目的:探究丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）通路中ASK1-P38α/JNK在除草醚诱导的先天性膈疝动物模型肺中的表达情况。方法:选取健康成年SPF级SD大鼠，怀孕后通过数字随机法分为空白对照组、膈疝组和膈疝+西地那非干预组3组，通过HE染色检测胎肺发育情况、免疫组织化学定位各组胎肺中的凋亡信号调节激酶1（apoptosis signalregulating kinase 1，ASK1）、丝裂原活化蛋白激酶14（mitogen activated protein kinase 14，P38α）、c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）蛋白和实时定量聚合酶链反应（quantificational real time polymerase chain reaction，qRT-PCR）检测各组胎肺的死亡结构域相关蛋白（Death domain associated protein，DAXX）、ASK1、MAPK激酶3（MAP kinase kinase 3，MKK3）、P38α、MAPK激酶4（MAP kinase kinase 4，MKK4）及JNK的mRNA相对表达量。结果:成功获得对照组4只孕鼠50只活胎、膈疝组6只孕鼠84只活胎，干预组4只孕鼠37只活胎。HE染色后与正常组相比，膈疝组的肺明显发育不良及血管重构，干预组明显改善。免疫组织化学显示，ASK1、P38α、JNK在各个组中均有表达，定位在肺叶边缘、气管、气管软骨、周围结缔组织、发育不良全肺叶及部分血管。qRT-PCR显示，ASK1与上游DAXX、下游P38α和JNK均呈正相关（ r=0.778、 P<0.001， r=0.816、 P<0.001和 r=0.284、 P=0.044），ASK1的mRNA表达量升高导致了下游P38α和JNK升高。DAXX中，膈疝组和干预组均较对照组升高（1.28±0.41、1.31±0.30比1.00±0.20， P<0.05）；ASK1中，膈疝组较对照组显著升高（1.51±0.70比1.00±0.23， P<0.05）；MKK3中，干预组较对照组和膈疝组升高（1.21±0.32比1.00±0.18、0.97±0.35， P<0.05）；MKK4中，干预组较对照组和膈疝组显著升高（1.52±0.40比1.00±0.25、1.19±0.38， P<0.05）。 结论:在除草醚诱导的先天性膈疝动物模型肺中，肺发育不良可能与MAPK通路中的ASK1-P38α/JNK上调有关。

目的:观察大黄灵仙方对肝内胆管细胞炎症模型大鼠MAPK/NF-κB信号通路中IKKα、ASK1、MKK3以及CX3CL1关键因子的表达水平的影响,探讨其缓解肝内胆管细胞炎症的改善作用及可能机制.方法:45只SD大鼠按照完全随机方法分为9组,每组大鼠5只,空白组、模型组、大黄灵仙颗粒组、NF-κB组、p38MAPK组、NF-κB+p38MAPK组、NF-κB+大黄灵仙颗粒组、p38MAPK+大黄灵仙颗粒组、NF-κB+p38MAPK+大黄灵仙颗粒组.除空白组外,余下各组大鼠均在胆总管注射5 mg/kg LPS制备肝内胆管炎症模型.第7天灌胃结束后取大鼠胆管树,采用HE染色观察其炎性程度,蛋白免疫印迹法和实时荧光定量PCR检测IKKα、ASK1、MKK3以及CX3CL1蛋白及mRNA表达量.结果:HE病理结果显示,模型组的肝内胆管组织和细胞炎症明显,加入大黄灵仙颗粒和信号阻断剂后肝内胆管炎症状态较前改善,总病理评分差异有统计学意义(P＜0.05).与模型组比较,大黄灵仙颗粒组、p38MAPK组、NF-κB组、NF-κB+p38MAPK组、NF-κB+大黄灵仙颗粒组、p38MAPK+大黄灵仙颗粒组、NF-κB+p38MAPK+大黄灵仙颗粒组的ASK1、CX3CL1蛋白及mRNA表达量下降,MKK3 mRNA表达量上升以及p38MAPK组IKKα和NF-κB+大黄灵仙颗粒组MKK3 蛋白表达量上升(P＜0.05).与大黄灵仙颗粒组比较,p38MAPK组、NF-κB组、NF-κB+p38MAPK组、p38MAPK+大黄灵仙颗粒组、NF-κB+大黄灵仙颗粒组、NF-κB+p38MAPK+大黄灵仙颗粒组的 ASK1、CX3CL1 mRNA表达量下降,p38MAPK+大黄灵仙颗粒组、NF-κB+大黄灵仙颗粒组、NF-κB+p38MAPK+大黄灵仙颗粒组的MKK3 mRNA表达量上升(P＜0.05).与大黄灵仙颗粒组相比,p38MAPK组的IKKα蛋白表达量下降,NF-κB+大黄灵仙颗粒组的ASK1、MKK3蛋白表达量上升(P＜0.05),而NF-κB+p38MAPK+大黄灵仙颗粒组的CX3CL1 蛋白表达量下降(P＞0.05).结论:大黄灵仙方可缓解LPS诱导的大鼠肝内胆管炎症反应,促进胆管细胞的修复,从而达到降低PIS发生及术后复发的目的,其作用机制可能与抑制NF-κB/MAPK信号通路中的IKKα、ASK1、MKK3以及CX3CL1关键炎症因子的活化有关.

目的 研究芒果苷对良性前列腺增生(BPH)腺体纤维化的改善作用和调节微小RNA(miRNA)-483-3p的作用机制.方法 雄性小鼠随机分为5 组:正常对照组、BPH模型对照组、非那雄胺组、芒果苷组和芒果苷+miRNA-483-3p拮抗剂组.通过摘除睾丸和皮下注射丙酸睾酮建立BPH模型.30d后通过前列腺组织切片行masson和天狼猩红染色,以及检测组织中羟脯氨酸含量评价胶原沉积,实时荧光定量PCR检测前列腺转化生长因子-β1(TGF-β1)、丝裂原活化蛋白激酶2(MK2)和丝裂原活化蛋白激酶6(MKK6)的mRNA水平以及miRNA-483-3p的水平,Western blotting检测前列腺TGF-β1、MK2、磷酸化MK2(p-MK2)、MKK6 和磷酸化MKK6(p-MKK6)的蛋白水平,并采用荧光素酶报告评估miR-NA-483-3p与MK2 的靶向结合能力.结果 与正常对照组比较,BPH模型对照组小鼠的前列腺胶原沉积明显,且 TGF-β1、MK2 和MKK6 的mRNA水平,以及TGF-β1、p-MK2 和p-MKK6 的蛋白水平均显著性升高(P＜0.01),而miRNA-483-3p水平显著性下降(P＜0.01).与BPH模型对照组比较,芒果苷组能够上调前列腺miRNA-483-3p的水平,降低TGF-β1、MK2 和MKK6 的mRNA水平,以及TGF-β1、p-MK2 和p-MKK6 的蛋白水平,减轻胶原沉积.与芒果苷组比较,联合使用芒果苷和miRNA-483-3p拮抗组显著性降低miRNA-483-3p水平,升高TGF-β1、MK2 和MKK6 的mRNA水平,以及 TGF-β1、p-MK2 和p-MKK6 的蛋白水平(P＜0.01).荧光素酶报告显示miRNA-483-3p能够靶向结合MK2.结论 芒果苷能够通过调节miRNA-483-3p,抑制MK2 减轻前列腺纤维化.

Aluminum(Al)toxicity can seriously restrict crop production on acidic soils,which comprise 40％of the world's potentially arable land.The zinc finger transcription factor STOP1 has a conserved and essential function in mediating plant Al resistance.Al stress induces STOP1 accumulation via post-transcriptional regulatory mechanisms.However,the upstream signaling pathway involved in Al-triggered STOP1 accu-mulation remains unclear.Here,we report that the MEKK1-MKK1/2-MPK4 cascade positively regulates STOP1 phosphorylation and stability.Mutations of MEKK1,MKK1I2,or MPK4 lead to decreased STOP1 stability and Al resistance.Al stress induces the kinase activity of MPK4,which interacts with and phos-phorylates STOP1.The phosphorylation of STOP1 reduces its interaction with the F-box protein RAE1 that mediates STOP1 degradation,thereby leading to enhanced STOP1 stability and Al resistance.Taken together,our results suggest that the MEKK1-MKK1/2-MPK4 cascade is important for Al signaling and con-fers Al resistance through phosphorylation-mediated enhancement of STOP1 accumulation in Arabidopsis.

Seed dormancy is nature's strategic pause in the plant life cycle,a purposeful interlude during which a seed,poised on the cusp of potential growth,bides its time in a state of quiescence.This period of dormancy is a crucial adaptation,allowing the seed to withstand unfavorable environmental conditions and syn-chronize germination with optimal circumstances for growth and survival(Née et al.,2017).Dormancy is orchestrated by a complex interplay of genetic,physiological,and environmental factors,including phytohormones like abscisic acid and gibberellins(GAs),as well as specific regulators like DELAY OF GERMINATION1 and others(Née et al.,2017;Liu et al.,2020).

Seeds establish dormancy to delay germination until the arrival of a favorable growing season.In this study,we identify a fate switch comprised of the MKK3-MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination.We show that dormancy-breaking factors activate the MKK3-MPK7 module,which affects the expression of some α-EX-PANSIN(EXPA)genes to control seed dormancy.Furthermore,we identify a direct downstream substrate of this module,ERF4,which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions.The activated MKK3-MPK7 module phosphorylates ERF4,leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs.Collectively,our work identifies a signaling chain consisting of protein phosphorylation,degradation,and gene transcrip-tion,by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.

目的:探讨丝裂原活化蛋白激酶4(MKK4)、生存素(Survivin)基因在喉鳞状细胞癌组织中的表达及其意义.方法:选择82例喉癌患为研究对象,取手术切除的喉癌组织及癌旁正常黏膜组织,采用逆转录聚合酶链反应(RT-PCR)法、免疫组织化学法检测MKK4、Survivin基因表达,分析其与临床病理特征的关系,随访记录喉癌患者5年生存率,并用Cox回归模型对喉癌患者预后因素进行分析.结果:喉癌组织中MKK4 mRNA表达较癌旁组织下降,喉癌组织中Survivin mRNA表达较癌旁组织升高(均P＜0.05).癌旁组织和喉癌组织MKK4蛋白阳性表达率分别为 71.95％(59/82)和 40.24％(33/82),Survivin 蛋白阳性表达率分别为 23.17％(19/82)和 71.95％(59/82)(x2=16.737,x2=39.117,均P＜0.01).不同肿瘤分化程度、TNM分期及有无淋巴结转移患者MKK4、Survivin蛋白阳性表达比较,差异有统计学意义(均P＜0.05).喉癌组织中MKK4和Survivin表达呈负相关(r=-0.403,P＜0.05).MKK4阳性患者5年生存率81.82％(27/33)高于阴性患者65.31％(32/49),Survivin阳性患者5年生存率62.71％(37/59)低于阴性患者82.61％(19/23),影响喉癌患者预后的因素为TNM分期和MKK4、Survivin表达(均P＜0.05).结论:喉癌组织中MKK4表达下调,Survivin表达上调,两者可能是潜在的预后标记物以及治疗靶点.

目的 探讨KCNQ1OT1基因敲除联合鸦胆子素D对乳腺癌MDA-MB-231细胞生物学行为的影响及其机制.方法 CCK8、划痕和Transwell侵袭实验分别检测鸦胆子素D和siKCNQ1OT1对MDA-MB-231细胞活力、迁移和侵袭能力的影响;qRT-PCR检测鸦胆子素D、siKCNQ1OT1对MDA-MB-231细胞中KCNQ1OT1表达的影响;Western blot法检测EMT相关蛋白及CDC42、p-MKK7、MKK7蛋白的表达情况.结果 鸦胆子素D和siKCNQ1OT1处理的MDA-MB-231 细胞活力、迁移和侵袭能力受到显著抑制,且二者联用时抑制作用更强( 均P < 0 . 0 5 ) ; 鸦胆子素D 处理后KCNQ1OT1在MDA-MB-231细胞中的表达下调(均P<0.05);鸦胆子素D联合siKCNQ1OT1处理组MDAMB-231细胞中CDC42、p-MKK7、N-cadherin和Vimentin的表达显著下调,E-cadherin的表达上调(均P<0.05).结论 鸦胆子素D联合siKCNQ1OT1抑制人乳腺癌MDA-MB-231细胞增殖、迁移、侵袭和EMT,其分子机制可能与CDC42/MKK7信号通路的阻断有关.

植物感受到重力刺激后可通过重力反应协调自身各器官的生长方向.在植物重力反应过程中,重力感应和信号转导一直都是植物学领域关注的焦点.经典的"淀粉-平衡石"假说认为植物对重力的感应是通过淀粉体(富含淀粉的质体)沉降来实现.此外,研究发现LAZY蛋白通过调控生长素的不对称分布参与植物重力反应.然而,淀粉体沉降引发的重力信号转导及其与LAZY蛋白之间协作的分子机制仍不清楚.近期,清华大学陈浩东研究团队发现重力刺激能够诱导拟南芥(Arabidopsis thaliana)MKK5-MPK3激酶途径,进而对LAZY蛋白进行磷酸化,LAZY蛋白的磷酸化增强其与淀粉体表面TOC蛋白的互作,促进LAZY蛋白在淀粉体表面富集.淀粉体沉降引导LAZY蛋白在新的底侧质膜极性再定位.该研究深入解析了植物重力信号转导的分子机制,建立了植物重力感应与LAZY蛋白介导的生长素不对称分布之间的联系,是植物向重力性研究领域的重大突破.

Mitogen-activated protein kinase(MAPK)cas-cades play pivotal roles in plant defense against phytopathogens downstream of im-mune receptor complexes.The amplitude and duration of MAPK activation must be strictly controlled,but the underlying mechanism re-mains unclear.Here,we identified Arabidopsis CPL1(C-terminal domain phosphatase-like 1)as a negative regulator of microbe-associated molecular pattern(MAMP)-triggered immunity via a forward-genetic screen.Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bac-terial peptide flg22.Furthermore,flg22-induced MPK3/MPK4/MPK6 phosphorylation was dra-matically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines,sug-gesting that CPL1 might interfere with flg22-induced MAPK activation.Indeed,CPL1 di-rectly interacted with MPK3 and MPK6,as well as the upstream MKK4 and MKK5.A firefly luciferase-based complementation assay in-dicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly re-duced in the presence of CPL1.These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.