Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepal-6 cells and its relationship with collagen type Ⅳ

AIM: To investigate the regulation of activin receptor-interacting protein 2 (ARIP2) expression and its possible relationships with collagen type Ⅳ (collagen Ⅳ) in mouse hepatoma cell line Hepal-6 cells.METHODS: The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate (PMA), forskolin and A23187. After pcDNA3-ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor (ActRII)and collagen Ⅳ expression were evaluated.RESULTS: The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation (2 or 4 h). TheARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 (25.3% ± 5.7% vS 48.1% ± 3.6%, P ＜ 0.01). ARIP2 overexpression could remarkably suppress the expression of ActRIIA mRNA in dose-dependent manner, but has no effect on ActRIIB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells.CONCLUSION: These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.     

内毒素血症时肝窦内皮细胞脂多糖受体CD14蛋白表达的观察

目的观察内毒素血症时肝窦内皮细胞(LSECs)中CD14蛋白合成和CD14 基因的表达,以及CD14蛋白在内毒素介导LSECs激活中的作用. 方法经尾静脉注入脂多糖(LPS,E coli O111B4)5mg/kg,建立大鼠内毒素血症动物模型,分别于术后0(对照组)、3、6、12、24h活杀取材.用兔抗鼠CD14抗体和异硫氢酸荧光素(FITC)标记的羊抗兔IgG对LSECs进行孵育后,流式细胞仪测定LSECs的平均荧光强度(MFI)及FITC阳性细胞数;用原位杂交法测定LSEC中CD14 mRNA的表达.用原位胶原酶灌注法分离大鼠LSECs,用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.并用CD14抗体阻断LSECs的 CD14蛋白后,再用不同浓度LPS(0、0.01、1、10、100μg/ml)刺激LSECs.测定LPS介导LSECs肿瘤坏死因子(TNF)-α及白细胞介素-6(IL-6)分泌及CD14抗体对LSECs细胞因子分泌的影响. 结果内毒素血症大鼠3、6、12和24h 时LSECs的MFI明显增加;FITC阳性细胞数也明显增多,分别为54.32%、65.83%、85.61%和45.65%,与对照组的4.45%比较差异有非常显著意义(P＜0.01).原位杂交显示,内毒素血症大鼠LSECs中CD14 mRNA的表达明显增强,而对照组CD14 mRMA无阳性表达.LPS组TNF-α的含量(pg/ml)分别为54.49±6.02、84.65±10.16、206.54±23.55、349.87±39.47和365.76±40.31;CD14阻断组TNF-α的含量(pg/ml)分别为55.93±6.95、63.32±7.81、85.34±9.72、112.75±13.54、198.66±21.54;两组间比较差异有非常显著意义(P＜0.01).LPS组IL-6的含量(pg/ml)分别为103.34±12.52、187.39±20.31、243.87±27.83、289.51±30.15、298.53±31.94;CD14阻断组IL-6的含量(pg/ml)分别为104.37±11.49、125.02±13.58、164.59±19.47、183.47±20.17、221.76±26.43;两组间比较差异有非常显著意义(P＜0.01). 结论内毒素血症时LSECs能合成CD14蛋白及表达CD14基因;抗CD14抗体对LPS诱导LSECs TNF-α和IL-6的分泌有抑制作用;CD14蛋白的表达在内毒素介导LSECs激活中可能起重要作用.     

Evaluation of serum zonulin level in prediabetic patients

BackgroundThis study aimed to investigate the relationship between lipopolysaccharide (LPS) and zonulin levels and also to show the effect of acute hyperglycemic stress induced by oral glucose tolerance testing (OGTT) on zonulin levels in pre-diabetic patients.MethodsFour groups were constituted according to the criteria of the American Diabetes Association (ADA), based on OGTT results: control group (n:40); prediabetic group (n:56), divided into two subgroups: impaired fasting glucose group (IFG) (n:36), and impaired glucose tolerance (IGT) + IFG group (n:20) and type-2 diabetes mellitus (T2DM) group (n:45).ResultsZonulin and LPS did not significantly differ between the prediabetes and control groups, but were significantly higher in the T2DM group compared to both the prediabetic and the control group (P < 0.001). After OGTT, zonulin and LPS were significantly higher in the prediabetes group compared to the control group (P < 0.01 and P < 0.05, respectively), and significantly lower in the IFG and IFG + IGT groups compared to the T2DM group (P < 0.001, P < 0.001 and P < 0.001, P < 0.001, respectively). A positive correlation was detected between fasting zonulin and 2-hour zonulin (r = 0.727, P < 0.001) and between fasting LPS (r = 0.555, P < 0.001) and 2-hour LPS (r = 0.567, P < 0.001) in the prediabetic group. Increased zonulin and LPS levels and the positive correlation between these levels during the prediabetic period although non significant suggests onset of intestinal permeability.ConclusionsDuring acute hyperglycemia in prediabetic patients, up-regulation of zonulin and LPS may affect intestinal function. The intestines may play a key role in up-regulation of glucose and the pathogenesis of diabetes.     

脂多糖和甲双吡丙酮诱发形成豚鼠运动性哮喘模型

目的建立一种接近临床特点的运动性哮喘动物模型。方法豚鼠２７只分４组。实验组（Ａ组）用脂多糖（１ｍｇ／ｋｇ）和甲双吡丙酮（５０ｍｇ／ｋｇ）腹腔注射，４天后测定气道阻力和动态肺顺应性基础值，８小时后进行运动试验并复测上述指标。对照组有３组（Ｂ、Ｃ、Ｄ组）；分别为腹腔注射脂多糖和甲双吡丙酮不运动组、腹腔注射生理盐水运动组和腹腔注射生理盐水不运动组。结果实验组豚鼠在运动后肺阻力增高、动态肺顺应性降低，而３个对照组上述指标变化均无统计学意义。结论脂多糖和甲双吡丙酮腹腔注射可诱发形成豚鼠运动性哮喘模型     

The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide北大核心CSCD

Objective To explore the effect of expression of protein kinase C receptor l(RACKl) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC). Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Annui province. Using immunocytochemistry method, the expression of RACK 1 protein in RPMVECs was detected, cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group, SAG(smoothened Agonist, a SHH signaling pathway specific agonist) dose-dependent group, LPS time-dependent group, SAG time-dependent group and LPS+SAG group. In LPS dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 mg/L LPS for 8 h. In LPS time-dependent groups, RPMVECs were cultured with 10 mg/L LPS for 0, 2, 4, 8, 12, 24 h. In SAG dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 u, mol/L for 8 h. In SAG time-dependent groups, RPMVECs were cultured with 1 u, mol/L SAG for 0, 2, 4, 8, 12, 24 h. In LPS+SAG group, RPMVECs were cultured with 1 u. mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h. In addition, blank group, LPS group and SAG group were set for control. Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention. Results Immunocytochemistry revealed that RACK1 were present in RPMVEC. 1. In LPS dose-dependent groups (0, 0.1, 1, 10 mg/L), the level of RACK 1 elevated as LPS dose increased correspondingly with inter-group difference (P<0.05); the relative expression levels of GLI-1 mRNA were (1.109 ± 0.063), (1.039 ± 0.135), (0.813 ± 0.066), (0.770 ± 0.105), (1 mg/L vs. 10 mg/L, P>0.05; the rest P<0.05). In LPS time-dependent groups, the relative expression level of RACK1 at 2 h (0.370 ± 0.010) was higher than that at 0 h (0.329 ± 0.008), peaked at 12 h (1.296 ± 0.048), and compared with 0 h, there was significant differences (F=l 272.204, P<0.05). The relative expression level of GLI-1 mRNA was decreased at 2 h (0.929 ± 0.007), and compared with 0 h(1.089 ± 0.042), there was significant differences (F=306.609, /><0.05). 2. In SAG dose-dependent groups, there was no significant difference in level of RACK1 between groups(all P>0.05). The relative expression levels of GLI-1 mRNA were (1.109 ± 0.063), (1.169±0.052), (3.468 ±0.128), (3.434±0.054), (0 μ.mol/L vs. 0.1 μ.mol/L and l μmol/L vs. 10 μ.mol/L, P>0.05, the rest P<0.05). Among SAG time-dependent groups, there was no significant difference in levels of RACK1 protein(P>0.05). The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065), and compared with 0 h (2.651 ± 0.123), there was significant differences (F= 132.841, P<0.05). 3. In LPS+SAG intervention groups, the expression of RACK1 was lower than that in LPS group (0.831 ± 0.040 vs. 1.189 ± 0.149, P<0.05), and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 ± 0.130 vs. 0.796 ± 0.082, P<0.05). Conclusions The LPS up-regulates the expression of RACK 1 in RPMVECs, and the activated SHH signaling pathway can down-regulate the expression of RACK 1 induced by LPS in RPMVECs. © 2018 Chinese Medical Association. All rights reserved.     

红景天苷对脂多糖诱导的小鼠心肌损伤保护作用的研究

目的:研究红景天苷对脂多糖诱导的小鼠心肌损伤的保护作用及其机制。方法:选取雄性Balb/c小鼠50只,随机分为对照组、模型组、地塞米松组、红景天苷低、高剂量组。地塞米松和红景天苷高、低剂量连续灌胃7 d,每天1次,最后一次灌胃后1 h,除对照组外,其余各组腹腔注射LPS,12 h后处死,取血和心脏。结果:红景天苷组小鼠心肌病理改变明显减轻于模型组;相比于模型组,红景天苷组能减少血清中TNF-α、IL-6的含量;减少心肌组织中TLR4、My D88和NF-κBp65蛋白的表达。结论:红景天苷减轻脂多糖诱导的小鼠心肌损伤,其机制可能通过调节TLR4/My D88/NF-κBp65信号通路,抑制炎症反应。     

Effect of Prostaglandin E Receptor Subtype EP4 Selective Agonist on the Secretion of Tumor Necrosis Factor-α by Macrophages in Acute Ethanol-Loaded Rats

Background: It is suggested that endotoxin and proinflammatory cytokines play an important role in the development and progression of alcoholic liver disease. Recently, a prostaglandin receptor subtype EP4 agonist with cytoprotective effect has been developed. We examined the efficacy of an EP4 agonist ONO-AE1-437 on tumor necrosis factor-α (TNF-α) secretion of Kupffer cells, splenic macrophages, and alveolar macrophages in acute ethanol-loaded rats.
Methods: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from control and acute ethanol-loaded rats (5 mg/g body weight of ethanol, intraperitoneally). After the preculture in the medium that containing 0, 0.1, 1, 10, or 100 nmol/liter of ONO-AE1-437, TNF-α secretion of these cells stimulated by 100 ng/ml of endotoxin was determined for 3 hr.
Results: The amount of TNF-α secreted from alveolar macrophages was largest in both the control and the acute ethanol-loaded rats. Acute ethanol load enhances TNF-α secretion of splenic macrophages. The addition of ONO-AE1-437 significantly inhibited TNF-α secretion of Kupffer cells and splenic macrophages in both the control and the acute ethanol-loaded rats. Alveolar macrophages were less affected.
Conclusions: An EP4 agonist ONO-AE1-437 suppresses excess TNF-α secretion from macrophages and seems promising for future trial in patients with severe alcoholic hepatitis.     

Cytokine-induced nitric oxide inhibits mitochondrial energy production and induces myocardial dysfunction in endotoxin-treated rat hearts

The mechanism responsible for cardiac depression in septic shock remains unknown. The present study examined whether nitric oxide (NO) overproduced by inducible NO synthase (iNOS) can inhibit aerobic energy metabolism and impair the myocardial function in endotoxin-treated rat hearts. Lipopolysaccharide (LPS) significantly decreased systolic blood pressure (BP) to 44% of control during the 48 h treatment. Hearts from control and LPS-treated rats were perfused in a Langendorff apparatus. After LPS injection, left ventricular (LV) developed pressure (LVDP) was significantly depressed, plasma NO2-/NO3- (NO(x)) concentration was markedly increased, and myocardial adenosine 5'-triphosphate (ATP), creatine phosphate (CrP), and the ratio of ATP/adenosine 5'-diphosphate were progressively decreased with time. Immunological examination showed a significant expression of iNOS protein in the LPS-treated myocytes. Aminoguanidine, an inhibitor of iNOS, significantly attenuated these LPS-induced functional and metabolic changes. Myocardial cyclic guanosine 3',5'-monophosphate (cGMP) content was significantly increased after LPS injection. Methylene blue, an inhibitor of soluble guanylate cyclase, blunted this increase in cGMP and significantly restored the LPS-induced contractile dysfunction 6 h after LPS injection. In addition, there was a significant negative correlation between LVDP and myocardial cGMP levels as well as a significant negative correlation between LVDP and plasma NO(x) levels. In contrast, 48 h after LPS injection, methylene blue no longer affected cardiac performance, and there was a significant positive correlation between LVDP and myocardial ATP content. Furthermore, the normalized activities (as a ratio of the citrate synthase activity) of mitochondrial NADH-CoQ reductase, succinate-CoQ reductase, and ATPase, were significantly inhibited, and the swelling or disruption of mitochondria cristae was seen in the 48 h LPS treatment. These LPS-induced functional and morphological disorders in the mitochondria were significantly improved by aminoguanidine. The findings suggest that sustained production of NO by iNOS leads to contractile dysfunction via cGMP in the early stage, but that it can directly impair the mitochondrial function, lower myocardial energy production, and contribute significantly to the myocardial dysfunction in the later stage of septic shock.     

Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.