氧化应激对PRRSV致PAM细胞TLR3/NF-κB分子转录的影响

为探讨氧化应激对猪繁殖与呼吸综合征病毒(PRRSV)感染猪肺泡巨噬细胞(PAM)TLR3/NF-κB信号分子转录的影响,体外分离培养PAM,分为对照组、PRRSV感染组、抗氧化剂NAC+PRRSV组,促氧化剂H2O2+RRRSV组。分别在培养6、12、24、48、72h收集细胞,观察各组的细胞病变、real-time PCR检测PRRSV、TLR3、TRIF和NF-κB mRNA转录量的变化。结果显示,PRRSV感染组PRRSV、TLR3、TRIF及NF-κB mRNA的转录量与对照组相比随感染时间的延长显著升高(P0.05),48h达到最大值;NAC处理接毒组各信号分子mRNA的转录量比PRRSV感染组同时间点略低;H_2O_2处理接毒组比PRRSV感染组的略高。结果表明,氧化应激可增强PRRSV致PAM细胞TLR3/NF-κB分子mRNA的转录量,NF-κB的活化可能是PRRSV导致细胞损伤的机制之一。     

抗菌肽Temprine-La(FS)抑制2型猪链球菌生物被膜形成的研究

试验旨在研究抗菌肽Temprine-La（S）（T-La（S））、Temprine-La（FS）（T-La（FS））、RGD-T-La（S）和RGD-T-La（FS）对2型猪链球菌（SS2）生物被膜形成的抑制作用。通过结晶紫染色法（CV）检测SS2生物被膜形成能力;微量稀释法测定抗菌肽对SS2生物被膜的最小生物被膜抑菌浓度（MBIC）和最小生物被膜杀菌浓度（MBEC）;结晶紫染色法和扫描电镜（SEM）检测抗菌肽对SS2生物被膜形成的影响;XTT法检测抗菌肽对SS2生物被膜代谢活性的影响;苯酚硫酸法检测抗菌肽对SS2生物被膜胞外多糖含量的影响;建立猪链球菌-斑马鱼感染模型,HE染色法观察T-La（FS）对斑马鱼脑组织病理变化的影响;实时荧光定量PCR法分析抗菌肽对SS2生物被膜相关基因及对斑马鱼炎性细胞因子基因转录水平的影响。结果显示,SS2具有良好的生物被膜形成能力;T-La（S）、RGD-T-La（S）、T-La（FS）和RGD-T-La（FS）对SS2生物被膜的MBIC分别为31.3、15.6、7.8和15.6 μg/mL;MBEC分别为62.6、31.2、15.6和31.2 μg/mL;结晶紫染色结果表明,抗菌肽对SS2生物被膜的形成有抑制作用;扫描电镜结果显示,抗菌肽使SS2生物被膜中的细菌数量和生物被膜的形态发生明显变化,细胞外基质大量减少;XTT法结果显示,抗菌肽可显著降低SS2生物被膜的代谢活性;苯酚硫酸法结果显示,抗菌肽能有效抑制SS2生物被膜合成胞外多糖;实时荧光定量PCR结果表明,抗菌肽作用后降低了SS2生物被膜基因的转录水平;T-La（FS）作用后TLR2、MyD88及促炎性细胞因子基因的转录水平显著或极显著降低（P<0.05;P<0.01）,抗炎性细胞因子基因的转录水平显著或极显著升高（P<0.05;P<0.01）。抗菌肽主要通过影响生物被膜相关基因转录水平和阻断胞外多糖的合成与分泌来抑制SS2生物被膜的形成,其中T-La（FS）可能通过抑制TLR2信号通路中TLR2和MyD88分子表达,抑制炎性细胞因子的释放,减轻脑膜炎的炎性反应。     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group, model （M） group, low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group, medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group, high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1, tail vein injection） group. After 7 days of intervention, the sleep deprivation model of rats in M group, Gen-L, Gen-M, Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）, and the levels of interleukin-1β （IL-1β）, IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR, and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group, the escape latency, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L, Gen-M and Gen-H groups （P<0.01）, and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Protective effects of DAPT on rat model with atherosclerotic ischemic brain stroke by blocking Notch pathway

AIM: To investigate the protective effects of DAPT on rat model with atherosclerotic (AS) ischemic brain stroke by blocking Notch pathway. METHODS: SD rats (n=24) were randomly divided into control group and model group, and the rats in model group were fed high-fat diet for 6 weeks to establish the AS model. The AS rats were randomly divided into 3 groups (n=6 in each group):AS-sham group, AS rats with ischemia (AS-ishemia) group, and DAPT administration (AS-ishemia-DAPT) group. The histopathological changes of carotid aorta were observed by HE staining. The serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by automatic biochemical analyzer. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The protein levels of Notch1 and Hes1 in rat artery, and nuclear factor-κB (NF-κB) and Toll-like receptor 4 (TLR4) in rat brain were determined by Western blot. RESULTS: Notch signaling pathway inhibitor DAPT significantly reduced intimal thickening, vascular stenosis and the formation of AS plaque. Compared with AS-ischemia group, the serum levels of lipids and inflammatory factors were decreased significantly in AS-ischemia-DAPT group, and the protein levels of Notch1 and Hes1 in the rat carotid artery and NF-κB and TLR4 protein expression in rat brain were also decreased significantly (P<0.05). CONCLUSION: Blocking Notch pathway by DAPT not only improves the blood lipid levels, but also inhibits the serum inflammatory cytokine release and NF-κB/TLR4 pathway activation.     

低氧胁迫对瘤背石磺Toll样受体4、血细胞和免疫酶活性的影响

为探究瘤背石磺面临低氧环境时固有免疫的应激机制,以RACE法对瘤背石磺TLR4 基因进行全长克隆,并进行生物信息学分析,测定了低氧胁迫下瘤背石磺TLR4基因的表达变化,以及分析了血细胞活力和溶菌酶（LSZ）活性、肝脏中超氧化物歧化酶（SOD）活性和碱性磷酸酶（ALP）活性。结果显示,瘤背石磺TLR4基因cDNA全长共3 605 bp,包括编码956 aa氨基酸的2 817 bp开放阅读框。系统进化树表明瘤背石磺TLR4基因与光滑双脐螺TLR4基因的进化地位较为接近。qRT-PCR结果显示TLR4基因在瘤背石磺7个组织中均有表达,其中表达量最高的是肝脏。低氧胁迫下,各组织中TLR4基因的表达量皆显著上升,其中神经节在4h时率先达到峰值。此外,血细胞活力、LSZ活性、ALP活性都是呈先下降后上升的趋势,而SOD活性呈先上升再下降再上升的趋势,波动幅度最为明显。研究结果初步说明了TLR4的生理功能,为探究潮间带生物免疫系统的低氧应激机制提供理论参考。     

C型凝集素受体在肿瘤免疫作用中的研究进展

先天免疫是宿主识别病原及消除病原感染的第一道防线。模式识别受体是参与识别病原入侵的主要分子,主要包括Toll样受体、RIG-I样受体、NOD样受体和C型凝集素受体等。模式识别受体在识别病原相关分子模式后,激活机体的先天免疫信号通路,诱导炎症细胞因子和干扰素的产生,从而启动抵抗病原入侵的免疫应答。越来越多的证据表明,免疫应答的激活、维持和终止受到了严格的调节,使机体在保持一定免疫强度的同时避免产生过度的免疫反应。microRNA是一类长度为18~23 nt的微小非编码RNA,是鱼类先天免疫应答网络中的重要调控因子。近年来,microRNA在鱼类免疫学领域已开展了大量的研究,但缺乏对其进行及时地全面性的总结。本文综述了近年来miRNA在鱼类先天免疫反应中的研究进展,以期为鱼类的分子抗病育种及疾病防控研究提供一些思路。