Summary： Excessive activation of macrophages is implicated in various inflammatory injuries. Salidroside （Sal）, one of the main bioactive components ofRhodiola Sachalinensis, has been reported to possess anti-infla...Summary： Excessive activation of macrophages is implicated in various inflammatory injuries. Salidroside （Sal）, one of the main bioactive components ofRhodiola Sachalinensis, has been reported to possess anti-inflammatory activities. This study aimed to examine the effect of Sal on the activa- tion of macrophages and the possible mechanism. The lipopolysaccharide （LPS）-stimulated phrobol 12-myristate 13-acetate （PMA）-differentiated THP-1 macrophage models were established. The changes in the inflammatory profiles of THP-l-derived macrophages were determined. The results showed that Sal significantly decreased the expression of inducible nitric oxide synthase （iNOS）, cyclooxygenase-2 （COX2）, interleukin-lbeta （IL-1β）, interleukin-6 （IL-6） and tumor necrosis fac- tor-or （TNF-a） at both mRNA and protein levels in THP-l-derived macrophages, and the effect was dose-depedent. Moreover, NF-B activation was significantly suppressed and the phosphorylation of ERK, p38 and JNK was substantially down-regulated after Sal treatment. The findings suggested that Sal can suppress the activation of LPS-stimulated PMA-differetiated THP-1 cells, as evidenced by the decreased expression of iNOS, COX2, IL-1β, IL-6 and TNF-a, and the mechanism involves the inhibition of NF-r,B activation and the phosphorylation of the MAPK signal pathway.展开更多
【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【...【目的】制备番鸭丝裂原活化蛋白激酶1(mitogen-activated protein kinase,MAPK1)蛋白多克隆抗体,鉴定其特异性与适用性,并探究MAPK1蛋白在卵泡颗粒细胞及卵泡中的表达定位,为研究其在番鸭卵泡发育与生殖调控中的功能提供试验工具。【方法】以番鸭卵巢组织cDNA为模板,通过PCR扩增MAPK1基因并测序,运用生物信息学工具分析MAPK1蛋白理化性质及主要抗原表位,筛选免疫原区段。对免疫原序列进行大肠杆菌密码子优化并人工合成,经同源重组克隆至原核表达载体pET-30a(+),构建重组表达载体pET30a-MAPK1并转化大肠杆菌BL21(DE3)感受态细胞。在HB-PET自诱导培养基中表达、纯化MAPK1重组蛋白,将纯化蛋白与QuickAntibody-Rabbit8W佐剂混合后免疫新西兰白兔制备多克隆抗体。采用ELISA、Western blotting、细胞免疫荧光(CIF)、组织免疫荧光(TIF)和免疫组化(IHC)等方法检测多克隆抗体的效价、特异性与适用性,并以颗粒细胞标志物促卵泡激素受体(FSHR)为参照分析MAPK1表达定位。【结果】成功克隆了番鸭MAPK1基因,大小约1107 bp,编码368个氨基酸。MAPK1蛋白理论分子质量约42 ku。生物信息学预测显示,MAPK1蛋白不含信号肽和跨膜结构;第50—100、200—300和250—350位氨基酸区段亲水性良好,第30—90和200—300氨基酸区段抗原指数较高,第30—70、120—150、200—260及320—360位氨基酸区段的氨基酸表面暴露概率较大。MAPK1蛋白二级结构以α-螺旋为主,包含典型激酶保守结构,活化环呈无规则卷曲状态,主要定位于细胞质和细胞核。综合蛋白特性,最终筛选了第4—364位氨基酸作为候选免疫原区段。成功构建原核重组表达载体pET30-MAPK1,MAPK1重组蛋白以可溶性形式表达,分子质量约48 ku。成功制备了兔抗鸭MAPK1蛋白多克隆抗体,ELISA结果显示该抗体效价达1∶102400;Western blotting、CIF、TIF和IHC结果显示,制备的多克隆抗体可特异识别MAPK1重组蛋白及番鸭卵泡颗粒细胞和卵泡中的内源性MAPK1蛋白。在颗粒细胞中,MAPK1主要定位于卵泡颗粒细胞的细胞质和细胞核;在卵泡中,MAPK1主要分布于卵泡颗粒细胞层,表达定位与FSHR基本一致。【结论】试验成功制备了番鸭MAPK1多克隆抗体,该抗体特异性良好,适用于番鸭卵泡相关细胞与组织样本中MAPK1的免疫学检测。研究结果为MAPK1在卵泡发育及生殖调控中的功能研究提供了技术支撑。展开更多
促分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase, MAPK)级联途径能够将细胞外刺激传导至细胞内,在植物生长发育和逆境响应中发挥重要作用。为进一步探究大豆MAPK基因在盐胁迫响应中的功能,本研究对大豆MAPK基因家族成员进行了...促分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase, MAPK)级联途径能够将细胞外刺激传导至细胞内,在植物生长发育和逆境响应中发挥重要作用。为进一步探究大豆MAPK基因在盐胁迫响应中的功能,本研究对大豆MAPK基因家族成员进行了系统进化分析、共线性分析、motif分析、基因结构分析、顺式作用元件分析、组织特异性表达分析以及NaCl、NaHCO_(3)、PEG和甘露醇胁迫下的表达分析,旨在解析MAPK基因家族的盐胁迫响应机制。通过比较不同非生物胁迫下的表达模式,证实该基因家族通过差异表达调控网络介导大豆对盐胁迫的适应性响应。结果表明:共筛选出19个大豆MAPK家族成员,分布于12条染色体上,其中16号染色体分布最多。理化性质分析表明,MAPK家族成员氨基酸长度为373~571 aa,分子量为41.03~62.81 kDa。系统进化树分析显示,大豆MAPK家族成员可分为4个亚家族。共线性、motif和基因结构分析表明,各亚家族成员间联系紧密,保守性高。启动子顺式作用元件分析发现,大豆MAPK家族成员启动子中含有多种激素和应激响应元件,推测该家族参与大豆非生物胁迫响应。已有研究证实B、C和D亚家族成员参与植物逆境响应机制,因此本研究未将这3族作为重点。表达模式分析显示,在A亚家族的6个成员中,GmMAPK23-4在NaCl、NaHCO_(3)、PEG和甘露醇胁迫下的表达量变化最为显著。本研究为MAPK基因家族的深入研究提供了理论依据,为进一步理解植物逆境适应性及提升作物耐逆性提供了重要参考。展开更多
基金supported by grants from the National Natural Science Foundation of China(Nos.81100282,81030007,81171558,81271808)Program for Changjiang Scholars and Innovative Research Team in University(No.PCSIRT1131)China Postdoctoral Science Foundation(No.2013M531700)
文摘Summary： Excessive activation of macrophages is implicated in various inflammatory injuries. Salidroside （Sal）, one of the main bioactive components ofRhodiola Sachalinensis, has been reported to possess anti-inflammatory activities. This study aimed to examine the effect of Sal on the activa- tion of macrophages and the possible mechanism. The lipopolysaccharide （LPS）-stimulated phrobol 12-myristate 13-acetate （PMA）-differentiated THP-1 macrophage models were established. The changes in the inflammatory profiles of THP-l-derived macrophages were determined. The results showed that Sal significantly decreased the expression of inducible nitric oxide synthase （iNOS）, cyclooxygenase-2 （COX2）, interleukin-lbeta （IL-1β）, interleukin-6 （IL-6） and tumor necrosis fac- tor-or （TNF-a） at both mRNA and protein levels in THP-l-derived macrophages, and the effect was dose-depedent. Moreover, NF-B activation was significantly suppressed and the phosphorylation of ERK, p38 and JNK was substantially down-regulated after Sal treatment. The findings suggested that Sal can suppress the activation of LPS-stimulated PMA-differetiated THP-1 cells, as evidenced by the decreased expression of iNOS, COX2, IL-1β, IL-6 and TNF-a, and the mechanism involves the inhibition of NF-r,B activation and the phosphorylation of the MAPK signal pathway.
