Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle

C. A. Martel; L. K. Mamedova; J. E. Minton; M. Garcia; C. Legallet; B. J. Bradford

摘要

Background: Associative data and some controlled studies suggest that the inflammatory cytokine tumor necrosis factor (TNF) α can induce fatty liver in dairy cattle. However, research demonstrating that TNFα is a necessary component in the etiology of bovine fatty liver is lacking. The aim of this work was to evaluate whether blocking TNFα signaling with a synthetic cyclic peptide (TNF receptor loop peptide; TRLP) would improve liver metabolic function and reduce triglyceride accumulation during feed restriction.Results: Capability of TRLP to inhibit TNFα signaling was confirmed on primary bovine hepatocytes treated with recombinant bovine TNFα and 4 doses of TRLP (0, 1, 10, 50 μmol/L) over 24 h. Next, 4 lactating Holstein cows (parity 1. 4 ± 0. 5, 433 ± 131 d in milk) in an incomplete Latin rectangle design (3 × 2) were subcutaneously administered with different TRLP doses (0, 1. 5, 3. 0 mg/kg BW) every 4 h for 24 h, followed by an intravenous injection of TNFα (5 μg/kg BW). Before and for 2 h after TNFα injection, TRLP decreased plasma non-esterified fatty acid (NEFA) concentration (P ≤ 0. 05), suggesting an altered metabolic response to inflammation. Finally, 10 non-pregnant, nonlactating Holstein cows (3. 9 ± 1. 1 yr of age) were randomly assigned to treatments: control (carrier: 57％ DMSO in PBS) or TRLP (1. 75 mg TRLP /kg BW per day). Treatments were administrated every 4 h for 7 d by subcutaneous injection to feed-restricted cows fed 30％ of maintenance energy requirements. Daily blood samples were analyzed for glucose, insulin, β-hydroxybutyrate, NEFA, and haptoglobin concentrations, with no treatment effects detected. On d 7, cows completed a glucose tolerance test (GTT) by i. v. administration of a dextrose bolus (300 mg glucose/kg BW). Glucose, insulin, and NEFA responses failed to demonstrate any significant effect of treatment during the GTT. However, plasma and liver analyses were not indicative of dramatic lipolysis or hepatic lipidosis, suggesting that the feed restriction protocol failed to induce the metabolic state of interest. Injection site inflammation, assessed by a scorer blinded to treatment, was enhanced by TRLP compared to control.Conclusions: Although the TRLP inhibited bovine TNFα signaling and altered responses to i. v. administration of TNFα, repeated use over 7 d caused apparent local allergic responses and it failed to alter metabolism during a feed restriction-induced negative energy balance. Although responses to feed restriction seemed atypical in this study, side effects of TRLP argue against its future use as a tool for investigating the role of inflammation in metabolic impacts of negative energy balance.

机译：背景：相关数据和一些对照研究表明，炎性细胞因子肿瘤坏死因子（TNF）α可以诱导奶牛脂肪肝。然而，缺乏证明TNFα是牛脂肪肝病因中必需成分的研究。这项工作的目的是评估用合成环肽（TNF受体环肽; TRLP）阻断TNFα信号传导是否可以改善饲料限制期间的肝脏代谢功能并减少甘油三酸酯的积累。结果：证实了TRLP抑制TNFα信号传导的能力在24小时内，用重组牛TNFα和4剂量的TRLP（0、1、10、50μmol/ L）处理了原代牛肝细胞。接下来，以不同的TRLP剂量（0、1、5、3、3）皮下注射4头不完全拉丁矩形设计（3×2）的泌乳荷斯坦奶牛（胎力为1. 4±0. 5，5，433±131 d）。每4小时0 mg / kg BW），然后静脉注射TNFα（5μg/ kg BW）。注射TNFα之前和之后2小时，TRLP会降低血浆非酯化脂肪酸（NEFA）浓度（P≤0. 05），表明对炎症的代谢反应发生了改变。最后，将10头未怀孕，不哺乳的荷斯坦奶牛（3. 9±1. 1岁）随机分配至以下处理组：对照（携带者：PBS中57％DMSO）或TRLP（每公斤体重1.75 mg TRLP / kg体重）天）。通过皮下注射，每4 h进行一次治疗，持续7 d，以饲喂限制饲喂30％维持能量需求的奶牛。分析每日血液样本中的葡萄糖，胰岛素，β-羟基丁酸酯，NEFA和触珠蛋白浓度，未发现治疗效果。在第7天，母牛通过i完成了葡萄糖耐量测试（GTT）。 v。给予右旋糖丸（300mg葡萄糖/ kg BW）。葡萄糖，胰岛素和NEFA反应未能证明在GTT期间任何显着的治疗作用。但是，血浆和肝脏分析未表明剧烈的脂解或肝脂质增生，提示进食限制方案无法诱导目标代谢状态。与对照组相比，由不知情的计分员评估的注射部位炎症通过TRLP得以增强。结论：尽管TRLP抑制牛TNFα信号传导并改变了对i的反应。 v。TNFα的施用，重复使用超过7天引起明显的局部过敏反应，并且在饲料限制引起的负能量平衡期间未能改变新陈代谢。尽管在本研究中对饮食限制的反应似乎是非典型的，但TRLP的副作用反对其将来用作研究炎症在负能量平衡的代谢影响中的作用的工具。

Effects of TNF receptor blockade on in vitro cell survival and response to negative energy balance in dairy cattle

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