ER membrane ubiquitin ligase Hrdl mediates tau degradation in proteosome-dependent and independe

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大豆黄酮对衰老小鼠脑组织神经递质含量的调节

中图分类号：Ｑ９６５文献标志码：Ａ文章编号：１７．５０２１）６０１．４６２６１（０１０ —０６０
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第２卷６
第６期
天津科技大学学报
Ｊｕｎｌｆｉｎｉｉｅｓｙｏｃｅｃ＆ＴｃｎｌｇｏｒａａｊｎＵｎｖｒｉｆｉｅｏＴｔＳｎｅｈｏｏｙ
、ｏ．６ＮＯ．，１２６Ｄｅ．ｃ２０１１
２１年１０１２月
大豆黄酮对衰老小鼠脑组织神经递质含量的调节
才金玲，单 Leabharlann ，陆军，郑元林（．天津市海洋资源与化学重点实验室，天津科技大学海洋科学与工程学院，天津３０５；１０４７２徐州师范大学生命科学学院，徐州２１１）．２１６
摘
要：研究大豆黄酮对Ｄ一乳糖致衰老小鼠脑神经递质水平的调节．续背部注射Ｄ一乳糖６周，立衰老模半连半建
ｂａｎｏｅａｅｉｅｗｅｅｅａｎｄＴｈｅｕｔｓｏｄｔａａｄｅｎｃｕｄｉｃｅｓｃｔｌｈｌｅａｅｙｃｏｉｅｔｒｓｒｉｆｔｇｄｍｃｒｘｍｉｅ．ｅｒｓｌｈｗｅｔｉｚｉｏｌｎｒａｅａｅｙｃｏｉ，ｃｔｌｈｌｓｅａｅ，ｈｓｈｄｎｎｄｐｉｅａｄ５ｈｄｏｙｒｐａｎｅｅｓｉｏｅｄｈｐｏａｕｆａｅｃ．ｉｚｉｏｌｃｅｓｅｏｒｓｔｏａｎ，ｍｎ－ｙｒｘｔｙｔｍｉｅｌｖｌｃｄｘａｉｐｃｍｐｓｏｇｄｍｉｅＤａｄｅｎｃｕｄｉｒａｅｎｕｔｎｎｎｒｎａｍｉ－ｔｒｅｅｓｏｅｍｉｅＳｒｉｄｈｖｎｉｅｅｅｃｏｅｄｇｅ．ｅｖｌｆｈｃ ’ ｂａｎａａｅａｔｓｎｃｎｅｉｓｍｅｒｅｌｔｎｎ

美国FDA批准Myrbetriq(mirabegron)用于治疗神经源性逼尿肌过度活动症

（来源：美国 FDA 政府公告，2021-03-25 夏训明编译）
Myrbetriq 片剂与 Myrbetriq 颗粒剂治疗 NDO 最常见的毒副作用包括尿路感染、鼻咽炎（普通感冒症状）、便秘、头痛、血压升高、血管性水肿等。
需要注意的是，Myrbetriq 片剂与 Myrbetriq 颗粒剂是 2 种不同的产品，不能相互按照 1 mg 对 1 mg 进行替代。 Myrbetriq 颗粒剂用于成年人的推荐剂量尚未进行研究。
Myrbetriq 片剂与 Myrbetriq 颗粒剂治疗儿童 NDO 的有效性经 1 项试验研究得到验证，该项试验有 86 名 3~17 岁的 NDO 患者参加。在用药 24 周后，受试者最大膀胱容量(Maximum cystometric capacity,MCC)、逼尿肌收缩次数、逼尿肌首次收缩时膀胱储尿量以及每日出现漏尿次数等指标都有改善。
（责任编辑：陈翔）
美国 FDA 批准 Myrbetriq(mirabegron)用于治疗神经源性
逼尿肌过度活动症
美国 FDA 于 2021 年 3 月 25 日批准美国安斯泰来制药公司(Astellas Pharma US,Inc.)的 Myrbetriq(mirabegron, CAS 登记号 223673-61-8)缓释片剂与 Myrbetriq 颗粒剂（mirabegron 缓释口服混悬液）用于年龄为 3 岁及以上儿童治疗神经源性逼尿肌过度活动症(neurogenic detrusor overactivity,NDO)。这是 FDA 批准 Myrbetriq 增加的新适用症。此前 FDA 于 2012 年 6 月 28 日批准 Myrbetriq(mirabegron)用于成人治疗膀胱过度活动症(overactive bladder, OAB)。

分子生物学词汇(中英文对照表 )

脑力苏胶囊对血管性痴呆小鼠脑组织NGF表达的影响

胶囊治疗Ｖ的部分作用机理。方法：用结扎双侧颈总动脉方法制备血管性痴呆小鼠动物模型，酶联免疫吸附法Ｄ采用
（ＬＳ法）定给药后Ｖ小鼠脑组织内神经生长因子（Ｇ）含量变化。结果：药１后，Ｄ小鼠脑内神经生长因ＥＩＡ测ＤＮＦ的给５ｄＶ子（Ｇ）模型对照组显著增高。结论：力苏胶囊可增加ＶＮＦ较脑Ｄ大鼠脑内神经生长因子（Ｇ）ＮＦ的含量，能与改善Ｖ小可Ｄ
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随着人口的老龄化，呆已成为老年人的常见病、痴多
［ｂｔｃ］ｊｃｉｅＴｔｙｈｆｃｏａＡｓａｔＯｂｅｔ：ｏｕｅｅｅｔｆｒｉｏｉａｓｌｏｒｉｎｒｅｒｗｈａｔ（Ｇ）ｅｐｅｓｎｉｖｓｕａｒｖｓｄｔｆｂｎ—ＳｖｔｐｕｅｎｂａｅｖｏｔｆｃｒＮＦｘｒｓｉａｃｌｒｅｃｎｇｏｏｎ
Ｊｎ２０ｕ．０１
Ｖ１３Ｎ．０．６ｏ３
现代临床 Nhomakorabea医
学
２１００年６月
ＪＯＵＲＮＡＬＯＦＭＯＤＥＩＣＡＬＭＥＤＩＮＥＲＮＣＬＮＩＣＩ

下丘脑调节肽名词解释

下丘脑调节肽名词解释下丘脑调节肽（Hypothalamic Regulating Peptide，HRP）是一类在下丘脑中产生，并通过下丘脑-垂体系统发挥调节作用的多肽物质。

以下将从功能、合成与分泌、作用机制等方面对下丘脑调节肽进行详细解释。

功能：下丘脑调节肽是一类重要的神经调节剂，主要负责调节下丘脑功能。

它们通过与下丘脑中的神经元结合，促进或抑制下丘脑神经元的活动，从而调节垂体前叶激素的合成和释放，调控内分泌系统。

下丘脑调节肽对于维持机体的内环境相对恒定至关重要，它们能够调控性腺、垂体、甲状腺、肾上腺等内分泌器官的功能。

合成与分泌：下丘脑调节肽由下丘脑中的特定神经元合成和分泌。

这些特定神经元存在于下丘脑的特定细胞核中，依赖于外源激素的刺激合成和分泌。

这些刺激可以是某些物质如甲状腺释放激素（TRH）和生长激素释放激素（GHRH）的作用，也可以是外界环境刺激的影响。

合成过程一般包括基因转录、翻译、后转录修饰和包装。

合成完成后，下丘脑调节肽通过轴突运输至垂体后叶或者其他特定的靶细胞进行释放，达到调节机体功能的目的。

作用机制：下丘脑调节肽通过作用于垂体前叶的相应受体发挥作用。

这些受体在垂体细胞膜上，与下丘脑调节肽结合后，可通过多种信号转导途径，调控细胞内的离子流、蛋白激酶的活性等。

具体作用机制的差异既受调节肽的种类和浓度的影响，也受垂体靶细胞自身特性的限制。

下丘脑调节肽通过调节垂体前叶细胞的器质性激素的合成和分泌，实现了对内分泌系统的调控。

总结而言，下丘脑调节肽是一类在下丘脑中产生的多肽物质，通过调节垂体前叶激素的合成和释放，调控内分泌系统的功能。

它具有调控性腺、垂体、甲状腺、肾上腺等内分泌器官的重要作用。

下丘脑调节肽的合成和分泌受到外源激素和环境刺激的影响，其作用机制涉及多种信号转导途径的调控。

对于正常机体内环境的维持和内分泌平衡的调节至关重要。

双氢青蒿素（DHA）通过调节自噬相关信号诱导PC12分化

分类号：R180.47单位代码：10719学号：*********密级：延安大学论文题目：双氢青蒿素（DHA）通过调节自噬相关信号诱导PC12分化论文作者：王艺臻指导教师、职称：王璐教授学科、专业名称：神经生物学提交论文日期：零一八年四月创新性声明本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成果。

尽我所知，除了文中特别加以标注和致谢中所罗列的内容以外，论文中不包含其他人已经发表或撰写过的研究成果；也不包含为获得延安大学或其它教育机构的学位或证书而使用过的材料。

与我一同工作的同志对本研究所做的任何贡献均已在论文中做了明确的说明并表示谢意。

申请学位论文与资料若有不实之处，本人承担一切相关责任。

本人签名：日期：关于论文使用授权的说明本人完全了解延安大学有关保留和使用学位论文的规定，即：研究生在校攻读学位期间论文工作的知识产权单位属延安大学。

本人保证毕业离校后，发表论文或使用论文工作成果时署名单位仍然为延安大学。

学校有权保留送交论文的复印件，允许查阅和借阅论文；学校可以公布论文的全部或部分内容，可以允许采用影印、缩印或其它复制手段保存论文。

（保密的论文在解密后遵守此规定）本人签名：日期：导师签名：日期：双氢青蒿素（DHA）通过调节自噬相关信号诱导PC12分化神经生物学专业研究生王艺臻指导老师王璐教授摘要：实验目的：双氢青蒿素(DHA)神经毒理学实验发现，DHA干预PC12后，细胞突触伸长，PC12分化速度加快，我们对DHA诱导PC12分化的机制进行探讨。

有望开发有效安全的治疗神经退行性疾病药物。

实验方法：K8比色法检测药物最适浓度，分别用0.01、0.1、1、10、25mg/L DHA干预PC12。

筛选DHA的最适浓度。

2.光学显微镜计数DHA干预PC12细胞形态学的变化以及突触的生长；3.流式细胞术（FACS）分析不同浓度DHA干预PC12细胞内ROS的；4.免疫荧光染色神经元标志物MAP2；5.RT-PCR法相对定量DHA干预PC12后，Beclin、LC-3、ATG5、MAPK基因表达水平检测。

血管紧张素

可被其他杂环取代，但咪唑环活性最佳
N NR
5位可变范围大，以可形成氢键的小基团为好
酸性基团
2位可引入2~6个碳的直链烷基、烯基和芳基，以正丙基和正丁基活性最好
对位取代有利活性
氯沙坦 Losartan
5、构效关系
H3C
Cl N
N
邻位酸性取代基有利，四氮唑增强活性；若为三氮唑则需在苯环上引入吸电子基团
含羧基的 ACEI
螺普利 spirapril
H3CH2CO
O CH3
S S
N
N
H O
COOH
1995年在荷兰上市最大降压作用出现在口服后4-8h 作用维持24h
赖诺普利Lisinopril
依那普利的赖氨酸衍生物，降压作用缓慢而长效
含磷酸基的 ACEI 福辛普利 fosinopril
0.5h起效，酯键在体内易水解，水解后产物仍具药理活性
血管紧张素I Angiotensin I
血管紧张素转化酶 ACE
ACEI ARB
Asp-Arg-Val-Tyr-Ile-His-Pro-
Phe 血管紧张素II Angiotensin II
Angiotensin II受体
血管收缩
醛固酮 Aldosterone
血容量增加
血压升高
卡托普利与 ACE 的相互作用
氯沙坦 Losartan
2、药理作用
Cl
N
H3C
OH
N
NN
N NH
药理作用：强力和持久性地降低血压，收缩压和舒张压均下降。
对各种组织中的AT1受体有较高的亲和力和选择性，直接阻断AⅡ分子与相应受体的结合，阻断所有已知AII效应。

Acta Biochim Biophys Sin-2012-Ma-948-56

Original ArticleA new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretionYi Ma,Tianjie Luo,Wenna Xu,Zulu Ye,and An Hong*Department of Cell Biology,Institute of Biological Medicine,Jinan University,Guangzhou510632,China*Correspondence address.Tel:þ86-20-85223266;Fax:þ86-20-85221983;E-mail:makesi8866@The recombinant peptide,DBAYL,a promising thera-peutic peptide for type2diabetes,is a new,potent,and highly selective agonist for VPAC2generated through site-directed mutagenesis based on sequence alignments of pi-tuitary adenylate cyclase-activating peptide(PACAP), vasoactive intestinal peptide(VIP),and related analogs. The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utiliza-tion.As much as28.9mg recombinant DBAYL peptide with purity over98%can be obtained from1l of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminal) were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about25folds compared with that of BAY55-9837in vitro.The bioactivity assay of DBAYL showed that it displaced[125I]PACAP38and [125I]VIP from VPAC2with a half-maximal inhibitory concentration of48.4+6.9and47.1+4.9nM,respective-ly,which were significantly lower than that of BAY55-9837,one established VPAC2agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2with a half-maximal stimulatory concentration (EC50)of0.68nM,whereas the receptor potency of DBAYL at human VPAC1(EC50of737nM)was only1/1083 of that at human VPAC2,and DBAYL had no activity toward human PAC1receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin re-ceptor substrate1(IRS-1)and glucose transporter4 (GLUT4)indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1and GLUT4expression more efficiently than BAY55-9837and VIP in adipocytes. Compared with BAY55-9837and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research(ICR)mice.These results suggested that DBAYL could efficiently improve glucose uptake and glucose-depend-ent insulin secretion by VPAC2-mediated effect.Keywords pituitary adenylate cyclase-activating peptide; type2diabetes;insulin;VPAC2-mediated effect;recombinant peptideReceived:June29,2012Accepted:August6,2012 IntroductionPituitary adenylate cyclase-activating polypeptide(PACAP) is a member of the superfamily of metabolic,neuroendo-crine,and neurotransmitter peptide hormones and belongs to the secretin,glucagons,and vasoactive intestinal peptide (VIP)family[1,2].PACAP exists as either a38-amino acid (PACAP38)or27-amino acid(PACAP27)peptide. PACAP27corresponds to the N-terminal27-amino acid portion of PACAP38and exhibits the same biological activ-ity as PACAP38[3,4].The action of PACAP is mediated through three G protein-coupled receptors,PAC1,VPAC1, and VPAC2.PAC1receptor exhibits high affinity for PACAP38and PACAP27,but much lower affinity for VIP. VPAC1and VPAC2receptors exhibit similar high affinity for PACAP38,PACAP27,and VIP[5].PACAP is widely distributed in the brain and peripheral organs,notably in the endocrine pancreas,gonads,respiratory,and urogenital tracts,which has been shown to have effects on many pathological states including Parkinson’s disease[6],dia-betes[7,8],ischemia[9],traumatic injury[10],immuno-logical disorders[11,12],myeloma kidney injury,and so on [13].Most of these neuroprotective actions of PACAP are mediated through the selective PAC1receptor whereas the effects on peripheral organs often involve VPAC1or VPAC2receptor.PACAP has been shown to increase insulin secretion from the pancreas through VPAC2recep-tor[14,15].But the role of PACAP in the control of glucose homeostasis is complex,because it also plays a role in increasing glucagon and catecholamine secretion,which increases glucose output from the liver through VPAC1-mediated effect[16].Therefore,PACAP derivative asActa Biochim Biophys Sin2012,44:948–956|ªThe Author2012.Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.DOI:10.1093/abbs/gms078.Acta Biochim Biophys Sin(2012)|Volume44|Issue11|Page948 at Jinan University on November 16, 2014 / Downloaded fromVPAC2-specific agonist,which would stimulate glucose-dependent insulin secretion from pancreatic b-cell without leading to increased glucose production by the liver could be used for clinical treatment of type2diabetes. Development of BAY55-9837,an established highly select-ive VPAC2agonist,as a potential peptide therapeutic for the treatment of type2diabetes was limited by its poor peptide stability[14].To overcome the limitation,the re-combinant peptide DBAYL with32amino acids was designed and generated through site-directed mutagenesis by gene-recombination technology.The recombinant DBAYL (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.DBAYL enhances the cAMP accumulation in VPAC2-CHO cells with higher bio-activity than BAY55-9837.DBAYL could more efficiently induce the expression of the key proteins of insulin receptor signaling pathway including insulin receptor substrate1 (IRS1)and glucose transporter4(GLUT4)than BAY55-9837in adipocytes[17,18].In addition,DBAYL treatment increased the insulin-stimulated GLUT4translocation to the plasma membrane.Corresponding to these results,glucose uptake activity of differentiated3T3-L1adipocytes treated with DBAYL were significantly improved,which was better than BAY55-9837.Thus,insulin signal transduction was more efficiently improved by DBAYL through VPAC2-mediated effect. DBAYL,a novel recombinant PACAP-derived peptide,as highly selective agonist for VPAC2,can hopefully be a peptide therapeutic for type2diabetes through efficiently promoting glucose uptake and glucose-dependent insulin secretion.Materials and MethodsMaterialsChitin beads and the plasmid pKYB-MCS were purchased from New England Biolabs(NEB,Ipswich,USA). Escherichia coli strain ER2566was kept in our laboratory. All the restriction enzymes were purchased from New England Biolabs.T4DNA ligase was obtained from TaKaRa (Dalian,China).Synthetic peptides were purchased from Sinoasis Pharmaceuticals(Guangzhou,China).Primer syn-thesis and DNA sequencing were performed by Invitrogen Company,Guangzhou Branch(Guangzhou,China).VPAC2-CHO cell line was constructed in our laboratory.3T3-L1 adipocytes were provided by Dr Zhang WJ(College of Life Sciences,Wuhan University,Wuhan,China). Construction and identification of the expression plasmid pKY-DBAYLThe DBAYL gene was designed according to the bias of E.coli for the codons to ensure its high expression.The gene was synthesized and amplified in two steps asdescribed previously[7]using three oligonucleotides primers:F1:50-GGTGGTCATATGCATAGCGATGCGGT GTTTACCGATCAGTATACCCGTCTGCGTAAA-30,con-taining an Nde I site(underlined);F2:50-CAGATATT TTTTCGCCGCCAGCTGTTTACGCAGACGGGT-30;F3:50-CCACCATGCTCTTCCGCAATAACGTTTCTGTTTAA TGCTCTGCAGATATTTTTT-30,containing an Sap I site (underlined);GGTGGT at the50end of F1and CCACCAat the50end of F3are the protecting bases.After polymer-ase chain reaction(PCR)products were purified by thePCR clean-up kit(Qiagen,Hilden,Germany)and digestedwith Nde I and Sap I,the DNA fragment was directly ligatedto a gel-purified Nde I/Sap I digested pKYB-MCS vector (NEB)to yield the expression plasmid pKY-DBAYL.pKY-DBAYL containing DBAYL gene was confirmed byDNA sequencing using the T7promoter as the sequencingprimer(Fig.1).Expression of fusion proteinThe recombinant expression vector pKY-DBAYL was transformed into the E.coli strain ER2566with the opti-mized procedure[19].Briefly,the cells were grown at378Cto a density of OD600¼0.8and induced by adding isopro-pyl b-D-thiogalactoside to a final concentration of0.5mM.Figure1The constructed recombinant expression vectorpKY-DBAYL(A)The amino acid sequence of RBAYL and rBAY.(B)The construction map of the expression plasmid pKY-DBAYL.PACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin(2012)|Volume44|Issue11|Page949at Jinan University on November 16, 2014/Downloaded fromThe induced cells were incubated for6h at358C and col-lected by centrifugation at10,621g for20min.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was used to identify the expression of the fusion protein.The cell pellet was resuspended in buffer A con-taining20mM Tris-HCl(pH8.0),500mM NaCl, and1mM EDTA by gentle shaking for20min,and then disrupted with JN-3000PLUS low-temperature ultra-high-pressure continuous flow cell crusher(JNBIO, Guangzhou,China)at the following conditions:diluted bac-teria concentration of18%by buffer A,crushing pressure of 1700bar and cooling temperature of38C.The lysate was then centrifuged at10,621g for30min at48C and the supernatant was subjected to purification and preparation of target peptide by chitin beads affinity chromatography. Preparation and identification of the recombinant peptide DBAYLThe supernatant(1.5l)was passed through a column (4.5cmÂ20cm)packed with25ml chitin beads at a flow rate of0.5ml/min.After the supernatant was loaded on the column,the flow rate was raised to2ml/min and the column was thoroughly washed with more than10bed volume of buffer A.Then80ml of buffer B containing 20mM Tris-HCl(pH8.0),500mM NaCl,1mM EDTA, and100mM b-mercaptoethanol was then quickly passed through the column to distribute b-mercaptoethanol evenly throughout the resin and the column flow was stopped.The column was incubated at258C for24h.Fractions contain-ing DBAYL were obtained by eluting the column with buffer A.Then the recombinant peptide DBAYL was puri-fied and prepared by reverse-phase high-performance liquid chromatography(HPLC)system using4.6mmÂ150mm 300SB-C18Sep-Pak column(Agilent Technologies, Beijing,China)through gradient elution with increasing concentration of acetonitrile from2%to55%for45min at 1ml/min.The eluate containing DBAYL was dried by ly-ophilization.Prepared DBAYL at a final concentration of 1mg/ml in45%acetonitrile containing0.1%trifluoroacetic acid was analyzed by4000Q TRAP electrospray ionization-mass spectrometry(ESI-MS;Applied Biosystems,Foster City,USA).Peptide concentrations were determined by com-paring the OD280values of peptide stock solutions in the assay buffer with the predicted extinction coefficient[20]. Stability assayDBAYL,BAY55-9837,PACAP38,or VIP at a final concen-tration of1mg/ml in20mM sodium phosphate buffer(pH 8.0)containing150mM sodium chloride were incubated at 378C.At different time points,samples were collected and analyzed by liquid chromatography mass spectrometry,a rapid and sensitive method to detect degradation of polypep-tide in these formulations.A2-ml sample was injected into HPLC-ESI-MS system containing 1.0mmÂ150mm300 SB-C18Sep-Pak(Agilent Technologies,Santa Clara,USA) column and analyzed under the condition of increasing con-centration of acetonitrile from2%to55%for55min at 0.05ml/min by HPLC-ESI-MS system.Competition receptor binding assayThe potential of DBAYL to displace[125I]PACAP38 and[125I]VIP by competitively binding to the human VPAC2receptor was examined in VPAC2-CHO cell mem-brane prepared previously[7].Briefly,10mg of membrane was incubated with0.1nM[125I]PACAP38(Phoenix Pharmaceuticals,Mountain View,USA)or[125I]VIP (PerkinElmer Life and Analytical Sciences,Boston,USA) in the presence of increasing concentrations of DBAYL peptide,in a total volume of100ml of20mM HEPES (pH7.4)containing150mM NaCl,0.5%BSA,2mM MgCl2,and0.1mg/ml bacitracin at378C.After being incu-bated for20min,the membrane was collected on GF/C filters pretreated with0.1%polyethylenimine.The filters were washed with25mM cold Na3PO4containing1% BSA and counted on a gamma counter.Non-specific binding was defined as the residual binding in the presence of1mM recombinant PACAP38(i.e.rPACAP38)or VIP and was always,20%of the total binding.The assay of PACAP38,VIP,and BAY55-9837were taken as the positive controls.[K15,R16,L27]VIP(1–7)/GRF(8–27),a VPAC1-specific agonist,was used as the negative control in the receptor binding assay[21].Each assay was per-formed at least three times.Assay of cAMP accumulation induced by DBAYL Human PACAP receptor-transfected cells,VPAC1-CHO, VPAC2-CHO,and PAC1-CHO cells,cultured in the Dulbecco’s modified Eagle’s medium at378C were scraped off with rubber policeman and washed with PBS twice. The density of the cells was adjusted to2Â106cells/ml. DBAYL or rPACAP38was added to the500-ml cell sus-pension,and the concentrations of the peptide were ranged from1Â10212to1Â1025M.The mixtures were incu-bated at378C for5min,then two volumes of0.2M HCl was added,and the mixtures were incubated at room tem-perature for another20min.Cells were lysed by pipetting up and down until the suspension was homogeneous.The precipitate was removed by centrifugation at225g for 10min,and the supernatant was transferred into test tube and cAMP concentrations were measured by using the cyclic AMP enzyme immunoassay kit(Cayman Chemical Company,Ann Arbor,USA).Western blot analysis of IRS-1and GLUT4induced by DBAYLCell culture and induction of3T3-L1adipocytes were carried out as described previously[22].DifferentiatedPACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin(2012)|Volume44|Issue11|Page950 at Jinan University on November 16, 2014 / Downloaded from3T3-L1adipocytes were incubated with100nM insulin for 20min[23].After being washed twice with PBS buffer, differentiated3T3-L1adipocytes were cultured for48h in medium,respectively,containing0and1m M of DBAYL, BAY55-9837or VIP.Then the total protein was extracted. After the total protein was separated by12%SDS-PAGE and transferred onto poly(vinylidene difluoride)membranes (Immobilon P;Millipore,Billerica,USA),the membranes were incubated with the IRS-1rabbit mAb(Cell Signaling Technology,Boston,USA)or anti-GLUT4antibody(Santa Cruz Biotechnology,Santa Cruz,USA)for2h at room temperature.The horseradish peroxide(HRP)-conjugated goat-anti-rabbit IgG(Immunology Consultants Laboratory, Portland,USA)or sheep-anti-mouse HRP-IgG(BioFX Laboratories,Owings Mills,USA)was used as the second antibody.Protein bands were visualized by using an ECL kit(Santa Cruz Biotechnology)and densitometric analysis of the results of western blot was performed with image analysis software[24].To evaluate the effect of DBAYL on GLUT4translocat-ing to the plasma membrane,differentiated3T3-L1adipo-cytes were incubated with100nM insulin for20min.After being washed twice with PBS buffer,differentiated3T3-L1 adipocytes were cultured for48h in medium containing 1m M of DBAYL.Another experiment group that differen-tiated3T3-L1adipocytes were cultured for48h in medium containing1m M of DBAYL without insulin treatment to determine whether the manner of the DBAYL effect on GLUT4translocating to the plasma membrane is insulin-dependent or non-insulin-dependent.Then plasma membrane lawns were prepared by sonic-ation as described previously[25].GLUT4contents of the plasma membrane lawns were determined by immunoblot-ting using anti-GLUT4antibody performed with an ECL kit. Effect of DBAYL on glucose uptake activity Differentiated3T3-L1adipocytes were incubated with 100nM insulin for20min[23].After being washed twice with PBS buffer,the3T3-L1adipocytes were cultured for 48h in medium,respectively,containing0,1,and5m M of DBAYL or BAY55-9837.The glucose level of the cell culture supernatants was determined with Glucose assay kit-glucose oxidase method(Applygen Technologies Inc., Beijing,China).Effect of DBAYL on insulin release and glucose disposal in ICR miceEighteen male ICR mice weighing25–30g were housed at room temperature on a12/12h light/dark cycle.ICR mice fasted over-night(12h)and were randomly divided into three groups according to their weight(six per group).The prepared DBAYL(0.5m g/kg)that is dissolved in the normal saline was intraperitoneally injected into the ICRmice and10min later,glucose dissolved in distilled water(2g/kg)was given to ICR mice by gavage.The experimen-tal groups with the same dose or volume of BAY55-9837and rPACAP38were as positive controls and the groupswith normal saline as a negative control.At15min after gavage,blood samples were collected from the tail vein andthe plasma glucose levels were determined using OneTouchUltra Meter(Johnson&Johnson,Johnson,USA)and the plasma insulin was measured using RIA kit(Linco Research,Charles,USA)in the First Affiliated Hospital ofJinan University(Guangzhou,China).ResultsExpression and preparation for DBAYLThe fusion proteins consisting of target peptide-,intein-and chitin-binding domain(i.e.DBAYL-intein-CBD)were expressed through a recombinant peptide expression vector,pKY-DBAYL,in E.coli strain ER2566.The fusion pro-teins were purified using chitin affinity column.The cleav-age of intein was induced by b-mercaptoethanol and thetarget peptide,DBAYL was released.Then the recombin-ant peptide DBAYL was further purified and prepared by reverse phase HPLC system.About28.9mg of recombin-ant DBAYL peptide over98%of purity can be obtainedfrom1l of Luria-Bertani medium.The prepared DBAYLwas analyzed and identified by ESI-MS.Figure2 showed that the molecular weight of DBAYL fromESI-MS was3916.6Da,which was consistent with the theoretical value(3916.5Da).The purity of prepared DBAYL was over98%by the analytical HPLC determin-ation method.Peptide stability improved by site-directed mutagenesisThe recombinant DBAYL was tested together withBAY55-9837,PACAP38,and VIP for stability at378C in20mM sodium phosphate buffer(pH8.0)containing150mM sodium chloride.After4weeks at378C,the main peptide peaks for BAY55-9837,PACAP38,and VIP were remarkably diminished and the slower migrating peak emerged,probably as a result of peptide degradation.Onthe other hand,DBAYL exhibited dramatic improvementin stability,losing only7.7%of the main peak.The sta-bility data in4weeks showed that the half-life of recom-binant DBAYL was about25folds compared with that ofBAY55-9837in vitro,and the half-life of wild-type PACAP38and VIP is slightly shorter than the BAY55-9837in vitro(Fig.3).DBAY L selectively binding to VPAC2receptorHuman VPAC2receptor-transfected cells,and VPAC2-CHO cells,were used for competition receptor binding petition binding of[125I]PACAP38or[125I]VIPPACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin(2012)|Volume44|Issue11|Page951at Jinan University on November 16, 2014/Downloaded fromon membranes purified from CHO cells identified DBAYL as a VPAC2-selective peptide (Fig.4).DBAYL competi-tively displaced [125I]PACAP38from VPAC2,with a half-maximal inhibitory concentration (IC50)of 48.4+6.9nM,and the IC50of the recombinant PACAP38,VIP,and BAY55-9837were 18.1+5.3,21.2+4.0,and 68.3+8.1nM,respectively [Fig.4(A)].DBAYL competitively displaced [125I]VIP from VPAC2with an IC50of 47.1+4.9nM,and the IC50for rPACAP38,VIP,and BAY55-9837at human VPAC2were 19.7+4.9,18.0+2.6,and 70.3+3.7nM,respectively [Fig.4(B)].Whereas the IC50for VIP(1–7)/GRF(8–27),an established human VPAC1-specific agonist,at human VPAC2was over 20m M.These results showed that DBAYL could competi-tively displace [125I]PACAP38and [125I]VIP by binding to human VPAC2receptor in VPAC2-CHO cells.In two com-petition receptor-binding experiments,the IC50of DBAYL was significantly lower than that of BAY55-9837,the established VPAC2-specific agonist.Receptor potency of DBAYL at PACAP receptors subtypesThe accumulation of cAMP in human PACAP receptor-transfected cells (VPAC1-CHO,VPAC2-CHO,andPAC1-Figure 2The ESI-MS of the prepared recombinant DBAYL Prepared DBAYL at 1mg/ml in 45%acetonitrile containing 0.1%TFA was analyzedby electrospray ionization time-of-flight massspectrometry.Figure 3Stability analysis of peptides at 1mg/ml in aqueous solution during incubation at 378C Sample (2-ml)was injected into HPLC-ESI-MS system and analyzed under the condition of increasing concentration of acetonitrile from 2%to 55%for 55min at 0.05ml/min.PACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin (2012)|Volume 44|Issue 11|Page 952at Jinan University on November 16, 2014/Downloaded fromCHO cells)was used as an index of the agonist activity.DBAYL was a potent agonist for the VPAC2receptor with a half-maximal stimulatory concentration (EC50)of 0.68nM.However,the receptor potency of DBAYL at human VPAC1(EC50of 737nM;Fig.5)was only 1/1083of that at human VPAC2,and DBAYL had no activity toward human PAC1receptor.Three receptors subtypes are both activated by rPACAP38.rPACAP38was a potent agonist at human PAC1with an EC50of 0.57nM,and the EC50for rPACAP38at human VPAC1and VPAC2receptor were 0.97and 0.99nM,respect-ively (Fig.5).These results showed that DBAYL was a VPAC2-spcific agonist with high potency and bioactivity,whereas rPACAP38could active human PAC1,VPAC1,and VPAC2receptor with different affinity.In vitro effects of DBAYL on the key proteins in insulin receptor signaling pathwayThe expression levels of IRS1,a key and essential protein for insulin signal transduction and GLUT4,an important rate-limiting factor of the glucose transport,were signifi-cantly increased in differentiated 3T3-L1adipocytes treated with DBAYL.Figure 6(A)showed that the insulin-stimulated IRS1and GLUT4expression levels in differentiated 3T3-L1adipo-cytes treated with DBAYL were 3.1and 2.9folds,respect-ively,of that in blank control group without DBAYL treatment.The effects of increasing IRS1and GLUT4ex-pression by DBAYL were significantly stronger than VIP and BAY55-9837.Simultaneously,GLUT4of translocating to the plasma membrane was significantly increased in differentiated 3T3-L1adipocytes treated with DBAYL.Figure 6(B)Figure 5Induced cAMP accumulation by DBAYL or PACAP38in CHO-VPAC2,CHO-VPAC1,and CHO-PAC1cells Results are expressed as the percentage of maximum cAMP accumulation by PACAP38.Data are the mean of three separateexperiments.Figure 6Effect of DBAYL on IRS-1and GLUT4protein expression (A)and GLUT4translocation (B)in 3T3-L1adipocytes Results of changes of GLUT4level by densitometric analysis.2,control group;þ,positive group;P ,0.01,(A):DBAYL(þ)group compared with DBAYL(2)group;(B):DBAYL(þ)/Insulin(þ)group compared with DBAYL(2)/Insulin(2)group;P ,0.05,(A):DBAYL(þ)group compared with BAY55-9837(þ)group or VIP(þ)group;(B):DBAYL(þ)/Insulin(2)group compared with DBAYL(2)/Insulin(2)group;DBAYL(þ)/Insulin(þ)group compared with DBAYL(2)/Insulin(þ)group.Figure 4Displacement of [125I]PACAP38(A)or [125I]VIP (B)by DBAYL,rPACAP38,VIP,BAY55-9837,and VIP(1–7)/GRF(8–27)in membranes purified from CHO cells expressing human VPAC2The results are expressed as percentage of maximum binding to [125I]PACAP38or [125I]VIP.PACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin (2012)|Volume 44|Issue 11|Page 953at Jinan University on November 16, 2014/Downloaded fromshowed that DBAYL treatment and combined treatment of insulin plus DBAYL significantly increased the GLUT4translocation to the plasma membrane by 51%,which was agreed well with the glucose uptake results.Lower doses of insulin could also increase GLUT4translocation,and com-bined treatment of insulin plus DBAYL could more effect-ively promote GLUT4translocation to the plasma membrane.As a VPAC2-specific agonist,DBAYL may sig-nificantly increase the GLUT4translocation to the plasma membrane in a non-insulin-dependent manner,and DBAYL had the biological synergistic effect with insulin on GLUT4translocation.DBAYL promoted glucose uptake activity of differentiated 3T3-L1adipocytesGlucose uptake activity of differentiated 3T3-L1adipocytes treated with different concentrations of DBAYL was all sig-nificantly improved in different degrees.Figure 7showed that 1and 5m M of DBAYL increased glucose uptake of differen-tiated 3T3-L1adipocytes by 43%and 49%,respectively.Improvement effect of DBAYL was better than BAY55-9837at the same concentration.As shown in Fig.7,1and 5m M of BAY55-9837increased glucose uptake of differ-entiated 3T3-L1adipocytes by 16%and 34%,respectively.In vivo effects of DBAYL on insulin release and glucose disposal in ICR miceAs shown in Table 1,compared with normal saline group,recombinant DBAYL (0.5m g/kg)obviously promoted the insulin release and decreased the level of plasma glucose after giving glucose by gavage in ICR mice.Furthermore,the results showed that the biological effects of DBAYL were significantly better than BAY55-9837and rPACAP38.Because of acting on three receptors subtypes,rPACAP38can not effectively decreased the plasma glucose level of ICR mice after glucose gavage.DiscussionAt present,the main approach to treat type 2diabetes is to maintain euglycemia through administration of sulfonylurea drugs that increase insulin levels or by injecting insulin itself.Both therapies produce significant bouts of hypoglycemia,because their onset of action is independent of the prevailing level of glucose.New therapies that retain or enhance glucose-dependent insulin secretion would be a significant advance,since they would avoid the risk of hypoglycemia.PACAP could activate both VPAC1and VPAC2.VPAC2activation enhances glucose disposal by stimulating insulin secretion while VPAC1activation elevates hepatic glucose output [26].Wild-type PACAP could not effective-ly lower blood sugar in vivo because the increase in glucose production may offset the increase in insulin secre-tion.Therefore,clinical treatment of diabetes requires a VPAC2-specific agonist that would enhance pancreatic b cell insulin release without causing increased glucose pro-duction [27].VPAC2-specific agonists such as BAY55-9837,Ro25-1553,and hexanoyl-VIP (C6-VIP)produced by chemical synthesis have been demonstrated to induce insulin secretion from b cells in a glucose-dependent manner [28].In this report,we provide a novel gene recombinant PACAP-derived peptide that is a VPAC2-specific agonist with high stability.Our previous studies had shown that BAY55-9837and some other polypeptides had potential in-stability due to either oxidation or deamidation because of several certain amino acid composition [7,19].Stability ana-lysis showed that the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminus)were much more stable than BAY55-9837,wild-type PACAP,and VIP.DBAYL lost only 7.7%of the main peak during the 4-week incubation at 378C,and the half-life of DBAYL was about 25folds compared with that of BAY55-9837in vitro .Compared with three previously studied VPAC2agonists,BAY55-9837,hexanoyl-VIP (C6-VIP),and Ro 25-1553[14,18],or wild-type PACAP and VIP,there was one methio-nine at the N-terminus of the recombinant DBAYL,which may effectively close the N-terminal sequence H-S-that is highly sensitive to the dipeptidyl peptidase IV that widely exists in organism.And closing of enzyme-sensitive sequences at the N-terminus may improve the stability and half-life of the peptide in vivo.From bioactivity assay of DBAYL,the methionine at the N-terminus should have no effect on the high flexibility of the N-terminal region of DBAYL,and the receptor potency of DBAYL at human VPAC2maintains highly selective activity.Except for the methionine at the N-terminus of DBAYL,other three muta-tions (N10Q,V18L,and N29Q)were simultaneously intro-duced into the peptide sequence by DNA recombination to avoid deamidation and improve the soluble (data notshown).Figure 7Effect of DBAYL or BAY55-9837on glucose uptake in differentiated 3T3-L1adipocytes Data are shown as the mean +SE of three independent experiments.*P ,0.05and **P ,0.01vs.DBAYL or BAY55-9837treatment (0m M).PACAP-derived peptide promotes glucose uptake and glucose-dependent insulin secretionActa Biochim Biophys Sin (2012)|Volume 44|Issue 11|Page 954at Jinan University on November 16, 2014/Downloaded from。

异泽兰黄素对脑出血小鼠的神经保护作用及氧化应激和神经元凋亡的影响

异泽兰黄素对脑出血小鼠的神经保护作用及氧化应激和神经元凋亡的影响申友奎;王雁秋;刘飞飞;徐良【期刊名称】《中国中医药科技》【年(卷),期】2024(31)2【摘要】目的:探究异泽兰黄素对脑出血(intracerebral hemorrhage,ICH)小鼠的神经保护作用及其机制。

方法:采用自体血注射法复制小鼠ICH模型,同时设立假手术组。

分别给予ICH小鼠1、3、10 mg/kg异泽兰黄素灌胃给药3 d。

Longa评分法评估小鼠神经缺损情况。

称重小鼠脑组织,测定含水量,免疫荧光染色观察脑组织神经元改变及应激诱导蛋白Sestrin2(Sestrin2)表达,ELISA测定脑组织丙二醛(MDA)水平及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,Western blot测定脑组织B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、天冬氨酸特异半胱氨酸蛋白酶-3(caspase-3)、AMP依赖性蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、肝醣合成酶激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)蛋白表达。

结果:异泽兰黄素干预后,ICH小鼠第1天、第3天神经缺损症状减轻(P<0.05或P<0.01),具有时间-剂量效应;此外,ICH小鼠脑部水肿程度降低(P<0.05或P<0.01),脑组织神经元细胞凋亡减少、尼氏小体数量增加(P<0.05或P<0.01),Sestrin2荧光表达下降(P<0.01),MDA水平降低(P<0.01),SOD、CAT活性提高(P<0.01),Bax、caspase-3蛋白表达下降(P<0.05或P<0.01),Bcl-2、p-AMPK/AMPK、p-GSK-3β/-GSK-3β表达升高(P<0.05或P<0.01),具有剂量效应。

结论:异泽兰黄素对ICH小鼠具有神经保护作用,抑制氧化应激和细胞凋亡是其部分作用机制。

藁本内酯通过抑制铁自噬延缓小鼠听皮层组织衰老

藁本内酯通过抑制铁自噬延缓小鼠听皮层组织衰老周颖东;张梦娴;王青玲;康浩然;张治成;王庆林;刘亚敏;郭向东【期刊名称】《中国药理学通报》【年(卷),期】2024(40)3【摘要】目的探究藁本内酯(ligustilide,LIG)延缓听皮层组织衰老,治疗中枢性老年性聋的机制。

方法将40只13月龄C57BL/6J小鼠随机分为LIG低剂量组(L-LIG)、LIG中剂量组(M-LIG)、LIG高剂量组(H-LIG)和衰老组(Age),同品系2月龄小鼠10只作为对照组(Ctrl)。

听性脑干反应测试检测给药前后小鼠听阈值;检测血清SOD活力和MOD含量了解氧化应激水平;HE染色观察听皮层病理变化;透射电子显微镜观察铁死亡情况;Western blot法检测听皮层组织NCOA4、GPX4和ACSL4蛋白的表达水平;免疫荧光法观察铁自噬情况;普鲁士蓝染色观察听皮层铁蓄积水平。

结果衰老组小鼠听阈值明显升高,并表现出明显的衰老与铁死亡特征。

中、高剂量LIG干预降低小鼠听阈值,改善听皮层组织病理损伤,保护神经元线粒体形态结构,减少铁聚集颗粒细胞数量,抑制铁自噬,降低NCOA4、ACSL4蛋白表达,升高GPX4的蛋白表达。

结论LIG可以通过抑制铁自噬,减少衰老小鼠听皮层神经元铁死亡,延缓听皮层衰老,提示LIG具有治疗中枢性老年性聋的作用。

【总页数】7页(P455-461)【作者】周颖东;张梦娴;王青玲;康浩然;张治成;王庆林;刘亚敏;郭向东【作者单位】河南中医药大学第一临床医学院;河南中医药大学第一附属医院耳鼻喉科;河南医学高等专科学校;河南中医药大学药学院【正文语种】中文【中图分类】R-332;R284.1;R322.81;R339.38;R591.1;R764.436【相关文献】1.益气活血法延缓衰老作用机理的理论与实验研究——大黄(庶虫)虫丸、当归补血汤对衰老小鼠皮肤组织中的脂褐质含量影响的对比研究2.电针耳穴通过SIRT1/PGC-1α途径延缓D-半乳糖致衰老豚鼠听皮层老化3.口服川芎挥发油后小鼠体内藁本内酯的组织分布特性4.人参皂苷Rg1通过AMPK/mTOR信号通路调控自噬延缓小鼠脑衰老的研究5.Ferrostatin-1通过抑制铁死亡延缓D-gal诱导的心肌细胞衰老的研究因版权原因，仅展示原文概要，查看原文内容请购买。

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维普资讯
军队老年人轻度认知损害的发病率及
向痴呆和Ａｚｅｒｌｉ病的转化率：ｈｍｅ基于人群的队列研究
于宝成田京利欧阳荔莎王玉敏王成章崔欣
王雪丽齐丽娟潘志刚魏士平医院
目的：研究军队老年人轻度认知损害（ＩＭＣ）的发病率及向痴呆和Ａｚｅｅ病（ＤｌｉｒｈｍＡ）的转化率，为进一步研究ＡＤ提供依据。方法：２０年石家庄市２个部队休干所ＭＣ患病率调查的２以０１６Ｉ
６４６岁及以上的离退休干部为研究对象，对患病率调查时诊断为ＭＣ的２６患者和２３２名７名ＯＩ１例０认知正常受试者进行为期３年的队列研究，比较ＭＣ患者和认知正常受试者痴呆和Ａ的平均年发ＩＤ病率。结果：认知正常的老年人ＭＣ的发病率为４８百人年，痴呆和ＡＤ的平均年发病率分别为Ｉ．／１３百人年和０８百人年；ＭＣ患者痴呆和Ａ．／．／ＩＤ的平均年发病率分别为８１百人年和５６百人年；．／．／男性和女性ＭＣ患者痴呆和ＡＩＤ的平均年发病率差别无显着性意义（＞Ｏ０）Ｐ．５；随着文化程度的提高，ＭＣ患者痴呆和ＡＤ的平均年发病率有降低的趋势（＜Ｏ０）；随着年龄的增长，ＭＣ患者ＩＰ．５Ｉ痴呆和ＡＤ的平均年发病率有增高的趋势（＜Ｏ０）Ｐ．１。ＭＣ转化为痴呆或ＡＩＤ的相对危险性分别为认知正常者的６４倍和７４。结论：．．倍军队老年ＭＣ患者转化为痴呆的危险性远远大于认知正常的Ｉ老年人。应加强对老年ＭＣ患者这一痴呆高危人群的监测。Ｉ关键词：轻度认知损害痴呆阿尔茨海默病队列研究发病率
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ＥＲｍｂａｅｕｉｕｔｎｌａｅＨｒｄａｅａｍｅｒｎｂｑｉｉｉｓｄｌｍｅｉｔｓｔｕｇｄｇａａｉｎｎｐｏｅｓｍｅｄｐｎｅｔａｄｉｄｐｎｅｔｍａｎｒｅｒｄｔｏｉｒｔｏｏ－ｅｅｄｎｎｎｅｅｄｎｎｅ