lowered alveolar protein and neutrophil infiltration as well as lung injury in response to LPS. Furthermore, MIL decreased the mRNA expression for HAS3 and MIP2 in lung tissue and decreased the protein content in BAL. MIL, a commonly used inotropic agent, inhibited LPS-induced lung inflammation and lung injury in mechanically ventilated rats. The anti-inflammatory properties of MIL may be mediated by inhibition of HAS3 and/or MIP2 and could be beneficial in the treatment of sepsis.     

Toll样受体(TLR)2、TLR4和TLR9在大鼠结肠炎模型结肠组织中的表达及其意义

Toll样受体（TLRs）家族可能在一系列免疫性疾病中发挥重要作用。目的：观察TLR2、TLR4和TLR9在大鼠结肠炎模型结肠组织中的表达，探讨三者在炎症性肠病（IBD）发病机制中的作用。方法：以三硝基苯磺酸（TNBS）＋乙醇灌肠制备大鼠结肠炎模型，观察和评估结肠黏膜的大体和组织学变化。分别以黄嘌呤氧化酶法和紫外分光光度法测定超氧化物歧化酶（SOD）和髓过氧化物酶（MPO）活性；以免疫组化方法检测TLR2、TLR4和TLR9的表达，以逆转录聚合酶链反应（RT-PCR）检测三者mRNA的表达。结果：造模后结肠组织中可见大量炎性细胞浸润，累及黏膜下层和固有层。与正常对照组相比，模型组结肠组织SOD活性显著降低（P〈0．01），MPO活性显著升高（P〈0．01）。正常对照组结肠黏膜下层和固有层炎性细胞胞膜和胞质仅有少量TLR2、TLR4表达，未见TLR9表达；模型组三者表达均显著增加（P〈0．01），此外还可见TLR2表达于肠上皮近肠腔侧胞膜，TLR4表达于肠上皮近肠腔侧胞膜和腺上皮近腺腔侧胞膜。模型组结肠组织可见TLR2、TLR4、TLR9 mRNA表达，而正常对照组未检出三者mRNA的表达。TLR2、TLR4、TLR9的表达与MPO活性呈正相关（P〈0．05），与SOD活性呈负相关（P〈0．01）。结论：大鼠结肠炎模型结肠组织中TLR2、TLR4和TLR9表达明显增加，可能与结肠的自身免疫损伤有关。     

Protective Effects of N-Acetyl- L -Cysteine and Platelet Activating Factor Inhibition Are Not Linked to Intercellular Adhesion Molecule-1 Expression after Intestinal Ischemia and Reperfusion Injury in Rats

Background: Intestinal ischemia and reperfusion (I/R) injury may result in development of the systemic inflammatory response syndrome (SIRS). The interactions between activated leukocytes and endothelial cells, mediated by adhesion molecules, seem to be pivotal in these conditions, leading as they do to extravasation of circulating leukocytes within the inflamed tissue. The intercellular adhesion molecule-1 (ICAM-1) mediating firm adhesion of activated leukocytes is upregulated in many organs after I/R injury, but the regulatory mechanisms are complex and have not been fully investigated. Methods: We evaluated whether ICAM-1 expression was linked with a potential protective effect of N-acetyl- l -cysteine (NAC) and the platelet activating factor (PAF) inhibitor (Lexipafant), administered 15 &#114 min after the start of reperfusion, in a model of intestinal ischemia (40 &#114 min) and reperfusion (12 &#114 h) in the rat. Results: ICAM-1 expression increased significantly in the ileum, colon, lungs and pancreas after intestinal I/R. Treatments with NAC and the PAF inhibitor did not affect this response. An increased endothelial albumin-leakage was observed in the same organs after I/R. Treatment with NAC reduced the endothelial leakage of albumin in the ileum, colon and lungs, whereas administration of the PAF inhibitor alone demonstrated a protective effect only in the ileum. Furthermore, neutrophil sequestration in the lungs and IL-1 &#35 levels in plasma increased significantly after I/R, and these changes were markedly reduced by both treatment regimes. Conclusion: The protective effect of NAC and the PAF inhibitor Lexipafant in intestinal I/R injury is not due to a decreased expression of ICAM-1.     

Gut Permeability and Intestinal Mucins,Invertase, and Peroxidase in Control and Diabetes-Prone BB Rats Fed Either a Protective or a Diabetogenic Diet

This study deals with the enteropathy recently identified in diabetes-prone BB rats (BBdp). Diabetes-resistant BB rats (BBc) and BBdp rats were fed from days 32–39 onward either a protective diabetes-retardant hydrolyzed casein diet (HC) or a plant-based diabetogenic (NTP) diet. The NTP diet decreased body weight and plasma insulin in BBc and BBdp rats. The BBdp rats displayed low intestinal invertase and increased intestinal peroxidase activity. In the BBdp rats fed the HC diet, the mucin content 30–35 cm below the pylorus was higher and the gut permeability lower than in the other three rat groups. There was a significant inverse correlation between gut permeability and the insulinogenic index in the BBdp rats fed the HC or NTP diet. Thus, in BBdp rats, the HC diet somehow prevents the increase in gut permeability and the decrease in the insulinogenic index otherwise found in some of these diabetes-prone animals.     

益气活血复方对内皮细胞TLR4及其下游MyD88、TRAF-6、TRAM、TRIF mRNA表达的影响

目的研究益气活血复方对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)Toll样受体4(TLR4)及其下游信号转导通路元件髓样分化因子88(MyD88)、肿瘤坏死因子受体相关因子-6(TRAF-6)、Toll样受体相关分子(TRAM)、Toll样受体相关的干扰素活化子(TRIF)表达的影响,探讨益气活血复方含药血清防治动脉粥样硬化(AS)的机制。方法选择新西兰大耳白兔20只,随机分为4组,即正常组、中药高浓度组、中药中浓度组、中药低浓度组,每组5只。以上各组白兔分别以生理盐水和高、中、低浓度益气活血复方连续灌胃7d。末次灌胃给药2h后,心脏采血,离心后分离血清。体外培养人脐静脉内皮细胞,用LPS刺激后,分别加入高、中、低浓度益气活血复方含药血清干预24h,收集细胞,用Real ti me PCR方法测定TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA的表达。结果用LPS刺激人脐静脉内皮细胞后,引起TLR4、MyD88、TRAF-6、TRAM及TRIF mRNA的高表达(与空白对照组比较,P0.01),用益气活血复方含药血清干预以后显著抑制TLR4、MyD88及TRAF-6 mRNA的高表达(与模型组比较P0.05或P0.01),对TRAM及TRIF作用不明显。结论益气活血复方可阻断TLR4高表达,同时阻断TLR4胞内信号转导的MyD88依赖性途径,而对MyD88非依赖性途径作用不明显,因此益气活血复方主要是通过阻断MyD88依赖性途径来发挥其抗动脉粥样硬化的作用。     

Insulin receptor and insulin-responsive glucose transporter (GLUT 4) mutations and polymorphisms in a welsh type 2 (non-insulin-dependent) diabetic population

Summary We have recently examined the exons encoding the insulin receptor tyrosine kinase domain and GLUT 4 in 30 subjects with Type 2 (non-insulin-dependent) diabetes mellitus using a molecular scanning approach. The variant sequences Val-Met⁹⁸⁵ and Lys-Glu¹⁰⁶⁸ of the insulin receptor and Val-Ile³⁸³ of GLUT 4 were each separately found in three different diabetic subjects. In a study of a Welsh population, the GLUT 4³⁸³ variant was found in three of 160 diabetic and none of the 80 control subjects. In this study, the same group of Welsh Type 2 diabetic and control subjects was analysed using allele-specific oligonucleotide hybridisation, single nucleotide primer extension and allele-specific restriction digestion to ascertain the frequency of the two insulin receptor mutations. The Val-Met⁹⁸⁵ mutation was found in none of the 160 Welsh Caucasian Type 2 diabetic subjects and two of 80 control subjects. The Lys-Glu¹⁰⁶⁸ mutation removes a Sty 1 site and digestion of amplified exon 18 with Sty 1 confirmed the presence of this mutation in the heterozygous state in the original subject. None of the Welsh diabetic or control subjects had the Glu¹⁰⁶⁸ mutation. The discovery of a very common silent polymorphism at codon 130 of GLUT 4 allowed examination of the association of this locus with Type 2 diabetes using allele-specific oligonucleotide hybridisation in a subset of the Welsh subjects. The genotypic frequencies (homozygous wild-type and heterzygous polymorphic (poly) sequences) were not significantly different between diabetic and control subjects (Type 2 diabetic subjects: wild-type/wild-type 40%, wild-type/poly 46%, poly/poly 14%; Control subjects: wild-type/wild-type 37%0, wild-type/poly 45 %, poly/poly 18 %;p > 0.05). In conclusion, in a British Caucasian population the examined insulin receptor tyrosine kinase domain mutations are uncommon. Also the GLUT 4 locus does not appear to be strongly associated with Type 2 diabetes.     

Expression of CXC Chemokines Gro/KC and SDF-1a in Rat H4 Hepatoma Cells in Response to Different Stimuli

Background: It is now well established that several environmental stress factors cause activation of p38 MAP kinase and JNK in various cell types to produce chemokines. Objective: To investigate the expression of CXC chemokines Gro/KC and SDF- 1a in rat's H4 hepatoma cells in response to heat shock, hyperosmolarity and oxidative stress. Methods: Hepatoma cells were maintained in MEM medium. Cells were subjected to different stresses [(H2O2 0.15% (w/v), manitol and NaCl (160 mM) and heat shock (42 °C for 20 minutes)]. Cells were harvested and RNA was extracted, purified and the CXC chemokine Gro/KC and SDF-1a expression was analysed by RT-PCR. cDNA was separated by gel electrophoresis on a 1% (w/v) agarose gel and visualized under a UV transilluminator. Results: There was detectable but low expression of both SDF-1a and Gro/KC in H4 hepatoma cells. Heat shock failed to induce expression of SDF-1a and Gro/KC in H4 hepatoma cells of rat. Hyperosmolarity also did not stimulate SDF-1a and Gro/KC expression. In this study we have also shown that oxidative stress did not induce expression of SDF-1a and Gro/KC. Overall, although detection is possible but regulatory responses were not observed in H4 hepatoma cells. Conclusion: Several known injurious conditions cause recruitment of macrophages, neutrophils and other immune cells to the liver. Immune cells are recruited to the hepatic vasculature following local liver injury and subsequent chemokine production. Our results demonstrated that failure to produce chemokines by hepatoma cells may be a way to escape from mechanism of immune surveillance.     

Insulin resistance in Type 2 (non-insulin-dependent) diabetic patients and their relatives is not associated with a defect in the expression of the insulin-responsive glucose transporter (GLUT-4) gene in human skeletal muscle

Summary To study whether insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus is due to a defect in the expression of the insulin-responsive glucose transporter gene (GLUT-4) in human skeletal muscle, we measured the level of GLUT-4 mRNA and (in some of the subjects) its protein in muscle biopsies taken from 14 insulin-resistant patients with Type 2 diabetes, 10 first-degree relatives of the diabetic patients and 12 insulin-sensitive control subjects. Insulin sensitivity was measured with a +45 mU· ·min^–1 euglycaemic insulin clamp in combination with indirect calorimetry and infusion of [3-³H]glucose. GLUT-4 mRNA was measured using a human GLUT-4 cDNA probe and GLUT-4 protein with a polyclonal antibody specific for the 15 amino acid carboxyterminal peptide. Both Type 2 diabetic patients and their relatives showed impaired stimulation of total-body glucose disposal by insulin compared with control subjects (29.5±2.1 and 34.0±4.8 vs 57.9±3.1 mol·kg lean body mass^–1·min^–1; p<0.01). This impairment in glucose disposal was primarily accounted for by a reduction in insulin-stimulated storage of glucose as glycogen (13.0±2.4 and 15.6±3.9 vs 36.9±2.2 mol·kg lean body mass^–1·min^–1; p<0.01). The levels of GLUT-4 mRNA expressed both per g of total RNA and per g DNA, were higher in the diabetic patients compared with the control subjects (116±25 vs 53±10 pg/g RNA and 177±35 vs 112±29 pg/g DNA; p<0.05, p<0.01, respectively). The GLUT-4 mRNA levels in the relatives were not significantly different from that observed in the control subjects (90±16 pg/g RNA and 117±23 pg/g DNA; p = NS). The GLUT-4 protein levels did not significantly differ between control subjects, diabetic patients and relatives (494±85, 567±133 and 323±80 cpm/100 g protein). No correlation was observed between the level of GLUT-4 mRNA andits protein. However, the level of GLUT-4 mRNA and the rate of total-body glucose disposal correlated positively in the control group and in the relatives (both p<0.05) but not in the diabetic subjects. A positive correlation between the level of GLUT-4 protein and total-body glucose disposal was also observed in the control subjects (r = 0.759; p<0.05) and in the relatives (r = 0.794; p<0.01) but not in the diabetic subjects. We conclude that insulin resistance in Type 2 diabetes is not related to a defect in the expression of the GLUT-4 gene in skeletal muscle. Nevertheless, the levels of GLUT-4 mRNA and GLUT-4 protein are related to the rate of total-body glucose disposal in subjects with normal fasting glucose concentrations.