Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

Role of IP3R in LH-EGFR-induced mouse oocyte meiotic resumption

AIM: To explore the effect of inositol 1, 4, 5-trisposphate receptor (IP₃R) in luteinizing hormone-epidermal growth factor receptor (LH-EGFR)-induced oocyte meiotic resumption. METHODS: Models of mouse cumulus-oocyte complexs (COCs) culture and follicle culture in vitro were generated to study the effects of 2-aminoethyl diphenyl borate (2-APB) and heparin (IP₃R specific inhibitors) on LH/EGF-induced oocyte meiotic resumption and EGF-induced cumulus cell expansion. Real-time PCR was used to detect the mRNA expression of cumulus expansion-related factors. The changes of the intracellular calcium level were monitored using Fluo 3-AM, and the cGMP level was measured by ELISA. RESULTS: The inhibitors of IP₃R, 2-APB and heparin, dramatically reversed EGF-induced oocyte maturation (P<0.05) and decreased cGMP levels in COCs (P<0.05). In addition, 2-APB and heparin reversed EGF-induced cumulus expansion, and significantly inhibited EGF-induced cumulus expansion-related factor expression (P<0.05). The activation of IP₃R increased intracellular calcium level, and the study found that 2-APB and heparin dramatically reversed EGF-induced elevation of calcium level in cumulus cells (P<0.05). Follicular culture in vitro showed that 2-APB and heparin significantly reversed the LH-induced oocyte maturation (P<0.05). CONCLUSION: LH-EGFR signaling pathway increases calcium level in cumulus cells through IP₃R, resulting in meiotic resumption.     

Achyranthes aspera (Prickly chaff flower) leaves- and seeds-supplemented diets regulate growth,innate immunity,and oxidative stress in Aeromonas hydrophila-challenged Labeo rohita

ABSTRACT

The immunostimulatory and disease-resistance properties of Achyranthes aspera were evaluated in rohu (Labeo rohita) challenged with Aeromonas hydrophila. Experimental diets were enriched with leaves at 0.25% (D1) and 0.5% (D2) and seeds at 0.5% (D3); the control diet (D4) was without any enrichment. Rohu (2.02 ± 0.23 g) were cultured for 75 days and then challenged with bacteria. The highest average weight was observed in the D3 diet-fed fish. The cumulative mortality rates were 70%, 60%, 40%, and 30% in the D4, D1, D2, and D3 diets fed to rohu respectively. Enriched diets significantly increased myeloperoxidase, nitric oxide synthase, and serum lysozyme levels and decreased malondialdehyde and carbonyl protein content. Expressions of lysozyme C and lysozyme G were significantly (P < .05) higher in the D3 diet-fed fish. In the kidney, IL-1β and TLR 4 were up-regulated in enriched-diet-fed fish. Supplementation of seeds and leaves at 0.5% showed a positive impact in fish.     

Gastrointestinal motility modulation by stress is associated with reduced smooth muscle contraction through specific transient receptor potential channel

Excessive stress response causes disability in social life. There are many diseases caused by stress, such as gastrointestinal motility disorders, depression, eating disorders, and cardiovascular diseases. Transient receptor potential (TRP) channels underlie non-selective cation currents and are downstream effectors of G protein-coupled receptors. Ca²⁺ influx is important for smooth muscle contraction, which is responsible for gastrointestinal motility. Little is known about the possible involvement of TRP channels in the gastrointestinal motility disorders due to stress. The purpose of this study was to measure the changes in gastrointestinal motility caused by stress and to elucidate the mechanism of these changes. The stress model used the water immersion restraint stress. Gastrointestinal motility, especially the ileum, was recorded responses to electric field stimulation (EFS) by isometric transducer. EFS-induced contraction was significantly reduced in the ileum of stressed mouse. Even under the conditions treated with atropine, EFS-induced contraction was significantly reduced in the ileum of stressed mouse. In addition, carbachol-induced, neurokinin A-induced, and substance P-induced contractions were all significantly reduced in the ileum of stressed mouse. Furthermore, the expression of TRPC3 was decreased in the ileum of stressed mouse. These results suggest that the gastrointestinal motility disorders due to stress is associated with specific non-selective cation channel.     

神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路

NMB/NMBR通过调节A型流感病毒（IAV/H1N1/PR8）感染诱导的细胞因子表达而参与抗IAV的先天性免疫反应。为探究其发挥抗IAV/H1N1感染的信号通路，本文用PR8和WSN毒株分别感染MLE-12细胞和小鼠，用NF-κB抑制剂BAY11-7028单独或联合NMB处理MLE-12细胞，小鼠后腿肌内注射NMB和NMBRA，采用RT-PCR和qRT-PCR分析NMB、NMBR、IL-6、IFN-α和NP基因表达变化，采用Western blot分析NMB、NMBR、P65/p-P65、IκBα和NP蛋白表达的变化。结果显示，BAY11-7028可促使PR8和WSN感染的MLE-12细胞中NMB、NMBR、IL-6和IFN-α基因表达水平均下降和NP基因表达水平上升，并降低NMB、NMBR和p-P65蛋白表达水平和提升IκBα和NP蛋白表达水平。然而，NMB联合BAY 11-7028诱导PR8或WSN感染后的细胞中IL-6和NP表达出现极显著下降和IFN-α显著上升。此外，NMB抑制PR8和WSN感染的小鼠肺组织内p-P65和NP蛋白表达水平和促进IκBα蛋白表达水平；NMBRA联合NMB抵消NMB对PR8或WSN感染后的这些蛋白表达水平的调节作用。综上表明，NMB/NMBR通过调节PR8和WSN感染的MLE-12细胞和小鼠体内的NF-κB信号通路上P65蛋白磷酸化和IκBα的表达，进而影响下游细胞因子IL-6和IFN-α基因的表达，从而发挥抗IAV/H1N1感染的先天性免疫应答反应。     

Kisspeptin对鱼类生殖轴的调控机制研究

Kisspeptin(简称Kiss或者Kp)是由KISS1/Kiss1基因编码的一种下丘脑神经肽,通过其受体KissR(也称作GPR54)的介导参与了多种生理过程,如抑制肿瘤转移和参与生殖调控。目前,尽管在鲤形目(Cypriniformes)、鲈形目(Perciforms)、鲽形目(Pleuronectiforms)、鲀形目(Tetraodontiforms)、颌针目(Beloniforms)、鲉形目(Scorpaeniformes)、鲑形目(Salmoniformes)及鳕形目(Gadiformes)等多种鱼类中均鉴定出了kiss/kissr基因,但Kiss/KissR系统在鱼类生殖调控中的精确作用及其分子机制尚未完全阐明。尤其是在鱼类中存在2种kiss及3种kissr基因,Kiss/KissR系统对鱼类生殖调控的作用方式更加复杂。本文简要总结鱼类Kiss及其受体的研究进展,并对Kiss的生理学功能、信号转导机制以及kiss/kissr表达调控研究进行概括讨论,旨在加深对鱼类Kiss/KissR系统的认识和了解,为后续研究指明方向。     

赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆、表达及功能

【目的】章鱼胺信号系统在调节昆虫行为和生理过程中具有至关重要的作用。赤拟谷盗(Tribolium castaneum)作为一种模式昆虫,被广泛用于解析昆虫生长发育及生理等调控机制的研究工作。本研究以赤拟谷盗为对象,旨在明确章鱼胺受体在调节赤拟谷盗行为和生理方面的功能。【方法】根据Gen Bank登录的相关序列信息(XP_008198078),利用RT-PCR技术克隆赤拟谷盗章鱼胺受体基因Tc OctβR3的c DNA序列。利用在线生物信息学分析软件预测该基因的开放阅读框、编码的氨基酸序列以及跨膜结构域等信息,基于邻接法构建该基因与其他昆虫相关序列的系统发育树,明确系统进化关系。分别提取赤拟谷盗各发育阶段(卵、幼虫、蛹和成虫)、不同组织(中枢神经系统、脂肪体、中肠、后肠、马氏管、精巢和卵巢)以及饥饿胁迫后的RNA,以赤拟谷盗核糖体蛋白S3(Tc RPS3)为内参基因,采用实时定量PCR技术分析该基因在赤拟谷盗不同发育阶段、不同组织以及在饥饿胁迫下的表达模式。运用哺乳动物异源表达系统在人胚胎肾细胞HEK293中瞬时表达Tc OctβR3,进而利用第二信使c AMP含量测定技术分析Tc OctβR3与配体的结合能力。最后,通过体外合成赤拟谷盗Tc OctβR3的双链RNA,利用RNA干扰以及轨迹球行为分析等技术探究该基因的生理功能。【结果】序列分析结果表明,赤拟谷盗Tc OctβR3开放阅读框全长1 305 bp,编码434个氨基酸,序列中含有G蛋白偶联受体典型的7个跨膜结构域。基于邻接法构建的系统发育树表明,该基因编码的蛋白质与小蜂甲(Aethina tumida)的OctβR3亲缘关系最近。实时定量PCR分析结果表明,Tc OctβR3在赤拟谷盗各发育阶段均有表达,尤其在低龄幼虫期转录水平最高,而在其他发育阶段表达量无显著差异;在赤拟谷盗不同组织中,Tc OctβR3在中枢神经系统的表达量显著高于其他组织;赤拟谷盗幼虫在经饥饿处理24 h的过程中,Tc OctβR3的表达量呈先下降后上升的趋势,且在处理6 h的表达量最低,为对照的0.47倍,在16 h表达量最高,为对照的1.80倍,最后恢复到正常水平。通过HEK293细胞异源表达Tc OctβR3后,c AMP测定结果表明章鱼胺(OA)呈浓度依赖性地激活Tc OctβR3,其EC50为8.68×10-7 mol·L-1,萘甲唑啉(NA)的激动活性最高,其有效中浓度EC50为8.56×10-8 mol·L-1。4种供试生物胺激动剂活性强弱为:萘甲唑啉酪胺(TA)章鱼胺多巴胺(DA)。进一步采用RNA干扰技术的分析结果表明,注射ds RNA能有效抑制Tc OctβR3的表达,沉默效率高达61.5%,但干扰该基因表达后不会影响赤拟谷盗成虫的爬行速度和产卵量。【结论】Tc OctβR3在赤拟谷盗中枢神经系统可能发挥重要作用,能调节幼虫对饥饿胁迫的响应。明确了Tc OctβR3的分子生物学特性,可为将来以其为靶标筛选高效激动剂和抑制剂提供理论依据。     

PMF模型的影响因素考察——以某铅锌矿周边农田土壤重金属源解析为例

正定矩阵因子分解模型(PMF)可用于污染源未知情况下的土壤中重金属来源解析,但对数据样本敏感,结果波动大。为探究PMF模型对土壤重金属源解析的适用性,本研究以湖南省水口山铅锌矿周边农田土壤为研究对象,考察成分谱元素种类和异常值剔除两个因素对PMF模型解析结果的影响。根据成分谱中有无地壳元素和是否剔除异常值,建立4种数据样本,对PMF模型结果进行比较分析。结果表明:根据散点图剔除2个异常样本后未改变分类结果,仅改变各源贡献率;引入6种地壳元素后PMF模型的源轮廓(源数量和贡献元素)均发生变化。在成分谱中增加地壳元素后,源解析结果受异常值影响小,结果更稳定且容易解释。因此,应将地壳元素引入成分谱并对数据进行预处理,可较好地保证源解析结果的稳定性和可信度。结合文献和该地区实际情况对模型结果进行解读,最终确定5个来源:Pb、Zn、Cd和Sb主要来自铅锌矿的采选及冶炼等工业活动源(26.81%),As和Hg主要来自污水灌溉和农药化肥施用等农业活动源(14.68%),Cr、Ni、Co和Mo主要来自土壤母质源(24.41%),Mn和Fe主要来自铁矿石开采和交通运输源(16.39%),Al和Ca主要来自矿石风化源(17.72%)。     

SCFAs对奶牛瘤胃上皮细胞Ca2+信号通路相关基因表达的影响

为探究短链脂肪酸(SCFAs)对奶牛瘤胃上皮细胞(BRECs)Ca~(2+)信号通路相关基因表达的影响,试验分为3个处理组,分别为野生型BRECs组,含20mmol/L SCFAs BRECs组,含20mmol/L SCFAs且通过CRISPR/Cas 9系统敲除GPR41基因的BRECs组,每个处理组3个重复,每组细胞均培养24h后,收集细胞提取总RNA,通过qRT-PCR对Ca~(2+)信号通路相关基因的mRNA表达量和细胞内Ca~(2+)浓度进行测定。结果表明,与野生型BRECs相比,添加20mmol/L SCFAs可极显著增加PLCB2的mRNA表达量(P0.01),显著增加IP3R1的mRNA表达量(P0.05),对PLCE1、PLCL1、PKCB和PKCG的mRNA表达量无显著差异(P0.05),可增加细胞内Ca~(2+)浓度但无显著差异(P0.05);敲除SCFAs的受体GPR41后,添加20 mmol/L SCFAs可显著降低IP3R1的mRNA表达量(P0.05),极显著上调PLCE1和PLCB2的mRNA表达量(P0.01),但对PLCL1、PKCB和PKCG的mRNA表达量无显著影响(P0.05),细胞内Ca~(2+)浓度有降低趋势但无显著差异(P0.05)。综上,SCFAs可以通过激活其受体GPR41来调控BRECs内Ca~(2+)信号通路中相关基因的表达和细胞内Ca~(2+)的释放。