NMTA_Bulletin_Vol_39
Notch_抑制剂DAPT_体外改善L02_细胞脂肪变研究
∗基金项目:国家自然科学基金资助项目(编号:81400610);上海交通大学医工交叉研究基金资助项目(编号:YG2016MS72)作者单位:200092上海市上海交通大学医学院附属新华医院消化内科(吴伟杰,丁雯瑾,杨蕊旭,范建高);福建省立医院南院消化内镜中心(吴伟杰)第一作者:吴伟杰,男,27岁,硕士研究生㊂主要从事代谢相关脂肪性肝病的诊断与治疗学研究㊂E-mail:weijie_wu96@ 通讯作者:丁雯瑾,E-mail:dingwenjin@ ㊃实验性肝炎㊃Notch抑制剂DAPT体外改善L02细胞脂肪变研究∗吴伟杰,丁雯瑾,杨蕊旭,范建高㊀㊀ʌ摘要ɔ㊀目的㊀探讨Notch抑制剂(DAPT)体外对脂肪变肝细胞细胞炎症因子及脂质相关基因水平的影响㊂方法㊀采用棕榈酸(PA)干预体外构建肝细胞脂肪变模型,采用不同浓度的DAPT干预㊂采用CCK-8检测细胞存活率,采用尼罗红染色了解细胞脂滴量,采用RT-qPCR法检测TNF-α㊁IL-1β和IL-6和脂肪相关因子ʌ固醇调节元件结合蛋白1c (SREBP1c)㊁FASN和ACACAɔmRNA水平,采用Western blot法检测细胞p65和SREBP1c蛋白表达㊂结果㊀在无PA干预的L02细胞,经1μM㊁2μM㊁5μM和10μM DAPT处理细胞24h,细胞存活率均较对照组显著下降(分别为85.2ʃ5.3%㊁84.6ʃ2.9%㊁84.4ʃ6.0%和84.5ʃ3.2%对100.0%,P均<0.05);经2μM和5μM浓度的DAPT干预,细胞TNF-αmRNA水平分别为(0.6ʃ0.01)和(0.5ʃ0.1),显著低于对照组ʌ(1.0ʃ0.0),P<0.01ɔ,IL-1β水平分别为(0.7ʃ0.2)和(0.4ʃ0.0),显著低于对照组ʌ(1.1ʃ0.1),P<0.01ɔ,IL-6水平分别为(0.8ʃ0.1)和(0.6ʃ0.1),显著低于对照组ʌ(1.0ʃ0.0),P<0.05ɔ,细胞p65蛋白表达量也显著降低(P<0.05);2μM㊁5μM和10μM DAPT处理细胞脂滴荧光强度较对照组显著减弱ʌ分别为(0.2ʃ0.1)㊁(0.3ʃ0.0)和(0.1ʃ0.0)对(0.7ʃ0.0),P均<0.001ɔ,2μM和5μM DAPT处理细胞FASN mRNA水平分别为(0.7ʃ0.0)和(0.4ʃ0.1),显著低于对照组ʌ(1.0ʃ0.0),P<0.001ɔ㊁ACACA mRNA水平分别为(0.6ʃ0.1)和(0.3ʃ0.0),显著低于对照组ʌ(1.0ʃ0.0),P<0.001ɔ;5μM DAPT处理细胞SREBP1c蛋白表达量显著低于对照组(P<0.01)㊂结论㊀DAPT能有效降低体外脂肪变细胞炎症因子表达量,缓解细胞内脂滴形成,其机制可能与调控脂肪相关因子的表达有关,提示抑制Notch信号通路有改善细胞脂肪变发生和进展的潜力㊂㊀㊀ʌ关键词ɔ㊀L02细胞;脂肪变;Notch抑制剂;炎症因子;脂质;体外㊀㊀DOI:10.3969/j.issn.1672-5069.2024.01.005㊀㊀Notch inhibitor DAPT ameliorates steatosis of L02cells in vitro㊀Wu Weijie,Ding Wenjin,Yang Ruixu,et al.Department of Gastroenterology,Xinhua Hospital,JiaoTong University School of Medicine,Shanghai200092,China㊀㊀ʌAbstractɔ㊀Objective㊀The purpose of this experiment was to investigate the effects of Notch inhibitor(DAPT)on cellular inflammatory factors and lipid related genes in L02cells in vitro.Methods㊀The steatosis of L02cells was established by palmitic acid(PA)incubation in vitro,and also intervened by DAPT at0μM,1μM,2μM,5μM and10μM concentration.The cell survival rate was detected by CCK-8,the cell lipid droplets was observed by nile red staining,and the cellular TNF-α,IL-1βand IL-6mRNA and fat related factors[sterol regulatory element-binding protein1c(SREBP1c),FASN and ACACA]mRNA loads were detected by RT-qPCR.The cell p65and SREBP1c expression was detected by Western blot.Results㊀The cell survival rates of L02cell without PA intervention at1μM,2μM,5μM and10μM DAPT incubation for24h were all decreased compared to in the control(85.2ʃ5.3%,84.6ʃ2.9%,84.4ʃ6.0%and84.5ʃ3.2%vs.100.0%,respectively,P<0.05);in2μM and5μM DAPT-intervened cells,the TNF-αmRNA levels were(0.6ʃ0.01)and(0.5ʃ0.09),significantly lower than in the control [(1.0ʃ0.0),P<0.01],the IL-1βmRNA levels were(0.7ʃ0.2)and(0.4ʃ0.0),significantly lower than[(1.1ʃ0.1),P< 0.01]in the control,and the IL-6mRNA levels were(0.8ʃ0.1)and(0.6ʃ0.1),significantly lower than[(1.0ʃ0.0),P< 0.05]in the control group;the p65expression showed also remarkably decreased(P<0.05);the lipid droplets in2μM,5μM and10μM DAPT-intervened cells were significantly weaker than in the control group[(0.2ʃ0.1),(0.3ʃ0.0),(0.1ʃ0.0) vs.(0.73ʃ0.0),P<0.001],the FASN mRNA loads in2μM and5μM DAPT-intervened cells were(0.7ʃ0.0)and(0.4ʃ0.1),much lower than[(1.0ʃ0.0),P<0.001]in the control group,and the ACACA mRNA load in2μM and5μM DAPT-intervened cells were(0.6ʃ0.1)and(0.3ʃ0.0),much lower than[(1.0ʃ0.0),P<0.001]in the control group;the expression of SREBP1c protein at5μM and10μM DAPT-intervened cells was significantly weaker than in the control group(P<0.01).Conclusion㊀The DAPT could effectively inhibit theexpression of inflammatory factors and ameliorate the formation ofintracellular lipid droplets in L02cells with PA-induced steatosisin vitro,hinting the mechanism might be related to the regulationof fat-related factors.Our findings suggest that the inhibition ofNotch signal pathway might have a potential to alleviate theoccurrence and progression of cell steatosis.㊀㊀ʌKey wordsɔ㊀L02cells;Steatosis;Notch inhibitors;Inflammatory cytokines;Lipids;In vitro㊀㊀Notch信号通路是高度保守的单次跨膜信号受体蛋白家族,包括四个受体(Notch1/2/3/4)和五个配体(Jagged1㊁Jagged2㊁DLL1㊁DLL3和DLL4)及靶基因编码转录因子(Hes1和Hey1),涉及多种组织和器官早期发育所必需的细胞间调节,在细胞增殖㊁分化㊁凋亡等方面具有重要作用[1㊁2]㊂近几年,有研究报道Notch信号的活化参与肝脏再生与修复㊁炎症和纤维化过程[3]㊂此外,Notch基因异常表达会导致胰岛素抵抗(insulin resistance,IR)㊁脂质异常和肥胖等多种代谢性疾病,它也能诱发非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)的发生[4,5]㊂我们之前的研究证实,无论是在蛋氨酸胆碱缺乏(methionine-choline deficient,MCD)小鼠模型还是棕榈酸(palmitic acid,PA)诱导体内肝细胞脂肪变模型,均存在Notch基因家族的异常表达,并随代谢障碍相关脂肪性肝病(metabolic dysfunction-associated fatty liver disease,MAFLD)不同发展阶段而改变[6]㊂随后,我们应用Notch信号通路抑制剂(DAPT)体外处理脂肪变肝细胞,发现DAPT能抑制Notch下游Hes-1和Hey-1基因水平,同时能有效降低甘油三酯(triglyceride,TG)和转氨酶水平[7]㊂本研究在体外应用DAPT处理脂肪变的L02细胞,观察了抑制Notch家族后细胞炎症因子和脂质代谢相关基因水平变化㊂1 材料与方法1.1细胞㊁试剂与仪器㊀人正常肝细胞L02由中国科学院上海细胞库提供;RPMI-1640培养基和杜氏磷酸盐缓冲液(Dulbecco's phosphate buffered saline, DPBS)均购于美国Hyclone公司;胎牛血清(fatal bo-vine serum,FBS)购于以色列Biological Industries公司;γ分泌酶抑制剂N-[N-(3,5-difluorophenacetyl)-L-alnyl]-S-phenylycine t-butyl ester(DAPT)和PA均购于美国默克公司;RPS -18内参引物购于中国上海生物工程公司;Trizol试剂和预混型定量用逆转录试剂盒均购于日本宝日医公司;qPCR SYBR Green Master Mix购于中国上海翊圣生物科技有限公司;CCK-8试剂盒㊁BCA蛋白浓度测定试剂盒㊁RIPA裂解液(强)和含DAPI的抗荧光淬灭封片剂均购于上海碧云天生物技术有限公司;尼罗红染料购于上海晶纯生化科技股份有限公司;抗GAPDH小鼠单克隆抗体购于美国Proteintech Group公司;抗固醇调节元件结合蛋白1c(sterol reg-ulatory element-binding protein1c,SREBP1c)小鼠单克隆抗体购于美国NOVUS公司;抗p65(NF-kappaB,NF-κB)兔单克隆抗体购于美国Abcam公司;ECL发光液购于美国Millipore公司;凝胶成像仪(美国Bio-Rad公司);Q3PCR扩增仪和Nano Drop 2000超微量分光光度仪(美国赛默飞公司);PCR逆转录仪(德国艾本德公司);Bio Tek Epoch全波长酶标仪(美国博腾仪器有限公司)㊂1.2体外细胞脂肪变模型的建立与DAPT干预㊀将5mM PA原液用RPMI-1640完全培养基(含10% FBS)稀释成含0.1mM㊁0.25mM和0.5mM PA的高脂培养基工作液㊁备用㊂取L02细胞,分为对照组(Con)㊁0.1mM PA组㊁0.25mM PA组和0.5mM PA组㊂待L02细胞融合度达到60%~70%时,弃培养基,用DPBS洗1遍,加入培养基或相应浓度的高脂培养基工作液处理L02细胞24h,收集细胞㊂根据前期实验结果确定0.25mM PA浓度作为体外细胞脂肪变的造模条件,分含或不含0.25mM PA处理细胞㊂采用0μM㊁1μM㊁2μM㊁5μM和10μM DAPT 处理细胞,其中0.25mM PA处理细胞在不加DAPT 处理组为模型组,处理L02细胞24h,收集细胞㊂1.3细胞存活率试验㊀使用CCK-8试剂盒进行细胞存活率测定㊂1.4细胞mRNA提取及水平检测㊀采用Trizol法提取细胞总RNA,逆转录后行实时荧光聚合酶链式反应(RT-qPCR),基因特异性引物序列为:SREBP1c-F:5 -CGGAACCATCTTGGCAACAGT-3 ; SREBP1c-R:5 -CGCTTCTCAATGGCGTTGT-3 ; FASN-F:5 -CCGAGACACTCGTGGGCTA-3 ; FASN-R:5 -CTTCAGCAGGACATTGATGCC-3 ; ACACA-F:5 -ATGTCTGGCTTGCACCTAGTA-3 ; ACACA-R:5 -CCCCAAAGCGAGTAACAAATTCT-3 ;TNF-α-F:5 -CCTCTCTCTAATCAGCCCTCTG-3 ;TNF-α-R:5 -GAGGACCTGGGAGTAGATGAG -3 ;IL-1β-F:5 -AGCTACGAATCTCCGACCAC-3 ;IL-1β-R:5 -CGTTATCCCATGTGTCGAAGAA -3 ;IL-6-F:5 -ACTCACCTCTTCAGAACGAATTG -3 ;IL-6-R:5 -CCATCTTTGGAAGGTTCAGGTTG -3 ㊂1.5细胞p65及SREBP1蛋白检测㊀采用Western blot法,用RIPA裂解法提取细胞总蛋白,经BCA试剂盒测定蛋白浓度,调节上样蛋白总量至相同㊂根据目的蛋白分子大小配制合适的SDS PAGE凝胶后开始上样㊁电泳㊁转膜,用5%脱脂牛奶封闭,按照抗体说明书相继加入一抗和二抗孵育后,经凝胶成像仪曝光成像,应用Image J软件对条带的灰度值进行定量分析㊂1.6细胞脂滴观察㊀在12孔细胞培养板,加入不同浓度的DAPT干预PA处理的L02细胞24h,弃培养基,用DPBS洗2遍,加入4%多聚甲醛固定15~30 min,用DPBS洗2遍㊂按照尼罗红原液:DPBS=1: 500的比例配制工作液,避光染色15min,弃去尼罗红染色工作液,用DPBS洗2遍㊂在每孔中加入含DAPI的抗荧光淬灭封片液100μL,置于荧光显微镜下观察㊂1.7统计学分析㊀应用GraphPad Prism8.0软件进行统计学分析和作图,计量资料以(xʃs)表示,多组间比较采用单因素方差分析,P<0.05被认为有统计学差异㊂2㊀结果2.1各组L02细胞存活率比较㊀在PA干预L02细胞8h㊁16h和24h,经CCK-8测定显示,L02细胞存活率随干预时间呈剂量依赖性下降趋势;与对照组细胞存活率为100%比,在干预8h㊁16h和24h 时,0.1mM㊁0.25mM和0.5mM PA干预L02细胞存活率均显著下降(P均<0.05,图1A);在无PA干预的L02细胞,经1μM㊁2μM㊁5μM和10μM DAPT 处理细胞24h,细胞存活率均较对照组显著下降(分别为85.2ʃ5.3%㊁84.6ʃ2.9%㊁84.4ʃ6.0%和84.5ʃ3.2%对100.0%,P均<0.05,图1B)㊂2.2不同浓度DAPT干预PA处理细胞相关细胞因子mRNA水平和p65蛋白表达变化㊀与模型(0μM DAPT处理)组比,各DAPT处理组TNF-α㊁IL-1β和IL-6mRNA水平均显著下调(P均<0.05,表1);与0μM DAPT处理组比,各DAPT处理组细胞p65蛋白均显著下调(P均<0.05,图2A㊁2B)㊂表1㊀不同浓度DAPT干预PA处理细胞相关细胞因子mRNA水平(xʃs)比较0μM1μM2μM5μM10μM TNF-α 1.0ʃ0.00.8ʃ0.10.6ʃ0.0③0.5ʃ0.1③0.4ʃ0.0③IL-β 1.1ʃ0.10.9ʃ0.10.7ʃ0.2②0.4ʃ0.0③0.4ʃ0.0③IL-6 1.0ʃ0.00.9ʃ0.10.8ʃ0.1①0.6ʃ0.1③0.6ʃ0.1③㊀㊀与模型组(0μM DAPT)比,①P<0.05;②P<0.01;③P<0.001图1㊀不同浓度PA和DAPT干预L02细胞存活率比较A:不同浓度的PA处理L02细胞;B:不同浓度的DAPT处理L02细胞24h;∗P<0.05图2㊀不同浓度DAPT干预PA处理细胞p65蛋白表达变化A:细胞p65蛋白表达(Western blot法);B:p65蛋白表达相对水平;∗P<0.05;∗∗P<0.012.3不同浓度DAPT干预PA处理细胞脂质代谢水平变化㊀与0μM DAPT组相比,各DAPT处理组细胞SREBP1c mRNA水平均无显著变化,在2μM㊁5μM和10μM DAPT处理组细胞FASN和ACACA mRNA水平均显著下降(P均<0.001,表2);在2μM㊁5μM和10μM DAPT处理组,细胞脂滴红色荧光强度较0μM DAPT组显著减弱[分别为(0.2ʃ0.1)㊁(0.3ʃ0.0)和(0.1ʃ0.0)对(0.7ʃ0.0),P均< 0.001,图3A);与0μM DAPT组相比,510μM和10μM DAPT处理组细胞SREBP1c蛋白水平均显著下调(P均<0.05,图3B和3C)㊂图3㊀不同浓度DAPT干预PA处理细胞脂质代谢变化A:细胞脂滴形成情况(尼罗红染色,400ˑ);B:细胞SREBP1c蛋白表达(Western blot法);C:细胞SREBP1c蛋白表达相对水平;∗P<0.05;∗∗P<0.01表2㊀不同浓度DAPT干预PA处理细胞脂质代谢相关基因mRNA水平(xʃs)比较0μM1μM2μM5μM10μM SREBP1c 1.1ʃ0.00.9ʃ0.10.9ʃ0.10.9ʃ0.10.9ʃ0.1 FASN 1.0ʃ0.00.9ʃ0.10.7ʃ0.0①0.4ʃ0.1①0.3ʃ0.0①ACACA 1.0ʃ0.00.9ʃ0.10.6ʃ0.1①0.3ʃ0.0①0.3ʃ0.1①㊀㊀与模型组(0μM DAPT)比,①P<0.053㊀讨论Notch基因的激活不仅与异常增殖㊁癌症疾病发生密切相关,也在细胞代谢过程中起着重要作用,其表达紊乱会导致多种代谢相关性疾病[8,9]㊂通常, MAFLD伴随代谢综合征的出现,目前普遍被认可的是 多次打击 学说,胰岛素抵抗㊁脂质过氧化㊁氧化应激等均参与其发病㊂在小鼠肝脏病理学研究发现,肝细胞特异性Notch功能丧失可缓解脂肪性肝炎相关肝纤维化,而增强Notch在肝细胞中的表达会通过上调Sox9依赖性骨桥蛋白,激活肝星状细胞并诱导肝脏纤维化[10]㊂也有研究使用Notch抑制剂可改善动物因高脂高糖饮食而导致的肝纤维化[11]㊂结合文献报道及我们团队先前的研究结果,有足够的证据表明Notch家族参与了MAFLD的发生和进展[6]㊂长期高脂饮食可以导致高脂血症㊁异位脂质沉积,过量的脂质沉积在肝细胞内而引起肝脏损伤,最后形成MAFLD[12]㊂有研究发现Notch在肝脏脂代谢过程中扮演重要角色㊂Notch1信号活化可以导致胰岛素抵抗加重,并与FOXO1协同增强葡萄糖-6-磷酸酶表达[13,14]㊂Notch信号可以激活mTOR信号通路,促使其下游SREBP1c表达,促进肝细胞脂肪生成㊂抑制Notch基因能通过降低mTOR的稳定性,弱化下游激酶核糖体蛋白S6激酶1活化,减缓SREBP1c依赖的脂肪从头合成,达到改善肝脂肪沉积的目的[15,16]㊂我们采用0.25mM PA浓度干预L02细胞24h成功构建体外细胞脂肪变模型,并可显著引起细胞内的脂质沉积和稳定激活Notch信号通路[7]㊂在体外肝细胞脂肪变细胞,以 不影响细胞活性 为基础,发现DAPT能缓解细胞炎症因子(TNF-α㊁IL-1β和IL-6)水平上升并下调p65蛋白表达,改善程度与DAPT浓度有一定的剂量依赖性㊂尼罗红染色可观察到细胞脂滴数量明显减少㊂TNF -α㊁IL-1β和IL-6与NF-κB核内易位活化密切相关㊂NF-κB信号通路激活可能是引起细胞内炎症反应的主要原因[17]㊂NF-κB的IKKα启动子序列与Notch信号下游转录因子Hes1存在部分重合,表明Notch信号可能从基因转录层面对炎症信号通路产生影响㊂因此,使用DAPT等Notch抑制剂或可抑制肝细胞内炎症反应和脂质沉积,具有防治MAFLD进展的潜力㊂作为γ-分泌酶底物的DAPT可以竞争性与γ分泌酶结合,从而实现间接抑制Notch信号转导[18]㊂在非酒精性脂肪性肝炎患者肝组织往往存在Notch 基因异常激活[19]㊂抑制Notch信号可改善糖尿病模型小鼠肝脏脂滴数量㊁降低血清肝酶水平并提高胰岛素敏感性,抑制Notch基因后可诱导AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)和ACACA磷酸化,从而激活AMPK信号通路,促进脂肪酸氧化并抑制脂肪合成[20]㊂本研究我们进一步检测脂质代谢相关性基因SREBP1㊁FASN和ACACA 表达情况,经WB检测结果显示SREBP1c蛋白表达水平经过DAPT处理后明显下调,下游的FASN和ACACA脂肪合成相关基因mRNA水平也相应出现了下降㊂故DAPT可能通过降低SREBP1c蛋白的表达而抑制脂肪合成,从而具有调控脂质紊乱的作用㊂综上所述,应用DAPT抑制Notch基因家族可改善MALFD的炎症反应和脂质代谢异常㊂ʌ参考文献ɔ[1]Liubomirski Y,Ben-Baruch A.Notch-inflammation networks inregulation of breast cancer progression.Cells,2020,9(7):1576.[2]Liubomirski Y,Lerrer S,Meshel T,et al.Notch-mediated tumor-stroma-inflammation networks promote invasive properties and CXCL8expression in triple-negative breast Cancer.Front Immunol, 2019,10:804.[3]Chen W,Liu Y,Chen J,et al.The Notch signaling pathway regu-lates macrophage polarization in liver diseases.Int Immunopharma-col,2021,99:107938.[4]Zhou B,Lin W,Long Y,et al.Notch signaling pathway:architec-ture,disease,and therapeutics.Signal Transduct Target Ther, 2022,7(1):95.[5]Richter L R,Wan Q,Wen D,et al.Targeted delivery of Notch in-hibitor attenuates obesity-induced glucose intolerance and liver fi-brosis.ACS Nano,2020,14(6):6878-6886.[6]Ding WJ,Wu WJ,Chen YW,et al.Expression of Notch family isaltered in nonalcoholic fatty liver disease.Mol Med Rep,2020;22(3):1702-1708.[7]吴伟杰,陈源文,丁雯瑾,等.体外脂肪变L02细胞Notch家族和脂质代谢变化研究.实用肝脏病杂志,2021,24(5):653-656. [8]Adams J M,Jafar-Nejad H.The roles of Notch signaling in liverdevelopment and disease.Biomolecules,2019,9(10):608. [9]Xu H,Wang L.The role of Notch signaling pathway in non-alco-holic fatty liver disease.Front Mol Biosci,2021,8:792667. [10]Zhu C,Kim K,Wang X,et al.Hepatocyte Notch activationinduces liver fibrosis in nonalcoholic steatohepatitis.Sci Transl Med, 2018,10(468):eaat0344.[11]Lee YA,Friedman SL.Inflammatory and fibrotic mechanisms inNAFLD-implications for new treatment strategies.J Intern Med, 2022,291(1):11-31.[12]Luna-Castillo KP,Olivares-Ochoa XC,Hernández-Ruiz RG,etal.The effect of dietary interventions on hypertriglyceridemia:from public health to molecular nutrition evidence.Nutrients,2022,14(5):1104.[13]Pajvani UB,Shawber CJ,Samuel VT,et al.Inhibition of Notchsignaling ameliorates insulin resistance in a Foxo1-dependent man-ner.Nat Med,2011,17(8):961-967.[14]Kitamoto T,Lee YK,Sultana N,et al.,Chemical induction of gutβ-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes.Mol Metab,2022,66:101624. [15]Zhang M,Wu P,Li M,et al.Inhibition of Notch1signaling re-duces hepatocyte injury in non-alcoholic fatty liver disease via auto-phagy.Biochem Biophys Res Commun,2021,547:131-138. [16]Bi P,Kuang S.Notch signaling as a novel regulator of metabolism.Trends Endocrinol Metab,2015,26(5):248-255. [17]Jz AL,Alshammari GM,Alfaris NA,et al.Ellagic acid protects a-gainst non-alcoholic fatty liver disease in streptozotocin-diabetic rats by activating AMPK.Pharm Biol,2022,60(1):25-37. [18]Aguayo-Ortiz R,Guzmán-Ocampo DC,Dominguez L.Toward thecharacterization of DAPT interactions withγ-secretase.Chem Med Chem,2019,14(10):1005-1010.[19]Auguet T,Bertran L,Binetti J,et al.Hepatocyte Notch signaling de-regulation related to lipid metabolism in women with obesity and nonal-coholic fatty liver.Obesity(Silver Spring),2020,28(8):1487-1493.[20]Lee YH,Yun MR,Kim HM,et al.Exogenous administration ofDLK1ameliorates hepatic steatosis and regulates gluconeogenesis via activation of AMPK.Int J Obes(Lond),2016,40(2):356-365.(收稿:2023-05-15)(本文编辑:陈从新)。
银杏叶提取物促进脑缺血半暗带神经元自噬提高神经保护作用
中国细胞生物学学报Chinese Journal of Cell Biology 2021,43(3): 552-560 DOI: 10.11844/cjcb.2021.03.0008银杏叶提取物促进脑缺血半暗带神经元自噬提高神经保护作用郭涛吴致远赵晓明陈学梅刘裕源黄志文何红云+邓仪昊*(昆明理工大学医学院解剖学教研室，脑卒中病理机制研宂实验室，昆明650500)摘要 银杏叶提取物(ginkgo biloba extract-761, EGb-761)注射液在中国常作为辅助药物被用于治疗脑卒中，但是，其潜在的细胞和药理机制尚未完全了解。
该研究旨在探讨EGb-761是否通 过调节缺血性脑卒中半暗带神经元的自噬从而发挥保护作用。
采用雄性S D大鼠大脑中动脉闭塞 (middle cerebral artery occlusion,MCAO)再灌注模型，将M CA0大鼠随机分为5组，分另1为Sham组、MCAO+saline组、MCAO+EGb组、MCAO+EGb+3-MA组和MCAO+3-M A组。
脑缺血大鼠用EGb-761药物腹腔注射7天后，并使用自噬抑制剂3-MA侧脑室注射进行干预，分别通过蛋白免疫印迹法 (WB)、实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和免疫荧光检测缺血半暗带的脑组 织，以检测自噬的表达。
另夕卜，根据脑梗死体积、神经功能缺损和TU N EL^r测神经元凋亡水平，以评估治疗效果。
结果表明，与MCAO+saline相比，MCAO+EGb组的EGb-761显著提高了神经元自噬 水平，同时，明显减轻了神经功能缺损、脑梗死面积和神经元凋亡。
此外，相对于MCAO+EGb组, MCAO+EGb+3-M A组中的3-MA抵消了 EGb增强神经元自噬的功效，并且仅使用3-MA继续加重了 神经损伤。
因此，EG B-761通过特异性促进脑缺血半暗带神经元自噬发挥神经保护作用。
依达拉奉右莰醇注射液对急性前循环脑梗死血管介入术后开通良好患者的脑保护作用
ing in treatment of chronic heart failure patient [J].Chinese Journal of General Practice,2020,18(9):1504-1507,1550.陈远园,刘庆生,彭伟献,等.益气化瘀汤辅助治疗对慢性心力衰竭患者微血管损伤和心室重构及代谢重构的影响[J].中华全科医学,2020,18(9):1504-1507,1550.[25]Blanda V ,Bracale UM,Di Taranto MD,et al.Galectin -3in cardio-vascular diseases [J].Int J Mol Sci,2020,21(23):9232.[26]Rabkin SW,Tang JKK.The utility of growth differentiation fac-tor -15,galectin -3,and sST2as biomarkers for the diagnosis of heart failure with preserved ejection fraction and compared to heart failure with reduced ejection fraction:a systematic review [J].Heart Fail Rev,2021,26(4):799-812.[27]Ye S,Luo W,Khan ZA,et al.Celastrol attenuates angiotensin Ⅱ-in-duced cardiac remodeling by targeting STAT3[J].Circ Res,2020,126(8):1007-1023.[28]Dai C,Luo W,Chen Y ,et al.Tabersonine attenuates angiotensin Ⅱ-in-duced cardiac remodeling and dysfunction through targeting TAK1and inhibiting TAK1-mediated cardiac inflammation [J].Phytomedi-cine,2022,103(1):154238.[29]Gu J,Qiu M,Lu Y ,et al.Piperlongumine attenuates angiotensin -Ⅱ-in-duced cardiac hypertrophy and fibrosis by inhibiting Akt-FoxO1sig-nalling [J].Phytomedicine,2021,82(1):153461.[30]Nie YY ,Song YL,Lu YH,et al.The effect of Xinbao pill and Wulingpowder on serum ang Ⅱand Gal -3protein levels in chronic heart fail-ure patients with Yang Deficiency and Water Pan syndrome [J].Chi-nese Archives of Traditional Chinese Medicine,2022,40(12):222-225.聂颖颖,宋业琳,卢英红,等.心宝丸合五苓散对阳虚水泛型慢性心力衰竭血清Ang Ⅱ、Gal -3蛋白水平的影响[J].中华中医药学刊,2022,40(12):222-225.(收稿日期：2023-09-26)依达拉奉右莰醇注射液对急性前循环脑梗死血管介入术后开通良好患者的脑保护作用高亚军1，宋倩2，刘春霞3，吴瑞1，郭改艳1延安大学附属医院神经内科1、检验科2、放射科3，陕西延安716000【摘要】目的研究依达拉奉右莰醇注射液对急性前循环脑梗死血管介入术后开通良好患者的脑保护作用。
少见脑干华勒氏变性1例
宁夏医学杂志2022年3月第44卷第3期Ningxia Med J,Mar.2022,Vol.44,No.3-207-[8]KIM D H,LEE D S,NAH H W,et al.Clinical and radiological factors associated with unfavorable outcome after intravenous thrombolysis in patients with mild ischemic stroke[J].BMC Neurology,2018,18(1)：30.[9]STRAMBO D,ZAMBON A A,ROVERI L,et al.Defining minorsymptoms in acute ischemic stroke[J].Cerebrovascular Diseases (Basel,Switzerland),2015,39(3/4)：209-215.[10]LIU D,SCALZO F,STARKMAN S,et al.DWI lesion patterns predict outcome in stroke patients with thrombolysis[J].Cerebrovascular Diseases(Basel,Switzerland),2015,40(5/6)：279-285.[11]MAZYA M V,COORAY C,LEES K R,et al.Minor stroke due tolarge artery occlusion.When is intravenous thrombolysis not enough?Results from the SITS International Stroke ThrombolysisRegister[J].European Stroke Journal，2018，3(1):29-38.[收稿日期]2021-03-26[责任编辑]马兴忠Doi：10.13621/j.1001-5949.2022.03.0207少见#干华&氏变）1例彭冬倩，何彩霞，王喜全［关键词］脑梗死；华勒氏变性；神经变性［中图分类号］R614［文献标识码］B•病例报告•1病例资料患者，女,69岁，因"头晕、头痛1周，加重1d"入院。
α1-MG和NAG／UCr在慢性HBV感染相关肝病患者早期肾损伤中的检测价值
!9:;<=!尿α１－微球蛋白及Ｎ－乙酰－β－Ｄ－氨基葡萄糖苷酶／尿肌酐在慢性ＨＢＶ感染相关肝病患者早期肾损伤中的检测价值张静怡，唐映梅，杨 娴，杨文霞昆明医科大学第二附属医院消化内科，昆明６５００００摘要：目的 探究尿α１－微球蛋白（α１－ＭＧ）、Ｎ－乙酰－β－Ｄ－氨基葡萄糖苷酶／尿肌酐（ＮＡＧ／ＵＣｒ）在慢性ＨＢＶ感染相关肝病患者肾损伤中的临床价值。
方法 纳入２０１９年８月—２０２０年８月就诊于昆明医科大学第二附属医院的慢性ＨＢＶ感染相关肝病患者８５例，根据患者核苷（酸）类似物（ＮＵＣ）治疗史，将患者分为ＮＵＣ治疗组（ｎ＝５７）和未经ＮＵＣ治疗组（ｎ＝２８），根据使用ＮＵＣ种类，将患者分为ＥＴＶ治疗组（ｎ＝３２）和ＴＤＦ治疗组（ｎ＝２５）；根据ＨＢＶ血清抗原、抗体标志物检测结果，将患者分为ＨＢｅＡｇ阴性组（ｎ＝５７）和ＨＢｅＡｇ阳性组（ｎ＝２８）；根据血清ＨＢＶＤＮＡ定量检测，将患者分为ＨＢＶＤＮＡ阴性组（ｎ＝４７）和ＨＢＶＤＮＡ阳性组（ｎ＝３８）；根据腹部影像学检查结果，将患者分为非肝硬化组（ｎ＝４７）和肝硬化组（ｎ＝３８），收集纳入患者的病史资料及实验室指标，并进行组间比较。
计量资料符合正态分布的两组间比较采用ｔ检验；偏态分布两组间比较采用Ｍａｎｎ－ＷｈｉｔｎｅｙＵ检验。
计数资料两组间比较采用χ２检验。
采用ＭｃＮｅｍａｒ检验比较不同指标检测的优劣性。
采用Ｓｐｅａｒｍａｎ相关性分析探讨各因素与α１－ＭＧ、ＮＡＧ／ＵＣｒ之间的相关性。
采用多元线性回归分析α１－ＭＧ、ＮＡＧ／ＵＣｒ的独立影响因素。
结果 未经ＮＵＣ治疗组、ＨＢｅＡｇ阳性组、ＨＢＶＤＮＡ阳性组的尿α１－ＭＧ水平分别较ＮＵＣ治疗组（Ｚ＝－２．０５４，Ｐ＝０．０４）、ＨＢｅＡｇ阴性组（Ｚ＝－２．２９３，Ｐ＝０．０２２）、ＨＢＶＤＮＡ阴性组（Ｚ＝－２．２２９，Ｐ＝０．０２６）明显升高。
ＨＢＶＤＮＡ阳性组、肝硬化组的ＮＡＧ、ＮＡＧ／ＵＣｒ水平分别较ＨＢＶＤＮＡ阴性组（Ｚ值分别为－２．９０８、－２．８２４，Ｐ值均＜０．０５）、非肝硬化组（Ｚ值分别为－３．２０４、－３．４１２，Ｐ值均＜０．０５）患者明显升高。
贝伐珠单抗注射液联合替莫唑胺同步放疗治疗脑转移的疗效及安全性
ｐｒｏｌｉｆｅｒａｔｉｏｎａｎｄｍｉｇｒａｔｉｏｎｏｆｌｕｎｇｃａｎｃｅｒｃｅｌｌｓｔｈｒｏｕｇｈｄｏｗｎ－ｒｅｇｕｌａｔｉｎｇｍｉｃｒｏＲＮＡ－２１７ｅｘｐｒｅｓｓｉｏｎ［Ｊ］．ＥｕｒＲｅｖＭｅｄＰｈａｒｍａｃｏｌＳｃｉ，２０２０，２４（１１）：６１５７－６１６５．［１８］ ＬＩＹ，ＧＯＮＧＹ，ＭＡＪ，ｅｔａｌ．Ｏｖｅｒｅｘｐｒｅｓｓｅｄｃｉｒｃ－ＲＰＬ１５ｐｒｅｄｉｃｔｓｐｏｏｒｓｕｒｖｉｖａｌａｎｄｐｒｏｍｏｔｅｓｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｇａｓｔｒｉｃｃａｎｃｅｒｖｉａｒｅｇｕｌａｔｉｎｇｍｉＲ－５０２－３ｐ／ＯＬＦＭ４／ＳＴＡＴ３ｐａｔｈｗａｙ［Ｊ］．ＢｉｏｍｅｄＰｈａｒｍａｃｏｔｈｅｒ，２０２０，１２７：１１０２１９．［１９］ ＰＥＩＸ，ＣＨＥＮＳＷ，ＬＯＮＧＸ，ｅｔａｌ．ＣｉｒｃＭＥＴｐｒｏｍｏｔｅｓＮＳＣＬＣｃｅｌｌｐｒｏｌｉｆｅｒａｔｉｏｎ，ｍｅｔａｓｔａｓｉｓ，ａｎｄｉｍｍｕｎｅｅｖａｓｉｏｎｂｙｒｅｇｕｌａｔｉｎｇｔｈｅｍｉＲ－１４５－５ｐ／ＣＸＣＬ３ａｘｉｓ［Ｊ］．Ａｇｉｎｇ（ＡｌｂａｎｙＮＹ），２０２０，１２（１３）：１３０３８－１３０５８．［２０］ ＬＩＹ，ＱＩＡＯＬ，ＺＡＮＧＹ，ｅｔａｌ．ＣｉｒｃｕｌａｒＲＮＡＦＯＸＯ３ｓｕｐｐｒｅｓｓｅｓｂｌａｄｄｅｒｃａｎｃｅｒｐｒｏｇｒｅｓｓｉｏｎａｎｄｍｅｔａｓｔａｓｉｓｂｙｒｅｇｕｌａｔｉｎｇｍｉＲ－９－５ｐ／ＴＧＦＢＲ２［Ｊ］．ＣａｎｃｅｒＭａｎａｇＲｅｓ，２０２０，１２：５０４９－５０５６．［２１］ ＣＨＥＮＭＳ，ＬＩＮＣＨ，ＨＵＡＮＧＬＹ，ｅｔａｌ．ＣｉｒｃＲＮＡＳＭＡＲＣＣ１ｓｐｏｎｇｅｓｍｉＲ－１４０－３ｐｔｏｒｅｇｕｌａｔｅｃｅｌｌｐｒｏｇｒｅｓｓｉｏｎｉｎｃｏｌｏｒｅｃｔａｌｃａｎｃｅｒ［Ｊ］．ＣａｎｃｅｒＭａｎａｇＲｅｓ，２０２０，１２：４８９９－４９１０．［２２］ ＣＨＥＨ，ＤＩＮＧＨ，ＪＩＡＸ．Ｃｉｒｃ＿００８０１４５ｅｎｈａｎｃｅｓｉｍａｔｉｎｉｂｒｅｓｉｓｔａｎｃｅｏｆｃｈｒｏｎｉｃｍｙｅｌｏｉｄｌｅｕｋｅｍｉａｂｙｒｅｇｕｌａｔｉｎｇｍｉＲ－３２６／ＰＰＦＩＡ１ａｘｉｓ［Ｊ／ＯＬ］．ＣａｎｃｅｒＢｉｏｔｈｅｒＲａｄｉｏｐｈａｒｍ，２０２０［２０２０－０９－２０］．ｈｔｔｐｓ：／／ｐｕｂｍｅｄ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／３２５９８１７０／．ＤＯＩ：１０．１０８９／ｃｂｒ．２０２０．３６００．（编校：张西敏）
魔芋甘露低聚糖增强肠道屏障功能改善酒精性中枢神经损伤的实验研究
网络出版时间：２０２４－０３－０７１０：４６：１８ 网络出版地址：ｈｔｔｐｓ：／／ｌｉｎｋ．ｃｎｋｉ．ｎｅｔ／ｕｒｌｉｄ／３４．１０８６．Ｒ．２０２４０３０６．１７２５．０１８魔芋甘露低聚糖增强肠道屏障功能改善酒精性中枢神经损伤的实验研究陈晶晶１，钟文艳１，２，沈文卓１，肖 莉１，２，袁成福１，２（三峡大学１．基础医学院、２．肿瘤微环境与免疫治疗湖北省重点实验室，湖北宜昌 ４４３００２）收稿日期：２０２３－１２－２０，修回日期：２０２４－０１－２６基金项目：国家自然青年科学基金资助项目（Ｎｏ８１９０３９１６）；三峡大学湖北肿瘤微环境与免疫治疗重点实验室开放基金项目（Ｎｏ２０２３ＫＺＬ０３５）作者介绍：陈晶晶（１９９８－），女，硕士，研究方向：代谢性疾病及药物干预，Ｅ ｍａｉｌ：ｊｉｎｇｊｉｎｇｃｈｅｎ０９２４＠１６３．ｃｏｍ；肖 莉（１９７９－），女，博士，副教授，硕士生导师，研究方向：代谢性疾病及药物干预，通信作者，Ｅ ｍａｉｌ：ｘｉａｏｌｉ ｃｎ＠１６３．ｃｏｍ；袁成福（１９７６－），男，博士，教授，硕士生导师，研究方向：代谢性疾病及药物干预，通信作者，Ｅ ｍａｉｌ：ｙｕａｎｃｆ４６＠ｃｔ ｇｕ．ｅｄｕ．ｃｎｄｏｉ：１０．１２３６０／ＣＰＢ２０２３１００７９文献标识码：Ａ文章编号：１００１－１９７８（２０２４）０３－０４４７－０８中国图书分类号：Ｒ ３３２；Ｒ３２２ ４５；Ｒ３２２ ８１；Ｒ３６４ ５；Ｒ５９５ ６摘要：目的 慢性酒精摄入导致的大脑过度神经炎症是中枢神经损伤的重要危险因素。
本实验主要研究魔芋甘露低聚糖（Ｋｏｎｊａｃｍａｎｎａｎｏｌｉｇｏｓａｃｃｈａｒｉｄｅｓ，ＫＭＯＳ）保护酒精喂养小鼠中枢神经炎症的作用及其机制。
方法 Ｃ５７ＢＬ／６Ｊ小鼠采用Ｇａｏ ｂｉｎｇｅ法制备慢性酒精喂养小鼠模型，同时使用不同剂量的ＫＭＯＳ灌胃干预，喂养６周。
评估脑组织皮层和海马区域神经元损伤和小胶质细胞活化情况，观察结肠组织损伤情况和炎症反应，检测血清ＬＰＳ浓度。
奈玛特韦原料药含量的定量核磁共振法测定
第42 卷第 7 期2023 年7 月Vol.42 No.7876~881分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）奈玛特韦原料药含量的定量核磁共振法测定胡炜琪1，刘秋芬1，丁娅1*，沈文斌2*（1．中国药科大学药学院药物分析教研室，江苏南京210009；2．中国药科大学药物科学研究院，江苏南京210009）摘要：建立了测定奈玛特韦原料药绝对含量的定量核磁共振氢谱法（1H qNMR）。
基于奈玛特韦在溶液中的构象稳定条件，以DMSO-D6和D2O（5∶1，体积比）为溶剂，δ4.96（1H，dd）和δ4.67（1H，dd）处的质子信号作为奈玛特韦的定量峰，δ6.22（1H，t）和δ5.84（1H，d）为拉米夫定的内标峰。
样品与内标摩尔比在0.5∶1.0 ~1.2∶1.0范围时的线性拟合方程为Y=0.456 4X - 0.000 5（r2 = 0.999 9），该方法专属性强，耐用性、重复性良好，回收率满足要求，稳定性可达48 h。
该方法测得的奈玛特韦样品的含量为99.23%（相对标准偏差为0.24%）。
1H qNMR法操作简单，具有无损、准确快速的特点，适用于新药奈玛特韦绝对含量的测定。
关键词：奈玛特韦；拉米夫定；定量核磁共振法；含量测定中图分类号：O657.61；R914.1文献标识码：A 文章编号：1004-4957（2023）07-0876-06Determination of Nirmatrelvir by QuantitativeNuclear Magnetic ResonanceHU Wei-qi1，LIU Qiu-fen1，DING Ya1*，SHEN Wen-bin2*（1．Department of Pharmaceutical Analysis，College of Pharmacy，China Pharmaceutical University，Nanjing 210009，China；2．Pharmaceutical Research Institute，China PharmaceuticalUniversity，Nanjing 210009，China）Abstract：A quantitative nuclear magnetic resonance（1H qNMR） method was developed for the de⁃termination of the absolute content of nirmatrelvir API．Based on the conformationally stable condi⁃tions of nemativir in solution，the proton signals at δ4.96（1H，dd），δ4.67（1H，dd） and δ6.22（1H，t），δ5.84（1H，d） were selected as the quantitative peaks for nirmatrelvir and lamivudine re⁃spectively in DMSO-D6 and D2O（5∶1，by volume） solvents on BRUKER AV－500 NMR instrument．The qNMR method exhibited a high specificity，and an linear fitting equation Y = 0.456 4X - 0.000 5（r2 = 0.999 9） was obtained in the range of 0.5∶1.0－1.2∶1.0 for the molar ratio of sample to inter⁃nal standard，and the results showed good durability，reproducibility，recovery and 48-hour stabili⁃ty．The content of nirmatrelvir was determined to be 99.23%（RSD = 0.24%）by qNMR．The 1HqNMR method is simple，non-destructive，accurate and rapid，which is suitable for determinationon the absolute content of nirmatrelvir API.Key words：nirmatrelvir；lamivudine；quantitative NMR；content determination奈玛特韦片/利托那韦片（Paxlovid）是辉瑞公司研制的口服新型冠状病毒肺炎（COVID－19）治疗新药，2021年12月，美国FDA正式发布了奈玛特韦片的紧急使用授权，用于治疗成人和儿童患者（12岁及以上，体重至少40 kg）的轻度至中度COVID－19［1］，以有效降低新冠患者的住院率和死亡风险。
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Industry News and Trends Banks Dabble in International Money Transfers September 04, 2006... By Gethan Ward... Gallatin News Examiner - Gallatin, TN, USA... "All banks … may be being conservative and rather do a front-end test to make sure what we're doing fits the Bank Secrecy Act guidelines," said Bob Byrd... http://www.gallatinnewsexaminer.com/apps/pbcs.dll/article?AID=/20060904/BUSINESS01/609040334/1309/MTCN04
B of A Ending Ties with Top Remitters (Paid subscription required) August 23, 2006... By David Breitkopf... American Banker Bank of America Corp.'s decision to cut its ties with the top two remittance companies - Western Union Financial Services Inc. and MoneyGram International Inc. - shows the intensity of banks' concern over a long-simmering issue. Federal regulators have sent mixed signals, on the one hand urging banks to continue to work with remittance companies but also insisting they pose a high risk of money-laundering activity and require greater scrutiny. http://www.americanbanker.com/
ICBA Fights Lowering of Wire Transfer Threshold August 2006... BankNet 360 - New York, NY, USA... and the Financial Crimes Enforcement Network yesterday, saying it opposes reducing or eliminating the threshold for recordkeeping on money wire transfers. http://www.banknet360.com/news/NewsAbstract.do?na_id=4847
Who Must Report Suspicious Transactions? August 22, 2006... Vive Le Canada - Canada... Money services businesses include alternative money remittance systems, such as Hawala, Hundi, Chitti, etc. Money services businesses... http://www.vivelecanada.ca/article.php/20060821232802907
Coleman, Kennedy Seek Hearing on Money Transfer Regulations August 25, 2006... By Frederic J. Frommer... Biloxi Sun Herald - MS, USA... Minnesota, Coleman and Kennedy wrote, "One way these immigrants have supported their friends and family back home is through cash remittances." The lawmakers... http://www.sunherald.com/mld/sunherald/news/breaking_news/15363635.htm Check out the following link to Senator Coleman‟s and Congressman Kennedy‟s press release. –ed. http://coleman.senate.gov/index.cfm?FuseAction=PressReleases.Detail&PressRelease_id=1076
Also see: Minnesota Somalis Fear Loss of Money Transfer Businesses August 22, 2006... By Gregg Aamot... Pioneer Press - St. Paul, MN, USA... summer. Several major banks that have been providing accounts for the businesses - known as hawalas - say they won't do that anymore. ... http://www.twincities.com/mld/twincities/news/15334909.htm
ABA Asks Treasury Paulson to Block Wire Transfer Reporting Plan August 02, 2006... Easy Bourse (Communiqués de presse) - Paris, France By Damian Paletta. Washington -(Dow Jones)- A top bank industry trade group asked US Treasury Secretary Henry M. Paulson Wednesday...
http://www.easybourse.com/Website/dynamic/News.php?NewsID=35807&lang=fra&NewsRubrique=2
Also see: ABA Calls For US Treasury to Scrap Wire Transfer Database Plan August 08, 2006... Finextra – UK... The American Bankers' Association (ABA) has called for the US government to block a massive data collection project that would require banks in the country to... http://www.finextra.com/fullstory.asp?id=15676
Opinion Please note that any opinion expressed is not necessarily the opinion of the NMTA. –ed. Five Ways to Transfer Money Abroad August 22, 2006... By Neil Faulkner (TMFVertigo)... Motley Fool UK - UK Every idiot in the world but for us Brits seems to be able to do cheap money transfers. The world... http://www.fool.co.uk/news/comment/2006/c060822c.htm?ref=foolwatch
Sending Money Home July 21, 2006... By Adolfo Pesquera... San Antonio Express (subscription) - San Antonio, TX, USA... neighborhoods. Remittance companies like Barri have been growing for about five years along with Texas' immigrant communities. "Immigrants... http://www.mysanantonio.com/business/stories/MYSA072306.01R.remittances.3b977b.html
Cost of Remittances Strains Immigrants July 19, 2006... By Carolyn Said... Kitsap Sun (Subscription) - WA, United States... Unfortunately, even while lowering remittance costs has become a priority, tough new Patriot Act laws designed to stamp out money laundering have made it harder than ever for small players to provide remittance services... http://www.kitsapsun.com/bsun/bu_business/article/0,2403,BSUN_19060_4857970,00.html