大豆异黄酮干预肥胖大鼠肝氧化应激及炎症反应

本研究旨在探究不同剂量大豆异黄酮(soy isoflavones,SIF)干预肥胖大鼠后肝的氧化应激及炎症反应情况。准备80只SD大鼠,普通饲料喂养16只SD大鼠做空白对照;高脂饲料喂养64只SD大鼠9周建立肥胖大鼠模型,将64只模型大鼠随机分成4组,每组16只,大豆异黄酮(0、150、300、450 mg·kg^-1)灌喂,每周定期测量体重,连续干预4周。取肝脏组织,苏木素-伊红(Hematoxylin-Eosin,HE)染色观察肝脏组织形态学结构;检测肝脏组织中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性;荧光定量PCR方法检测肝脏组织中促炎因子白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达。组织病理学发现,肥胖大鼠出现典型的肝细胞脂肪变性伴肝组织炎性损伤,肝SOD、CAT和GSH-Px水平降低,IL-6、IL-1β和TNF-α mRNA表达水平升高。大豆异黄酮干预后,各剂量组肝组织病理损伤得到改善,抗氧化因子活性显著提高并呈剂量效应,促炎因子表达水平显著下调。大豆异黄酮可逆转肥胖致肝组织病理损伤和提高肝脏抗氧化活性,下调肥胖相关促炎因子基因的表达,上述结果可为大豆异黄酮的应用开发提供参考。     

Dietary high protein-induced diarrhea and intestinal inflammation by activation of NF-κB signaling in piglets

The present study aimed to investigate whether inflammation-associated responses in piglets are induced by high protein (HP) through activating nuclear factor kappa B (NF-κB) signaling. Sixteen piglets (35 d of age, Duroc × [Landrace × Yorkshire], weaned at d 21, initial BW = 9.70 ± 0.11 kg) were allocated to 18% and 26% CP (HP group) at random, comprising 8 replicate pens per treatment. The piglets were slaughtered to collect intestinal tissues when apparent, persistent, and stable diarrhea syndromes happened (on d 12). No significant differences were observed in their growth performance (P > 0.05), but reduction by 19.11%, 25.31%, 23.64% of ADFI, ADG, and G:F, respectively was detected in the HP group. The HP group had greater (P = 0.002) diarrhea rates. Furthermore, dietary HP had lower ileal villus height (VH; P = 0.048), ratio of villus height to crypt depth (VH/CD ratio; P = 0.016), and colonic CD (P = 0.034), as well as had the trend (P = 0.075) to reduce the ileal villus absorptive area. Moreover, HP diets significantly elevated the goblet cell numbers in the ileal villi (P = 0.016) and colonic crypts (P < 0.001) and up-regulated (P = 0.012) the mRNA expression of mucin2 (Muc2) in the ileum. In addition, HP diets increased the myeloperoxidase concentration in the ileum (P = 0.002) and colon (P = 0.007) of piglets. Dietary HP significantly down-regulated the mRNA expression of tumor necrosis factor-α (TNF-α; P < 0.001) in the ileum, induced nitric oxide synthase (iNOS; P = 0.040) and interleukin-22 (IL-22; P = 0.008) in the colon, and inclined to down-regulate interleukin-1β (IL-1β; P = 0.076) expression in the colon. The relative protein abundance of Galectin-3 (P = 0.046) in the colon and the ratio of phosphorylation NF-κB to NF-κB (p–NF–κB/NF-κB ratio) in the ileum of HP piglets were also greater (P = 0.038). These results suggest that dietary HP may cause diarrhea in piglets by activating NF-κB signaling induced intestinal inflammation.     

基于猪小肠上皮细胞炎症模型分析产肠毒素大肠杆菌诱导的转录组表达变化

【目的】分析产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）诱导猪小肠上皮细胞（IPEC-J2）炎症反应的主要宿主调控基因及相关分子通路，为进一步揭示ETEC感染引发炎症反应的作用机制提供理论依据。【方法】用感染复数（multiplicity of infection，MOI）为100的ETEC菌液感染IPEC-J2细胞，18 h后收集细胞上清，通过ELISA方法检测炎性因子白细胞介素1β（interleukin 1 β，IL-1β）的分泌水平。由Illumina PE150测序平台进行高通量转录组测序，通过edgeR v 3.12.1软件对测序结果进行差异表达分析，利用GOseq和KOBAS 2.0软件对得到的差异表达mRNA进行GO功能和KEGG通路富集分析，随机挑选5条差异表达mRNA，利用实时荧光定量PCR对其验证。【结果】试验成功建立了ETEC感染诱导的IPEC-J2细胞炎症模型。与对照组相比，炎症模型组共发现529条差异表达mRNA，其中236条表达上调，293条表达下调，包括HSP90AA1、CGNL1、CADPS2、EHBP1等免疫相关基因。GO功能分析表明，ETEC感染后差异表达mRNA显著富集于细胞组分和生物过程，包括细胞内成分（intracellular part）、细胞质（cytoplasm）、核成分（nuclear part）、胞内膜结合细胞器（intracellular membrance-bounded organelle）、膜结合细胞器（membrance-bounded orgabelle）及细胞进程（cellular process）等。KEGG通路分析表明，炎症模型中差异表达基因大多数被注释到疾病相关通路及Toll样受体信号通路、mTOR信号通路、细胞周期、黏附连接等免疫相关信号通路。利用实时荧光定量PCR验证随机挑选的5条差异表达mRNA，结果与测序报告中差异表达mRNA变化趋势一致，证明测序结果可信。【结论】HSP90AA1、CGNL1、CADPS2和EHBP1等基因可能在ETEC黏附IPEC-J2细胞并诱导炎症反应中发挥关键作用，并通过Toll样受体、mTOR等信号通路进行免疫调控。研究结果为进一步揭示ETEC感染诱导炎症反应的分子机制及相关调控网络提供参考依据。