Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.     

牛磺酸对急性氨中毒的鲫和草鱼抗氧化及炎症相关基因表达影响的比较

为了比较牛磺酸对急性氨中毒的鲫和草鱼缓释作用的差异,实验分别构建了4个处理组,组1实验鱼通过腹腔注射生理盐水,组2注射醋酸铵(鲫7 mmol/g,草鱼9 mmol/g),组3注射醋酸铵和牛磺酸(100μg/g),组4注射牛磺酸。毒性实验持续96 h。结果显示,组2鲫肝脏中SOD、CuZnSOD和CAT基因mRNA表达量显著低于组1和组3;组2和组3鲫肝脏中GPx基因mRNA表达量显著低于组1;组1鲫大脑中SOD、CuZnSOD、CAT和GPx基因mRNA表达量最高;组2草鱼肝脏中SOD、CuZnSOD、CAT和GPx基因mRNA表达量显著高于其他组;组2和组4草鱼大脑中SOD和CuZnSOD基因mRNA表达量显著低于组1和组3;组3草鱼大脑中CAT和GPx基因mRNA表达量最高;此外,组2鲫和草鱼肝脏及大脑中TNF和IL基因mRNA表达量均显著高于其他组。研究表明,鱼类氨中毒会扰乱机体的抗氧化酶系统和免疫应答,引起氧化损伤和炎症反应;草鱼通过提高抗氧化相关基因表达以应对氨中毒;牛磺酸能够有效缓解氨中毒对鲫和草鱼造成的氧化伤害,但牛磺酸并不能降低氨中毒对鲫和草鱼造成的炎症反应。     

低聚半乳糖干预缓解肠道炎症的研究进展

肠道炎症已成为我国社会健康的难题和挑战，其发病率在我国迅速增长。肠道炎症发病原因复杂，目前尚缺乏有效的缓解药物，因此加强肠道炎症有效缓解物质的研发至关重要。低聚半乳糖（galactooligosaccharide，GOS）是一种食疗益生性较优的乳源功能性低聚糖，能够有效促进肠道内益生菌的增殖，改变肠道菌群结构，刺激免疫应答，进而改善肠黏膜屏障功能，缓解肠道炎症。本文综述近年来国内外有关肠道炎症及GOS干预缓解肠道炎症作用的研究进展，并对其应用前景进行展望，为此领域的研究提供科学依据。     

TFDG对IL-1β体外诱导大鼠软骨细胞炎性损伤的保护作用研究

体外分离培养SD雄性大鼠膝关节软骨细胞,经甲苯胺蓝及Ⅱ型胶原(Col Ⅱ)免疫荧光染色鉴定后,加入白介素-1β(IL-1β)诱导建立骨关节炎(OA)细胞模型,探讨茶黄素双没食子酸酯(TFDG)对OA软骨细胞的保护作用。细胞形态观察发现TFDG能明显改善OA软骨细胞形态。实时荧光定量PCR(Real-time PCR)结果显示,TFDG不仅可以上调软骨细胞分子标志物Col Ⅱ mRNA的表达,还可以下调炎症因子IL-1β、IL-6mRNA的表达。酶联免疫吸附实验(ELISA)检测结果进一步表明TFDG可明显降低炎症因子的分泌。免疫印迹(Western blot)检测结果证明,TFDG预干扰可降低炎症诱导酶环氧化酶COX-2蛋白表达量。这些结果说明TFDG通过减弱炎症反应,从而对IL-1β体外诱导大鼠软骨细胞炎性损伤起到保护作用。     

Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno‐histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post‐seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II⁺ cells in heart, pancreas and pyloric caeca, IgM⁺ cells in heart and pancreas, and CD3⁺ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD‐affected tissues were MHC II⁺ and CD3⁺ cells suggesting that antigen‐presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.     

Liver of the fish Gymnotus inaequilabiatus and nematode larvae infection: Histochemical features and expression of proliferative cell nuclear antigen

Histopathological lesions due to third‐larval stage of nematode Brevimulticaecum sp. within the liver of a subpopulation of 31 Gymnotus inaequilabiatus from the Pantanal Region (Brazil) were studied with histochemical and immunohistochemical methods. In 93.5% of fish, livers harboured nematode larvae and the intensity of infection ranged from 8 to 293. In livers with highest number of larvae, the hepatic tissue was occupied primarily by the nematodes. Each larva was encircled by focal inflammatory granulomatous reaction. Within the thickness of the granuloma, three concentric layers were recognized: an inner layer of densely packed epithelioid cells, a middle layer of mast cells (MCs) entrapped in a thin fibroblast‐connective mesh and an outer layer of fibrous connective tissue with fibroblasts. Epithelioid cells and fibroblasts within the thickness of the granuloma wall were positive for proliferative cell nuclear antigen (PCNA). Moreover, several hepatocytes in infected liver were immunoreactive to PCNA. Occurrence of rodlet cells and MCs in parenchyma, in close proximity to the encysted nematode larvae and near the blood vessel of infected liver, was observed. Macrophage aggregates (MAs) were numerous within the granulomas and scattered in parenchyma of the infected liver. High quantity of haemosiderin was encountered in MAs and hepatocytes of infected liver.     

乙酰乙酸抑制脂多糖诱导的奶牛中性粒细胞炎症信号通路的激活

为研究乙酰乙酸(Acetoacetic acid,AcAc)对奶牛中性粒细胞炎症信号通路的影响,以脂多糖(Lipopolysaccharide,LPS)诱导的奶牛中性粒细胞炎症模型为研究对象,利用qRT-PCR、生化和酶联免疫吸附法(ELISA)测定IL-1β、IL-6、TNF-α和IKKβ激酶活性。结果表明:1)qRT-PCR结果显示,与对照组相比较,LPS处理组IL-1β、IL-6和TNF-αmRNA的表达显著增强(P0.01)。与LPS处理组比较,LPS+AcAc混合处理组,其表达量显著下调(P0.05);2)生化检测结果显示,与对照组相比较,LPS处理组中性粒细胞IKKβ激酶活性显著增强(P0.01)。与LPS处理组比较,LPS+AcAc混合处理组IKKβ激酶活性则显著降低(P0.05);3)ELISA结果显示,与对照组相比较,LPS增加促炎因子TNF-α、IL-6和IL-1β的释放。相对于LPS处理组,LPS+AcAc混合处理组促炎因子的释放则显著降低(P0.01)。综上,AcAc可以抑制LPS诱导的奶牛中性粒细胞炎症信号通路的激活,具有一定的抗炎功能。     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.