月桂酸单甘油酯对花鲈脂质代谢的影响

为评价月桂酸单甘油酯(Glycerol monolaurate,GML)对花鲈(Lateolabrax maculatus)脂质代谢的影响,文章通过基础饲料拌喂0 mg·kg^-1(对照组)、1000 mg·kg^-1(低剂量组)、2000 mg·kg^-1(中剂量组)和4000 mg·kg^-1(高剂量组)的GML,养殖均质量为(230±20)g的花鲈8周。结果显示:1)添加GML组的腹脂率均显著降低,肝体比第4周明显降低(P<0.05);2)低剂量和中剂量组的血清甘油三酯、总胆固醇、低密度脂蛋白胆固醇浓度和天冬氨酸转氨酶活性均低于高剂量组(P<0.05);3)中剂量组的高密度脂蛋白胆固醇浓度均高于其他组(P<0.05);4)添加GML组肝脏中超氧化物歧化酶、谷胱甘肽过氧化物酶活性和还原型谷胱甘肽浓度均有升高,而丙二醛浓度显著降低(P<0.05);5)添加GML组的肝脏中脂肪酸合成酶和乙酰辅酶A羧化酶活性降低,且脂质沉积明显减少(P<0.05);脂蛋白脂酶活性均显著下降,中剂量组显著高于其他组(P<0.05)。综上,饲料中添加GML能够显著改善花鲈脂质代谢,该实验条件下,GML最适拌料量为2000 mg·kg^-1。     

Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

CnAβ基因对大鼠RBL-2H3细胞脱颗粒的影响

过敏是人和养殖动物常见的疾病，而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制，本研究利用CRISPR/Cas9基因编辑技术，构建钙调磷酸酶A beta （calcineurin A beta，CnAβ）基因敲除的大鼠RBL-2H3细胞株，并探讨CnAβ基因对RBL-2H3细胞生长及脱颗粒的影响。选取大鼠CnAβ基因第一外显子为敲除靶点，设计并合成3个单导向RNA （single guide RNA，sgRNA），构建pX459-CnAβ-sgRNA质粒，并用脂质体3000将构建好的质粒转染到RBL-2H3细胞内；利用嘌呤霉素对转染细胞进行筛选，通过DNA测序验证获得CnAβ基因敲除的RBL-2H3细胞株；并检测CnAβ基因缺失对细胞增殖和脱颗粒的影响。结果表明，成功构建了CnAβ单基因敲除的大鼠RBL-2H3细胞株；CnAβ基因缺失对RBL-2H3细胞增殖、细胞的颗粒形成以及颗粒含量无显著影响，但显著抑制由细胞表面受体介导的RBL-2H3细胞脱颗粒作用。该研究结果有助于深入了解动物过敏性疾病的发生机制，为动物过敏性疾病的预防提供了理论基础。     

黄曲霉毒素B1、玉米赤霉烯酮和呕吐毒素的累加细胞毒性研究

由于饲料中多种霉菌毒素并存的几率比较高，本研究以仔猪肠上皮细胞（IPEC-J2）为模型，研究黄曲霉毒素B1（AFB1）、玉米赤霉烯酮（ZEA）和呕吐毒素（DON）的叠加细胞毒性。细胞毒性试验选用AFB1、ZEA和DON三种毒素作为响应面Box-Behnke设计的三个因素，以AFB1：10、20、30 μg/L，ZEA：150、300、450 μg/L，DON：500、1000、1500 μg/L作为Box-Behnke设计的三个编码水平。利用响应面设计构建得到17组复合霉菌毒素组合，以其对IPEC-J2细胞活力的影响作为参考指标，得到对细胞损伤程度最高和最低的霉菌毒素添加比例。结果表明：经方程预测后，得到细胞活力最低（霉菌毒素毒性最高）的AFB1、ZEA和DON组合为30、150 μg/L和1500 μg/L，经测定细胞活力为32.32%|得到细胞活力最高（霉菌毒素毒性最低）的AFB1、ZEA和DON组合为10、150 μg/L和600 μg/L，经测定细胞活力为53.01%。该结果为多种霉菌毒素叠加毒性的研究提供了依据。 [关键词] IPEC-J2细胞|黄曲霉毒素B1|玉米赤霉烯酮|呕吐毒素|细胞毒性     

斑马鱼幼鱼嗜中性粒细胞对副溶血弧菌清除的动态变化

副溶血弧菌(Vibrio parahaemolyticus)是人-兽-鱼共患的致病菌,严重威胁食品安全和人类的健康。为了研究宿主嗜中性粒细胞清除细菌感染的动态变化过程,利用红色荧光蛋白标记的副溶血弧菌Vp57~(RFP)菌株感染受精后48 h(hours post fertilization,hpf)的斑马鱼(Danio rerio)幼鱼耳泡,建立局部感染模型,并以大肠杆菌(Escherichia coli)Ec01菌株为对照,系统比较了副溶血弧菌和大肠杆菌的半数致死剂量(median lethal dose,LD_(50))、存活曲线以及和嗜中性粒细胞相互作用的动态过程,RT-qPCR验证炎症相关基因il1b和il10在感染前后的表达量变化。结果显示副溶血弧菌和大肠杆菌感染斑马鱼幼鱼的LD_(50)分别为7.90×10~8 CFU/mL和1.14×10~(11) CFU/mL,与感染大肠杆菌相比,斑马鱼感染副溶血弧菌后招募嗜中性粒细胞的速度更快,且数量更多。RT-qPCR结果发现斑马鱼感染副溶血弧菌后在相同时间点能够激活更高的il1b和il10基因的表达,引起强烈的炎症反应。基于以上研究,我们建立的副溶血弧菌-斑马鱼幼鱼耳泡局部感染模型,为进一步研究嗜中性粒细胞清除副溶血弧菌感染的动态过程,以及深入揭示天然免疫应答机制提供了依据。     

甘草总黄酮对高脂条件下罗非鱼肝损伤的保护作用

为了研究甘草总黄酮对高脂饲料造成的罗非鱼(Oreochromis niloticus)脂肪肝损伤是否具有保护作用,随机将180尾健康无伤罗非鱼分成6组,即对照组、高脂饲料模型组和4个不同浓度甘草总黄酮组(0.05、0.10、0.50、1.00 g·kg^-1甘草总黄酮),每组30尾。饲喂90 d后,采集罗非鱼各实验组肝,测定肝匀浆中超氧化物歧化酶(SOD)活性、还原型谷胱甘肽(GSH)活性、丙二醛(MDA)含量、甘油三酯(TG)含量、总胆固醇(TC)含量、总抗氧化能力(T-AOC)水平,Western blotting法检测肝中核转录因子-κB P65(NF-κB P65)和核转录因子-κB C-Rel (NF-κB C-Rel)蛋白表达水平,RT-PCR法测定肝中微粒体甘油三酸酯转移蛋白(MTTP)和载脂蛋白B100(ApoB100)基因表达情况。结果表明:与对照组相比,高脂饲料模型组罗非鱼肝匀浆中TG、TC、MDA含量极显著(P<0.01)升高,GSH活性、SOD活性、T-AOC水平均极显著(P<0.01)降低;MTTP和ApoB100 mRNA表达水平降低(P<0.01或P<0.05);Western blotting结果显示,NF-κB P 65和C-Rel蛋白表达水平升高。饲料中加入甘草总黄酮后,与高脂饲料模型组相比,罗非鱼肝中GSH活性、SOD活性、T-AOC水平不同程度提高,TC、TG、MDA含量和NF-κB P65、C-Rel蛋白表达水平不同程度降低,MTTP和ApoB100的mRNA表达水平升高,以0.50 g·kg^-1甘草总黄酮组效果较好。说明甘草总黄酮对于罗非鱼脂肪肝损伤具有一定的保护作用,可以缓解高脂饲料造成的脂肪肝损伤。     

褪黑激素对长毛兔毛囊细胞凋亡的影响

为研究褪黑激素对皖系长毛兔毛囊细胞凋亡的影响及相关机制,选择60只皖系长毛兔,随机均分成4组,1组不埋植褪黑激素(对照),另3组每只兔按25 mg(低剂量组)、40 mg(中剂量组)和55 mg(高剂量组)分别埋植褪黑激素,饲养70 d后,进行组织病理学观察和RNA的转录组高通量测序及q PCR表达分析。结果表明:通过组织切片观察,证实55 mg褪黑激素处理能有效抑制毛囊细胞凋亡,维持细胞正常形态;通过对高剂量组和对照组测序,得到25个差异表达基因,其中有14个基因上调,11个基因下调,涉及通路包括嗜T细胞病毒感染、坏死性凋亡、甲状腺癌、铁死亡、I型糖尿病和胰岛素信号通路,其中坏死性凋亡、铁死亡通路与长毛兔毛囊凋亡相关;选取4个与凋亡相关的差异表达基因进行荧光定量验证,得到新基因MSTRG.3561表达显著上调,SLC25A5表达下调,但差异无统计学意义。     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.