Chronic hypoxia enhances endothelin-1-induced intracellular calcium elevation in rat carotid body chemoreceptors and up-regulates ETA receptor expression

Endothelin-1 (ET-1) excites carotid body (CB) chemoreceptors and induces mitosis of the chemoreceptors in chronic hypoxia. The aim of the present study was to examine the hypothesis that up-regulation of both ETA receptor and endogenous ET-1 expression in CB chemoreceptors enhances the response of intracellular Ca2+ to ET-1 following adaptation to chronic hypoxia (10% inspired O2 for 3-4 weeks). Cytosolic free [Ca2+] ([Ca2+]i) in type-I (glomus) cells freshly dissociated from rat CBs was measured by spectrofluorometry. Application of exogenous ET-1 (1-100 nM) concentration-dependently elevated [Ca2+]i in the glomus cells. This response to ET-1 (100 nM) was 49% greater in the chronically hypoxic (CH) group. The ET-1 response was abolished completely by the ETA receptor antagonist BQ610 (1 microM), but not by the ETB antagonist BQ788 (1 microM). The transient [Ca2+]i elevation induced by caffeine (30 mM) in the normoxic group was similar to that in the CH group, suggesting no differences in the intracellular Ca2+ stores. In situ hybridization with a digoxigenin-labelled antisense ETA receptor mRNA oligonucleotide probe revealed very intense and ubiquitous specific expression of ETA receptors in the lobules of glomus cells in the CH group, whereas staining in normoxic controls was light. Immunohistochemical studies revealed intense cytoplasmic staining for ET-1-immunoreactivity in most of the cell clusters in glomera in the CBs of CH rats but was faint in normoxic CBs. These findings indicate increased expression of both the ETA receptor and ET-1 in CB chemoreceptors during chronic hypoxia. Taken together, our results suggest that the [Ca2+]i response to ET-1 in rat CB chemoreceptors is augmented by up-regulation of ETA receptors and ET-1 expression. The enhancement of the paracrine/autocrine effect of ET-1 on the chemoreceptors is consistent with an excitatory and mitogenic role of the ET-1 and ETA receptor in the CB during chronic hypoxia.     

A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury

Background: The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol‐induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. Methods: Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair‐fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa‐B (NF‐κB) and mRNA levels for tumor necrosis factor‐alpha (TNF‐α), cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. Results: All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol‐fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 ± 1.2 vs. 1.2 ± 0.4; necrosis (foci/mm²), 3.9 ± 2.3 vs. 0.4 ± 0.3; inflammation (cells/mm²), 19.0 ± 6.3 vs. 1.8 ± 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF‐κB and mRNA of pro‐inflammatory mediators (TNF‐α, COX‐2, iNOS) and procollagen‐I were increased (p < 0.05) in ethanol‐fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol‐fed rats. Conclusions: A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF‐κB and mRNA and protein levels of the pro‐inflammatory mediators TNF‐α, COX‐2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.     

BMP2,3,4,5 mRNA在三叉神经中的表达分析

目的：明确 Ｂ Ｍ Ｐｓ 与外周神经的关系。方法：用原位杂交方法观察 Ｂ Ｍ Ｐ２,３,４,５ 的ｍ Ｒ Ｎ Ａ在三叉神经中的表达。结果：在外周神经（三叉神经）中均有 Ｂ Ｍ Ｐ２,３,４,５ ｍ Ｒ Ｎ Ａ 分布, Ｂ Ｍ Ｐ２,３,４,５ 的分布主要位于构成神经纤维髓鞘的雪旺细胞中;此外发现 Ｂ Ｍ Ｐ２ 在部分神经纤维中也有表达。结论：首次确定了 Ｂ Ｍ Ｐ２,３,４,５ 在外周神经中的表达和分布,揭示了 Ｂ Ｍ Ｐ２,３,４,５可能在外周神经的发育和再生以及形态结构的维持中发挥着调控作用。     

细胞周期素依赖激酶4在颌骨软骨肉瘤的表达及意义

目的：分析细胞周期素依赖激酶４（ＣＤＫ４）在颌骨软骨肉瘤的表达及意义。方法利用免疫组化法检测ＣＤＫ４在２０例颌骨软骨肉瘤、８例骨软骨瘤和８例骨质增生中的表达。结果６５．０％（１３／２０）的软骨肉瘤中ＣＤＫ４呈阳性表达ＣＤＫ４表达率随病理学分级的升高呈增高趋势,但未见统计学差异（Ｐ〉０．０５）;ＣＤＫ４在骨软骨瘤和骨质增生中的阳性表达率分别为１２．５％（１／８）和０（０／８）,且均与软骨肉瘤有显性     

颌骨骨肉瘤中p~(27)的表达及意义

目的 :分析细胞周期素依赖激酶抑制物 (CKI)p2 7蛋白在颌骨骨肉瘤的表达及意义。方法 :采用免疫组织化学ABC法检测p2 7蛋白在 2 0例颌骨骨肉瘤和 8例骨软骨瘤中的表达。结果 :2 0 % (4 2 0 )的骨肉瘤中p2 7呈阳性表达 ,87 5 % (7 8)的骨软骨瘤呈p2 7阳性 ,阳性表达率与骨肉瘤相比有显著性差异 (p <0 0 5 )。结论 :p2 7在颌骨骨肉瘤中低表达并可作为骨肉瘤发生和发展过程中的一个抑癌基因标志物     

Macrophages, neutrophils and tumour necrosis factor-alpha expression in bronchiectatic airways in vivo

Bronchiectasis is increasingly being recognized as an inflammatory condition of the airways in which pathological permanent dilation occurs. We have obtained endobronchial biopsies in 14 patients with stable bronchiectasis and 15 control subjects. Airway neutrophils, macrophages and tumour necrosis factor-alpha (TNFalpha)-positive cells were stained with monoclonal antibodies and the densities of positive cells in the lamina propria were determined by using a computer image analyser. There was significantly higher neutrophil, macrophage and TNFalpha-positive cell densities in the lamina propria of bronchiectatic than control airways (P < 0.001, P < 0.001 and P=0.0002, respectively). Airway neutrophil density in bronchiectasis but not in controls, correlated with TNFalpha-positive cell density (r=0.71, P=0.004). A significant correlation between airway macrophage and TNFalpha-positive cell densities was demonstrated in both control and bronchiectatic airways (r=0.63, P=0.016 and r=0.60, P=0.02 respectively). Neutrophil density negatively correlated with per cent forced vital capacity (FVC%) predicted among patients with bronchiectasis (r=-0.53, P=0.04). Bronchiectasis patients who were regular sputum producers had a significantly higher macrophage, but not neutrophil density than their counterparts (P=0.02 and P=0.48 respectively). Our original findings suggest that airway macrophages could contribute to neutrophil influx into airway walls through their production of TNFalpha and therefore play an important role in the pathogenesis of bronchiectasis.     

Overexpression of matrix metalloproteinase-8 and -9 in bronchiectatic airways in vivo.

The progressive bronchial dilatation in bronchiectasis is likely to be the result of continued airway matrix destruction, although little is known about the role of neutrophil matrix metalloproteinases (MMPs) in this process. Immunohistochemistry has been used to investigate the expression and cellular localisation of MMP-8 and MMP-9 in bronchiectatic airways in vivo. Endobronchial biopsies were taken from 25 bronchiectatic patients, and from the right lower lobe in 14 control subjects. MMP-8, MMP-9, neutrophils and macrophages were stained with monoclonal antibodies and quantified as positive cell x mm(-2) of the lamina propria by using an image analysis system. There were significantly higher densities of MMP-8 and MMP-9 positive cells in the lamina propria of bronchiectatic than control airways. In bronchiectatic airways, the densities of MMP-8 and MMP-9 positive cells correlated with each other and with neutrophil density, but not with macrophage density. In control airways, a significant correlation was found between MMP-8 with neutrophil and MMP-9 with macrophage densities. An overexpression of neutrophil matrix metalloproteinases in bronchiectatic airways could help explain the continuation of airway destruction in bronchiectasis. In view of the clinical availability of matrix metalloproteinase antagonists, the results presented here could have a significant impact on the development of novel therapies of this untreatable disease.     

Green tea polyphenols as an anti-oxidant and anti-inflammatory agent for cardiovascular protection

Our review aims to examine the cellular and molecular mechanisms of cardiovascular protection of green tea polyphenols, particularly epigallocatechin gallate (EGCG), which focuses on the anti-oxidative and anti-inflammatory effects. EGCG is the major and the most active component in green tea. Studies have shown that EGCG protects cellular damage by inhibiting DNA damage and oxidation of LDL. One of the protective properties of EGCG is its ability to scavenge free radicals. EGCG can also reduce the inflammatory response associated with local tissue injuries such as the hepatocellular necrosis in acute liver injury induced by carbon tetrachloride. The protective effect of EGCG is due to its ability to decrease lipid peroxidation, oxidative stress and the production of nitric oxide (NO) radicals by inhibiting the expression of iNOS. EGCG also ameliorates the overproduction of pro-inflammatory cytokines and mediators, reduces the activity of NF-kappaB and AP-1 and the subsequent formation of peroxynitrite with NO and reactive oxygen species. Thus, EGCG effectively mitigates cellular damage by lowering the inflammatory reaction and reducing the lipid peroxidation and NO generated radicals leading to the oxidative stress. Green tea is proposed to be a dietary supplement in the prevention of cardiovascular diseases in which oxidative stress and proinflammation are the principal causes.