Septicemia of grass carp is a systemic inflammatory reaction caused by bacterial infection. More and more evidences show that long non-coding RNAs (lncRNAs) can participate in the regulation of inflammatory response. In the present study, lncRNA-WAS and lncRNA-C8807 were confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila. LncRNA-WAS and lncRNA-C8807 could interact with miR-142a-3p. LncRNA-WAS and lncRNA-C8807 interact with miR-142a-3p to effect pro-inflammatory genes and NF-κB pathway. Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by lncRNA in grass carp.

为了评估养殖条件下青鱼(Mylopharygodon piceus)不同组织的状态和分析青鱼不同肠段的功能分化,使用组织学半定量系统对青鱼的鳃、肝、脾和肠进行评估,并使用酶活检测分析青鱼不同肠段中消化酶和抗氧化酶的分布.结果 表明:青鱼的鳃、肝和脾的状况良好,处于Ⅰ级和Ⅱ级变化,肠道是最受影响的器官,达到Ⅲ级变化,在肠道的组织学分析中发现肠黏膜上皮细胞增生和固有层易碎和坏死.青鱼的Fulton状态指数(fulton's conditional factor,CF)介于1.01到2.30之间,均值为1.19;肝体指数(hepatic somatic index,HSI)介于0.29到0.93之间,均值为0.58,表明被检测的青鱼状态良好.在青鱼的肠道中,消化酶和抗氧化酶在不同肠段的分布存在显著差异,前肠淀粉酶、脂肪酶和胰蛋白酶的活性显著高于后肠,后肠中超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽还原酶(glutathione reductase,GR)、过氧化氢酶(catalase,CAT)活性和总抗氧化能力(total antioxidant capacity,T-AOC)显著高于前肠.结果 可以为养殖鱼类组织状态评估和肠道不同部分功能差异分析提供一定的参考资料.

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.

为探究miR-462在嗜水气单胞菌感染草鱼肾脏细胞(Ctenopharyngodon idella kideny,CIK)后的调控机制,实验利用荧光定量技术检测了CIK细胞感染嗜水气单胞菌后miR-462表达水平的变化;运用RNAhybrid软件预测miR-462的靶基因,利用双荧光素酶报告基因系统进行确定;此外还分析了miR-462对靶基因下游基因的调控作用.结果 显示,在CIK细胞感染嗜水气单胞菌的过程中,miR-462的表达发生显著变化;cx32.2、slc9a3.1和tbk1的表达先降低后升高,与miR-462的表达模式呈负相关.双荧光素酶报告系统显示,miR-462可靶向cx32.2、slc9a3.1和tbk1的3′非编码区抑制其表达,过表达miR-462可以显著抑制cx32.2、slc9a3.1和tbk1的表达.转染miR-462模拟物后,下游slc4a4a、tnfrsf5、cxcl9和cxcl11基因的表达受到抑制.研究表明,miR-462参与调控嗜水气单胞菌感染后草鱼CIK细胞中的免疫应答.cx32.2、slc9a3.1和tbk1被鉴定为miR-462的靶基因.miR-462可通过靶向slc9a3.1和tbk1影响下游基因的功能.

Ctenopharyngodon idellagrowth arrest and DNA damage-inducible 45aa (CiGadd45aa) is a member of the Gadd45 family of immune-related proteins in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory responses and apoptosis. However, little is known about the regulatory effects of miRNAs on CiGadd45aa expression in grass carp. In the present study, CiGadd45aa was identified as a target gene of miR-731 based on miRNA expression profiling and dual-luciferase reporter assay. Our study revealed that miR-731 targets CiGadd45aa and regulates the expression of proinflammatory factors, thereby regulating immune response in grass carp. In addition, CiGadd45aa and miR-731 were both found induced apoptosis. Hence, our findings provide a theoretical basis for exploring the molecular mechanism by which miR-731 regulates inflammation and apoptosis in grass carp.

Grass carp septicemia is a systemic inflammatory response that develops following a bacterial infection. The hyperinflammatory state develops could lead to septic shock and lethality. There is increasing evidence that microRNAs are involved in the regulation of the inflammatory response. In the present study, miR-21 was confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila and LPS stimulation. Both jnk and ccr7 were identified as target gene of miR-21 by overexpression, inhibition, and dual luciferase reporter assays experiments. Meanwhile, miR-21 targets the jnk and ccr7 to modulate downstream pro-inflammatory factors tnf-α, il-1β, il-6, and il-12. Our results provide a theoretical basis for exploring the molecular mechanism of grass carp miR-21 regulating inflammation.

c-Jun N-terminal kinases (JNKs) and p38s are central components of signal transduction pathways, which are stimulated mainly by environmental stress and inflammatory cytokines. Manipulation of JNK and p38 dependent immune responses either boosts or subdues immune responses to infectious diseases or inflammatory disorders. In this study, we analyzed the whole-genome database of the grass carp and identified 4 JNK and 6 p38 genes. JNK and p38 genes of grass carp were distributed in 7 out of 24 chromosomes. All JNK and p38 proteins contained characteristic dual-phosphorylation site. The JNKs contain a specific dual-phosphorylation consensus ((Thr-Pro-Tyr) that is different from that of the p38 proteins (Thr-Gly-Tyr). Deduced gene secondary structure analyses as well as the syntenic analyses further supported their annotation and orthologs. Results of tissue distribution detection revealed that JNK and p38 genes exhibited lower expression in health grass carp. The mRNA expression levels of JNK and p38 genes were significantly up-regulated in tissues and CIK cells after bacterial infection, indicating their potential roles in bacterial-regulated immune responses. These findings in our study will facilitate the further evolutionary characterization of JNK and p38 genes in teleost species and provide a theoretical basis for their functional study.