Effects of prenatal exposure to alcohol on the release of adenocorticotropic hormone, corticosterone, and proinflammatory cytokines

Prenatal alcohol exposure has been shown to produce hyperresponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis to immune challenges. Because cytokines, which are released in response to immune challenges, are known to activate the HPA axis, this study determined whether altered release of cytokines contribute to the HPA hyperresponsiveness to immune challenges observed after prenatal alcohol exposure. Pregnant dams were exposed to alcohol vapors (6-7 hr daily) between days 7 and 18 of gestation. At postnatal days 45 and 60, control (C) and prenatal alcohol-exposed (E) offspring were subjected to three different types of immune challenges: injections of interleukin-1beta or endotoxin (lipopolysaccharide), or turpentine-induced tissue injury. We observed the expected higher plasma adrenocorticotropic hormone and corticosterone levels in E compared with C rats, and this HPA hyperresponsiveness was greater in E females compared with E males. Plasma tumor necrosis factor-alpha or interleukin-6 responses were comparable in the C and E groups. Females exhibited significantly higher corticosterone, tumor necrosis factor-alpha, and interleukin-6 responses than males. These results indicate that (1) prenatal alcohol exposure produces HPA hyperresponsiveness to immune challenges; (2) prenatal alcohol treatment does not influence the release of cytokines to immune challenges; and (3) there are gender differences in the secretory pattern of corticosterone and cytokines to immune challenges. Therefore, these data do not support the hypothesis that cytokines play a role in the hyperresponsiveness of the HPA axis to immune challenges observed after prenatal alcohol exposure.     

Suppression of Tumor Necrosis Factor Production by Alcohol in Lipopolysaccharide-Stimulated Culture

Many studies have shown that alcohol consumption is associated with alteration in immune responses and increased incidence of infection in the host. Tumor necrosis factor (TNF) is a potent soluble mediator of immunoregulation and inflammation, and plays a very important role in host's defenses against infection and tumor. We propose that one of the mechanisms of alcohol-mediated immunosuppression may be due to a defect in the synthesis and release of the TNF. To determine this, we studied the direct effect of alcohol on lipopolysaccharide (LPS)-induced TNF production by whole blood and total mononuclear cell from normal subjects. Aliquots of blood samples (1 ml) or ficollhypaque separated total mononuclear cells (1 × 10⁶/ml) were cultured with different concentrations of either ethanol or acetaldehyde in the presence or absence of LPS for 4 hr at 37°C. Plasma samples and culture supernatants were assayed for TNF levels in a bioassay using a TNF-sensitive WEHl 164 sub-clone 13 cell line. LPS at 10 μg/ml produced a maximal level of TNF compared with lower (1 μg/ml) or higher concentration (50 μg/ml) of LPS. Kinetics studies showed that an incubation time of 4 hr with LPS produced a maximum level of TNF production by blood. Alcohol, as low as 0.1% concentration, produced significant suppression of LPS-inducted TNF production by whole blood, whereas alcohol at 0.2 and 0.3% concentrations were required to produce a significant suppression of TNF production by separated mononuclear cells. Anti-TNF-α antibodies significantly neutralized the LPS-induced TNF that suggests that blood monocytes may be the primary source of TNF production. Further, significant correlation between TNF production and monocyte numbers was observed. Acetaldehyde one of the primary metabolites of alcohol, did not suppress the LPS-induced TNF production by whole blood. These studies suggest that alcohol-induced inhibition of TNF may be one of the mechanisms for immunosuppression in alcoholic patients.     

大鼠肝纤维化模型肝细胞凋亡及肝细胞坏死的动态分析

目的　观察肝细胞凋亡和肝细胞坏死在肝纤维化形成过程中的变化 ,探讨其在肝纤维化发病机制中的作用。方法　雄性Wistar大鼠 32只 ( 85～ 95克 )随机分为 4组。A ,B ,C为实验组 ,采用腹腔注射CCl4 的方法造模 ;D为对照组 ,代之以生理盐水。于 2 ,4,6周末分别处死A ,B ,C三组大鼠 ,D组同期处理。取肝脏石蜡包埋 ,连续切片做病理学检查、细胞凋亡原位检测。测定血清ALT。结果　病理学检查A ,B ,C ,D四组分别符合肝纤维化的S1,S3 ,S4 ,S0 期。各实验组 (A ,B ,C)的肝细胞坏死记分值均高于对照组 (D) ( 1 6 7± 0 82 ) ,( 2 83± 0 75 ) ,( 2 5 0± 1 0 5 )vs( 0 16± 0 4 1) ,(P <0 0 1) ,肝细胞凋亡级数也均高于对照组 (D) ( 2 5 0± 0 5 5 ) ,( 2 83± 0 4 1) ,( 2 0 0± 0 6 3)vs( 0 33± 0 5 2 ) ,(P <0 0 1)。各实验组 (A ,B ,C)的ALT均高于对照组 (D) ( 76 83± 2 1 87) ,( 117 0 0±15 94) ,( 10 3 6 7± 36 6 8)vs( 39 16± 2 32 ) ,(P <0 0 5 )。结论　肝细胞凋亡和肝细胞坏死在肝纤维化发生过程中存在动态变化 ,二者在其不同时期发挥的作用不同。     

酒精性肝硬化患者血清肿瘤坏死因子受体的检测及其临床意义

同特异性免疫测定ELISA法检测了32例酒精性肝硬化患者和10名健康人血清中肿瘤坏死因子受体的水平。酒精性肝硬化患者两种可溶性肿瘤坏死因子受体(P55、P75)均明显高于健康组(P<0.01),肝硬化代偿期与失代偿期患者比较有显著差异(P值分别<0.005,<0.01)。这些结果提示循环中可溶性肿瘤坏死因子受体(STNFR)的水平与肝硬化和疾病的进展程度呈正相关。     

-238 G>A polymorphism of tumor necrosis factor alpha gene (TNFA) is associated with alcoholic liver cirrhosis in alcoholic Spanish men

BACKGROUND: The tumor necrosis factor alpha gene (TNFA) has been recently associated to alcoholic steatohepatitis. We have analyzed the distribution of genotypes and alleles of two polymorphisms at positions -238 and -308 in the promoter region of the TNFA gene in a Spanish male population of alcoholics with and without alcoholic liver cirrhosis. METHODS: 149 male alcoholics (84 without alcoholic liver disease, and 65 with alcoholic liver cirrhosis) and 90 control subjects were included. Genotyping was done by polymerase chain reaction and digestion with restriction enzymes. RESULTS: No significant differences in the distribution of genotypes and alleles of the -308 TNFA gene polymorphism were observed between alcoholics and non-alcoholics, or between alcoholics with liver cirrhosis and those without liver disease. However, we found an association between the -238 TNFA polymorphism and alcoholic liver cirrhosis; the frequency of the heterozygous genotype being significantly higher in alcoholics with cirrhosis than in those without liver damage. CONCLUSION: The -238 TNFA-A allele is associated with a higher risk to develop alcoholic liver cirrhosis. This polymorphism could be considered as a genetic factors that confer predisposition to suffer liver cirrhosis in the alcoholic population of Castile and León.     

Hepatic microvascular dysfunction in endotoxemic rats after acute ethanol administration

BACKGROUND: A high concentration of ethanol is reported to cause hepatic microvascular dysfunction. However, little is known about the effect of ethanol on hepatic microcirculation in endotoxemic animals. The objective of this study was to determine whether endotoxemia enhances the hepatic microvascular dysfunction induced by acute ethanol administration. METHODS: Intravital videomicroscopy was used to monitor leukocyte recruitment, number of nonperfused sinusoids, and flow velocity of erythrocytes (RBC) labeled with fluorescein isothiocyanate (FITC) in the livers of male Wistar rats that were administered ethanol (20%, 3 g/kg; or 40%, 6 g/kg) from a gastric tube. Flow velocity of RBC in sinusoids was measured with an off-line velocimeter. Plasma tumor necrosis factor (TNF)-alpha levels were also measured. In some experiments, rats were injected with 2 mg/kg of lipopolysaccharides (LPS) intraperitoneally at 16 hr before the experiments, and the same protocol was performed. RESULTS: Although FITC-RBC velocity was initially increased by both 20% and 40% ethanol in control rats, it was reduced by only 40% ethanol at 60 min. In LPS-treated rats, the FITC-RBC velocity also was increased initially but was reduced at 60 and 30 min by 20% and 40% ethanol, respectively. Only 40% ethanol caused leukostasis in the pericentral region of control rats. In LPS-treated rats, however, leukostasis was noted in the midzonal and pericentral regions of liver after both 20% and 40% ethanol administration. Ethanol increased plasma TNF-alpha levels only in LPS-treated rats. CONCLUSIONS: These results suggest that LPS synergistically enhances ethanol-induced hepatic microvascular dysfunction and liver injury, especially in the midzonal region via coagulation, which may be mediated by TNF-alpha.     

慢性乙醇中毒所致大鼠肝损伤和肝细胞凋亡

目的:研究肝细胞凋亡在实验性大鼠乙醇性肝病(ALD) 发生、发展中的作用．方法:给大鼠饮用220 g／L的乙醇,并结合540 g／L乙醇分次、少量灌胃的方法建立大鼠肝损伤模型;常规HE 染色,光镜观察大鼠病理学形态改变,用TUNEL法检测肝细胞凋亡,速率法检测血清ALT,AST水平．结果:灌乙醇5 wk大鼠肝脏出现轻到中度脂肪变性,脂滴为以大泡型为主的混合脂滴,脂变率为40％(8／20); 10 wk,85％(17／20)大鼠发生肝脂肪变,45％(9／20)大鼠出现乙醇性肝炎的病理变化．5,10 wk大鼠血清 ALT,AST均较同期对照组有显著升高(5 wk:1017 ±267 VS 550±133,P<0．05;1350±333 vs 967±150, P<0．05;10 wk:1500±267 vs 767±250．P<0．05;2167 ±533 vs 850±183,P<0．05);灌乙醇10 wk血清ALT, AST较5 wk升高(P<0．05),有统计学意义．5,10 wk大鼠 TUNEL指数分别为0．33±0．49％、2．03±1．61％,与同期对照组相比,5 wk无显著差异,10 wk有显著性差异 (P<0．05);灌乙醇10 wk TUNEL指数较5 wk高,有显著性差异(P<0．05)．按病理损伤程度,将实验组分成乙醇性肝炎(AH)组和乙醇性脂肪肝(AFL)组,结果显示AH 组TUNEL指数也较AFL组高(3．24±1．50％ vs 1．12± 0．63％,P<0．05),差异显著．结论:过量饮用乙醇可以引起中毒性肝脏疾病,饮用乙醇及持续时间与肝损伤的发生有密切关系．细胞凋亡在乙醇诱发肝损伤时可能起着重要作用．     

克罗恩病患者促炎性细胞因子肿瘤环死因子—α和白细胞介素—6促炎作用的研究

背景：近年来许多研究证明肿瘤坏死因子(TNF)—α和白细胞介素(IL)—6在炎症的发生中起重要作用。目的：研究克罗恩病(CD)患者血清TNF—α和IL—6水平的变化，探讨它们在CD发病中的作用。方法：以健康成人作为对照，用酶联免疫吸附测定(ELISA)方法测定CD活动期和缓解期患者的血清TNF—α和IL—6水平，同时测定血沉(ESR)、血小板计数和C反应蛋白(CRP)。结果：活动期CD患者的血清TNF—α和IL—6水平均显著高于缓解期患者和健康成人(TNF—αP＜0．05，IL—6：P＜0．01)，与ESR、血小板计数和CRP的变化一致。结论：TNF—α和IL—6在CD患者的炎症发生中起重要作用，可作为CD活动期的新标志物。     

大鼠内毒素性急性肝损伤后肝细胞凋亡与炎性因子的表达

目的 探讨脂多糖诱导D-氨基半乳糖胺致敏大鼠急性肝损伤肝细胞凋亡、炎性因子表达情况及其发生机制.方法 56只大鼠分为0 h对照组与1、2、4、6、24和48 h脂多糖+D-氨基半乳糖胺处理组.在相应时间点处死大鼠后收集肝组织及血清,肝组织苏木精-伊红染色后光学显微镜下观察ELISA法检测血清细胞因子表达;反转录(RT)-PCR法检测TNF-α、IL-β、诱导型一氧化氮合酶(iNOS)和p53基因表达;收集24 h肝组织用底物显色法检测Caspase-3、8、9,12活性.组间比较用方差分析.结果 经药物处理后,肝组织出现碎片状坏死、大量炎性细胞浸润等表现,从6 h开始,24 h和48 h显著加重.血清TNF-α浓度在1 h处理组为(727.8±261.3)ng/L,显著高于对照组及其他处理组(F=49.82,P<0.01),2 h处理组为(156.4±52.2)ng/L,显著低于1 h组,但高于对照组(F:30.23,P<0.01);血清IL-β浓度逐渐上升,24 h处理组最高,为(360.5±121.6)ng/L(F=18.61,P<0.01).24 h处理组肝组织Caspase-3、8、9、12活性明显高于对照组(F=84.96,P<0.01).iNOS基因在对照组无表达,药物作用后6 h达最高,24 h和48 h则显著下降(F=34.07,P<0.01);p53基因在24 h和48 h处理组表达明显增高(F=37.43,P<0.01);TNF-α和IL-1β基因表达均较对照组升高(F=2.94,P<0.05),其峰值均出现在1 h处理组.结论 小剂量脂多糖可诱导D-氨基半乳糖胺致敏大鼠发生急性肝损伤;Caspase-3、8、9、12活性明显增强是其特征性改变之一;肝损伤的发生与TNF-α、iNOS和p53基因早期高水平表达有密切关系.