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.

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.

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.

目的:探讨二苯乙烯苷(TSG)通过调控MKK7/JNK通路改善阿尔茨海默病大鼠神经元损伤中的作用机制.方法:采用24月龄的雄性老年大鼠42只,随机分为正常组,假手术组,模型组,阳性药组(0.15 mg/kg石杉碱甲),以及TSG低剂量组(0.033 g/kg)、TSG中剂量组(0.1 g/kg)、TSG高剂量组(0.3 g/kg),每组6只.模型组、TSG各剂量组采用立体定位Aβ25-35溶液构建老年痴呆模型,于造模28 d后经被动回避实验筛选造模成功的大鼠.筛选后阳性药物组、TSG各剂量组按照对应剂量灌胃给药,给药28 d后进行实验.采HE染色镜下观察每组大鼠大脑内海马区神经元细胞形态学变化;IHC 检测各组大鼠脑组织 MKK7、JNK蛋白的表达情况;qRT-PCR检测各大鼠脑组织内MKK7、JNK mRNA的表达情况.结果:(1)HE染色观察,与正常组,假手术组相比,模型组神经细胞数量减少且排列紊乱无规则,细胞膜发生皱缩,细胞核溶解变形.与模型组比较,阳性药组以及TSG各组神经细胞数量增多且排列相对整齐.(2)TUNEL染色阳性细胞比值结果:与正常组、假手术组相比,模型组凋亡细胞数量明显上调;与模型组相比,阳性药物组、TSG各组染色凋亡细胞减少(P<0.05).(3)免疫组化测定结果表明:与正常组和假手术组相比,模型中MKK7、JNK在大鼠脑中的蛋白表达含量显著增加(P<0.05);与模型组比较,阳性药及TSG各剂量组蛋白表达量均有不同程度降低(P<0.05).(4)qRT-PCR结果显示,与正常组、假手术组相比,模型组大鼠脑组织中MKK7、JNK mRNA水平明显上升(P<0.05);与模型组相比,各组给药大鼠脑组织中MKK7、JNK mRNA的表达含量均明显下调(P<0.05).结论:二苯乙烯苷对神经元的损伤修复有一定的作用效果,其作用机制可能与下调MKK7、JNK激酶的mRNA转录及蛋白的表达有关.

目的 探讨"双固一通"电针法通过调控miR-133a-3p-丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MKK)3/MKK 6-p38 丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38 MAPK)通路对心肌缺血再灌注(ischemia reperfusion,I/R)损伤(MIRI)模型大鼠的保护作用.方法 将 50 只大鼠随机分为 5 组:假手术组(sham group,S 组)、模型组(ischemia reperfusion group,I/R组)、电针预处理组(electroacupuncture pretreatment group,EA组)、miR-133-3p抑制剂组(antagomir group,Ant组)、电针预处理+miR-133-3p抑制剂组(EA+Ant组).采用全自动生化分析仪检测各组大鼠血清学指标,透射电镜观察各组大鼠心肌梗死区组织超微结构,荧光定量PCR检测大鼠心肌梗死组织相关基因表达,免疫组化法检测相关蛋白表达水平.结果 与S组比较,I/R组大鼠血清肌酸激酶心肌型同工酶(creatine kinase isoenzymes,CK-MB)和乳酸脱氢酶(lactic dehydrogenase,LDH)水平增加,总抗氧化能力(total antioxidant capacity,T-AOC)水平降低(P<0.05);心肌梗死组织 B 淋巴细胞瘤(B-cell lymphoma,Bcl)-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸天冬氨酸蛋白酶(cystein-asparate protease,Caspase)-3,MKK3,MKK6,p38MAPK表达增加,Bcl-2 表达下降(P<0.05).与I/R组比较,EA组大鼠血清CK-MB和LDH水平降低,T-AOC水平升高(P<0.05);心肌梗死组织Bax、Caspase-3,MKK3,MKK6,p38MAPK表达下降,Bcl-2 表达升高(P<0.05).结论"双固一通"电针预处理可有效改善心肌缺血再灌注损伤,其机制可能与通过调节miR-133a-3p-MKK3/MKK6-p38MAPK通路抑制细胞凋亡有关.

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes.Serine pal-mitoyltransferase(SPT)is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway.The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1(LCB1)and LCB2 subunits.SPT activity is in-hibited by orosomucoid proteins and stimulated by small subunits of SPT(ssSPTs).However,whether LCB1 is modified and how such mod-ification might regulate SPT activity have to date been unclear.Here,we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3(MPK3)and MPK6 by upstream MKK9 and treat-ment with Flg22(a pathogen-associated molec-ular pattern)increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana,with activated MPK3 and MPK6 phosphorylating AtLCB1.Phosphor-ylation of AtLCB1 strengthened its binding with AtLCB2b,promoted its binding with ssSPTs,and stimulated the formation of higher order oligo-meric and active SPT complexes.Our findings therefore suggest a novel regulatory mechanism for SPT activity.