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??This review provided the research progresses of the strategies of the modification and application of poly (lactic-co-glycolic acid, PLGA) nanoparticles . The preparation and modification METHODS of PLGA nanoparticles and their application according to the reported foreign literature were analysed and summaried. PLGA is a biodegradable and biocompatible polymer which is one of the most widely used materials in preparation of sustained and controlled microparticles as drug vectors. This review focuses on the METHODS of modified nanoparticles and the advantages. Emulsification method, nanoprecipitation, emulsification solvent diffusion, solvent injection method and supercritical anti-solvent technique are the main five preparation METHODS of PLGA nanoparticles.The strategies of modifition include covalent cross-link, electrostatic interaction and hydrophobic interaction. The applications of modified nanoparticles to the optimal formulation, sustained release profile, improved cellular uptake, and targeting to specific organs or cells are introduced.     

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??OBJECTIVE To rapidly identify the related substances in doxycycline hyclate tablets and investigate the possible degradation pathways of doxycycline hyclate solution by 2D-LC-IT-TOF/MS. METHODS Firstly,the chromatography was carried out using the 1stD InertSustain^TM C₁₈(4.6 mm??150 mm,5 ??m) column and a mobile phase containing a mixture of buffer solution(0.25 mol??L^-1 ammonium acetate solution-0.1 mol??L^-1 ethyldiaminetetraacetic acid disodium: triethyiamine=100??10??1)-acetonitrile(85??15). Solution pH was adjusted to 8.8 with glacial acetic acid. Then a InertSustain^TM C₁₈(2.1 mm??50 mm,2 ??m) column was used with 0.1% formic acid-water as mobile A and 0.1% formic acid-acetonitrile as mobile B in a gradient elution mode in 2ndD. Electrospray ionization (ESI) source was tested in both positive and negative ion modes. Nebulized gas flow was 1.5 L?? min^-1. Dry gas flow was 10 L?? min^-1. The desolvation tube temperature was kept at 200 ??. Related substances were characterized according to multi-level MS behaviors. The 2D-LC-IT-TOF/MS method was employed to identify the structures of impurities in forced degradation solutions and illuminate the degradation pathways of doxycycline hyclate solution. RESULTS A total of eight related substances were detected in doxycycline hyclate tablets. Four of them had a content of more than 0.1%, and their structures were identified to be 4-epidoxycycline, metacycline, ??-epidoxycycline and 2-acetyl-2-decarbamoyldoxycycline. The solution of doxycycline hyclate easily degraded under alkaline condition and generated an open loop compound taken off the keto group. The solution was sensitive to heat, and generated 4-epidoxycycline with a little amount of 4-epi-6-epidoxycycline; but the solution was stable under illumination and acidic condition. CONCLUSION The established method is suitable for rapid identification of impurities in doxycycline hyclate, which can be applied as a useful analytical tool for quality control and drug process optimization of doxycycline hyclate.     

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??OBJECTIVE To prepare small interfering RNA(siRNA) lipid complexes (TPOS-L/siRNA) modified by D-α-tocopheryl poly (2-ethyl-2-oxazoline) succinate (TPOS).METHODS The conventional siRNA lipid complexes (CLs/siRNA) and PEGylated CLs/siRNA (PEG-L/siRNA) were used as controls. CLs/siRNA was prepared by mixing blank CLs and siRNA directly of equal volume according to the electrostatic interaction of positive and negative charges. The encapsulation efficiency, morphology, stability, in vitro release and cell uptake of TPOS-L/siRNA were investigated.RESULTS The CLs/siRNA had obvious lipid bilayer structure, the encapsulation efficiency (EE) was (86.68±1.41)%, and the particle size of CLs/siRNA was less than 200 nm. The modification with TPOS or PEG-DSPE had no significant effect on the EE and particle size of CLs/siRNA, which could endow the lipid complexes with good stability. In addtion, TPOS-L/siRNA had good pH-sensitive property, and could respond to slightly acidic environment, which significantly enhanced the cell uptake.CONCLUSION TPOS can construct good siRNA carrier and increase the stability and pH sensitivity of the nanocarrier.     

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??OBJECTIVE To establish an analytical method for simultaneous determination of scutellarin ethyl ester and its related substances by RP-HPLC and identify the main related substances by UPLC-MS.METHODS The assay was performed on a Kromasil C₁₈ column(4.6 mm??250 mm,5 ??m),and gradient elution was used with methanol-water containting 0.2% formic acid. The flow rate was 1.0 mLmin^-1and the detection wave length was set at 335 nm.The structure of the main related substance was identified by high resolution MS.RESULTS Under the separation condition,the related substances were completely separated from the principal components.The calibration curve of scutellarin ethyl ester showed good linearity in the concentration range of 5.005-1 001 mgL^-1(r²=0.999 9). The average recovery was 99.6%. The main related substance was identified as scutellarin by LC-MS. The contents of total impurities and scutellarin were 1.14% and 0.89%, respectively.CONCLUSION The method is simple, efficient and accurate, and can be used for the quality control of scutellarin ethyl ester.     

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??OBJECTIVE To develop an HPLC-ELSD method for determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection. METHODS The column was Waters Symmetry 300 C₁₈(4.6 mm??150 mm, 5 ??m;pore size:300). The mobile phase was methonal-tetrahydrofuran-0.17 mol??L^-1 ammonium acetate(93??6??1). The flow rate was 1.0 mL??min^-1. The column temperature was 25 ?? and the injection volume was 10 ??L. The ELSD conditions were as follows: Alltech 2000ES ELSD detector; drift temperature: 110 ??; rate: 2.6 L??min^-1. RESULTS This method had good specificity. The linear ranges of the calibration curves for MPEG-DSPE and HSPC were 0.03-0.48 mg??mL^-1 (r=0.999 8) and 0.1-1.0 mg??mL^-1 (r = 0.999 8), respectively. The average recovery rates of MPEG-DSPE and HSPC were 100.0% (n=3??3) and 101.0% (n=3??3), respectively. The LODs of MPEG-DSPE and HSPC were 13 and 52 ng, respectively. The repeatability and intermediate precision of MPEG-DSPE were 0.9% (n=5) and ??1.9%(n=3), respectively. The repeatability and intermediate precision of HSPC were 1.1%(n=5) and ??1.3%(n=3) , respectively. CONCLUSION The established method is accurate, reliable, repeatable and suitable for the determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection.     

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??OBJECTIVE To develop a comprehensive strategy integrateded with chemometrics METHODS for quality evaluation of Glechoma longituba(GL) from different geographical origins. METHODS The chemical differentiation of 22 batches of GL were performed by UPLC/QTOF-MS^E coupled with UNIFI^TM software. To evaluate the quality of GL from different geographical origins, the principal component analysis (PCA) was developed to analyze the MS^E data of 22 batches of GL. To find the markers, the orthogonal partial least squares discriminant analysis (OPLS-DA) was adopted to analyze the MS^E data of 22 batches of GL. RESULTS A total of 31 compounds including 8 phenolic acids, 14 flavones, 7 terpenes, 1 organic acid and 1 coumarin were unambiguously or tentatively identified in the GL from Guangxi province. And 14 compounds were reported for the first time. Twenty-two batches of GL were well gathered and segregated into two different groups scattering in the score plot of PCA by MarkerLynx XS. The result of PCA showed that the chemical compositions of GL from Anhui province had obvious difference with those from other provinces. Based on the S-Plot from the score plot of OPLS-DA, the differential component of GL from Anhui province was tentatively identified as terpene. CONCLUSION The integrated strategy facilitates authentication of herbal medicines of different origins in a more efficient and more intelligent manner.     

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??OBJECTIVE To establish an HPLC-MS/MS method for the determination of melatonin in human plasma. METHODS The plasma samples were extracted with ethyl acetate, using melatonin-D7 as the internal standard (IS). Then the ethyl acetate layer was evaporated in vacuum concentrator. Dried samples were redissolved in mobile phase (0.1% formic acid-methanol= 56:44, V/V), vortexed and centrifuged. The redissolved solution was transferred to an auto sampler vial and the supernatant was injected to the HPLC-MS/MS system. The MS/MS analysis was carried out in positive ionization mode by multiple reactions monitoring (MRM) at m/z 233.1??174.1 for melatonin and m/z 240.2??178.0 for IS, respectively. RESULTS The calibration curve of melatonin in human plasma was linear over the concentration range of 0.020 00-30.00 ng??mL^{- 1}. The lower limit of quantitation was 0.020 00 ng??mL^{- 1}. The RSDs of within-day and between-day were less than 15%. The extraction recoveries were between 59.0%-65.0%. The matrix effects were between 95.5%-98.9%. CONCLUSION The method is proved to be convenient, sensitive and accurate. It can be applied to study the pharmacokinetics of melatonin prolonged-release tablets in healthy Chinese volunteers.     

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??OBJECTIVE To establish an HPLC-MS/MS method for determination of ASC-J9 in rat blood. METHODS After liquid-liquid extraction, ASC-J9 was separated on a Symmetry C₁₈colume, with mobile phase of acetonitrile-water containing 0.1% formic acid and 10 mmol??L^-1 ammonium formate.The flow rate was 1.0 mL??min^-1, mass shunt was 0.4 mL??min^-1, and column temperature was main tained at 35 ??. Quantification was performed in positive ion multiple-reaction-monitoring(MRM) mode. RESULTS The calibration curve of ASC-J9 had good linearity in the concentration range of 3.54-1 180 ng??mL^-1. The extraction recovery rate was within 83.19% to 87.27%,and the intra-day and inter-day RSDs were both less than 8.89%. CONCLUSION This method is specific, sensitive and suitable for determination of ASC-J9 in rat blood.
     

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??OBJECTIVE To assess the cost-effectiveness of clinical pharmaceutical care in the H. pylori(HP) eradication in outpatients with peptic ulcer.METHODS Ninty-six Outpatients with HP positive peptic ulcer from July 2015 to June 2016 were prospectively collected, and randomly divide into control group and intervention group. Patients in the control group were given the traditional outpatient service. The intervention group patients were given with pharmaceutical education and follow-up by clinical pharmacist. The score of compliance with medication, HP eradication rate and the improvement of gastrointestinal symptom were selected as outcome indicator. Time cost of intervention and fee of clinical pharmacist training were estimated and recorded as the cost of clinical pharmacy intervention. RESULTS ??Improvement of the score of compliance with medication of intervention group patients was significantly higher than the control group (1.71 vs. 0.44, P<0.01). Forty-four patients of the intervention group, while 36 patients in the control group reported less gastrointestinal symptoms (91.67% vs. 75.00%, P=0.028). The HP eradication rate of the intervention group was significantly higher than the control group (91.67% vs. 72.92%, P=0.016). ??For the hospital, the cost of pharmacy service of control group was 292.31 yuan, and 821.61 yuan of the intervention group. ??The cost-effectiveness ratio of the score of compliance with medication of control group was 664.34, while the intervention group was 480.47, which was superior to the control group. It was cost-effective. However, the cost-effectiveness ratio of HP eradication rate and the improvement of gastrointestinal symptom of the intervention group were both higher than the control group. CONCLUSION Clinical pharmaceutical care increase the cost of the hosipital. However, clinical pharmaceutical care can result in cost-effective improvement of the medication compliance.     

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??OBJECTIVE To identify rapidly the chemical constituents in Tanreqing injection and Tanreqing capsules by ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS^E).METHODS The separation was performed on a Waters Acquity UPLC BEH C₁₈ column (1.0 mm??100 mm, 1.7 ??m) with acetonitrile-0.1% formic acid as mobile phase in gradient elution. ESI ion source was employed in negative ion mode. The differences of chemical compositions between the two preparations and the sources of these compounds were illustrated based on their retention time, accurate mass measurements and the mass fragments by comparison with those in the literature or database and the reference standards.RESULTS A total of 111 compounds including 13 unknown components were identified or tentatively characterized. Among these compounds, 14 were derived from Scutellariae Radix (SR) intermediate, 36 were from Bear Bile Powder(BBP) intermediate, 7 were from Caprae Hircus Cornu(CHC) intermediate, 34 were from Lonicerae japonicae Flos(LJF) intermediate and 22 were from Forsythiae Fructus(FF) intermediate. Moreover, quinic acid and rutin were simultaneously detected in LJF and FF intermediates, 28 constituents were unambiguously confirmed by their reference standards. However, 71 compounds were observed both in injection and capsules, while 24 compounds were only found in Tanreqing injection and 16 compounds only in the capsules.CONCLUSION The differences of chemical constituents between Tanreqing injection and capsules are effectively characterized by UPLC/Q-TOF-MS^E method, which will facilitate the quality control of the two preparations.     

VCC-1慢病毒表达载体的构建及其对胶质瘤细胞增殖的影响

目的:构建小趋化因子VCC-1的慢病毒表达载体并研究其对人胶质瘤细胞株U251增殖的影响.方法:以人结肠癌细胞为模板扩增VCC-1基因,并连入pMX载体,挑取测序正确的克隆在293 T细胞中表达,并用realtime-PCR和Westernblot进行验证.结果:成功构建了人VCC-1的慢病毒表达载体,并感染了星形胶质瘤细胞系U251.结论:证明了VCC-1基因在胶质瘤细胞的增殖过程中起促进作用,并为后续研究VCC-1和胶质瘤形成发展和浸润的机制提供了良好的基础.     

稳定表达人寡肽转运体1的细胞模型构建及应用

 目的 构建稳定表达人寡肽转运体1（hPepT1）的马丁-达比犬肾细胞模型(MDCK-hPepT1),并将其应用于中药单体中人寡肽转运体1抑制剂的筛选。方法 构建重组质粒pcDNA3.1(+)-hPepT1,将其转染马丁-达比犬肾系细胞,经G418筛选后以有限稀释法挑选单克隆细胞。以人寡肽转运体1荧光底物β-Ala-Lys(AMCA)和经典底物Glysar对单克隆细胞转运活性进行验证;通过反转录聚合酶链式反应验证人寡肽转运体1在转录水平的表达。以中药单体对Glysar摄取的影响实现人寡肽转运体1抑制剂的筛选。结果 获得的两株单克隆细胞（D19、A11）对β-Ala-Lys(AMCA)摄取均显著高于转染空载体的马丁-达比犬肾系细胞,对Glysar的摄取分别为转染空载体细胞的24、11倍;D19、A11中hPepT1的mRNA水平亦显著高于转染空载体的细胞;已知的人寡肽转运体1抑制剂（底物）显著减少单克隆细胞D19和A11对Glysar的摄取;部分中药单体对Glysar摄取产生不同程度的抑制。结论 本实验成功构建了稳定高表达人寡肽转运体1的马丁-达比犬肾系细胞模型,并应用该模型筛选出对人寡肽转运体1具有潜在抑制作用的中药单体。     

稳定表达人有机阳离子转运体1的细胞模型构建

 目的 建立能稳定表达人有机阳离子转运体1（human organic cation transporter1, hOCT1）野生型及两个突变体的马丁达比狗肾上皮（Madin-Darby canine kidney, MDCK）细胞模型。方法 从人肝组织中提取hOCT1野生型基因,经定点突变获得hOCT1_P341L,hOCT1_M420del两个突变型基因,构建表达质粒pcDNA3.1(+)-hOCT1,pcDNA3.1(+)-hOCT1_P341L,pcDNA3.1(+)-hOCT1_M420del。将质粒转染MDCK细胞,通过G418筛选获得抗性克隆,并通过hOCT1的荧光底物(4-二甲氨基苯乙烯基)-N-甲基吡啶 (ASP+)筛选得到具有较高活性的MDCK-hOCT1细胞。经反转录聚合酶链式反应(RT-PCR)及经典底物N-甲基-4-苯基吡啶(MPP+)的摄取实验,鉴定筛选得到的单克隆细胞株hOCT1 mRNA的表达及功能。结果 本实验获得的hOCT1野生型及2个突变体细胞模型与转染空载体的MDCK细胞相比,其 hOCT1 mRNA表达量显著增高,对经典底物MPP+的摄取为转染空载体细胞的12.5,13.7,12.0倍,hOCT1抑制剂可显著抑制细胞对MPP+的摄取。结论 建立的细胞模型可用于hOCT1抑制剂及底物的筛选。     

小鼠RPS20基因miRNA干扰载体的构建及鉴定

目的 构建小鼠RPS20 miRNA干扰载体,进行载体质粒的提取及鉴定,以进行细胞转染实验。方法 针对小鼠RPS20基因miRNA序列,设计合成阴性对照及4对目标基因oligo,采用载体构建试剂盒进行重组克隆,经过退火、连接及转化后,进行测序及质粒提取鉴定。结果 测序结果提示序列构建完全正确,电泳结果提示质粒大小正确,纯度较高。结论 成功构建了针对于小鼠RPS20基因的miRNA干扰载体,测序及电泳结果提示,所构建序列可用于后续转染实验。     

RPS20RNA干扰慢病毒的构建及其对CT26细胞生长的影响

目的 研究脾虚证差异表达基因——核糖体蛋白S20 (RPS20)RNA干扰慢病毒转染对小鼠结肠癌细胞(CT26)细胞指数的影响,验证前期筛选的RNA干扰序列的有效性,为后续在小鼠体内以RNA干扰进行RPS20的功能鉴定提供参考.方法 根据前期筛选的RPS20 RNA干扰有效序列,进行质粒载体的重组,包装RPS20干扰慢病毒并转染CT26细胞,采用xCELLigence细胞动态分析仪实时监测转染后细胞的细胞指数以反映细胞的生长情况.结果 测序图谱和Blast比对结果表明重组慢病毒包装载体构建成功,慢病毒滴度为1.2×105 TU/mL,RNA干扰慢病毒转染后CT26细胞指数的增长受到明显抑制,转染后30 h细胞指数抑制率达到82.24％.结论 前期筛选的RPS20 RNA干扰序列可成功构建包装干扰慢病毒,转染后能有效抑制CT26细胞的生长,可为后续研究提供参考.     

RNA干扰抑制生存素基因对肝癌细胞凋亡的影响

目的将生存素(survivin)基因特异性小干扰性RNA(smallinterferingRNA,siRNA)直接转染肝癌hepG2细胞系,观察其对肝癌细胞survivinmRNA水平及细胞凋亡的影响。方法合成survivin特异性的siRNA序列1、序列2和对照序列,通过Lipofectamin2000进行直接转染。试验细胞随机分为4组,第1组为空白对照组,第2组以对照序列进行转染,第3组以序列1进行转染;第4组以序列2进行转染。转染后48h,通过半定量RT-PCR的方法检测survivinmRNA的水平。转染后72h,通过流式细胞仪检测各组的细胞凋亡指数。结果转染后48h,1组、2组、3组、4组survivinmRNA相对表达率(%)分别为:48.5±10.5、43.4±6.4、34.5±8.6、23.7±6.2(P1:3、P1:4、P2:4、P3:4均<0.05)。转染后72h,1组、2组、3组、4组细胞凋亡指数(%)分别为:10.7±3.1、11.8±3.5、15.9±3.5、18.6±3.2(P1:3、P1:4、P2:3、P2:4均<0.05)。结论survivin特异性siRNA直接转染肝癌细胞可抑制survivinmRNA的表达,促进肝癌细胞凋亡的发生。     

知母总皂苷对β淀粉样蛋白诱导的PC12细胞Bcl-2表达的影响

目的: 观察知母总皂苷对β淀粉样蛋白(β-amyloid,Aβ)Aβ_25-35诱导的PC12细胞中抗凋亡基因Bcl-2表达的影 响。方法: 培养PC12细胞,分为6组:正常组、模型组、知母低、中、高剂量组、盐酸多奈哌齐组。模型组加入终浓度为20 μmol·L^-1 Aβ_25-35 的培养基,作用48 h;知母低、中、高剂量组和盐酸多奈哌齐分别加入含有低、中、高剂量知母总皂苷 (5,10,20 mg·L^-1) 及盐酸多奈哌齐(1 μmol·L^-1)和Aβ_25-35(终浓度为20 μmol·L^-1)的培养基共同作用于PC12细胞48 h。然后采用MTT检测各组细胞活力的变化;采用RT-PCR检测各组细胞中Bcl-2的mRNA表达变化;采用Western blotting技术检测各组细胞中Bcl-2蛋白的表达变化。结果: 和正常组比较,模型组细胞活力降低(P<0.01);和模型组比较,高、中、低剂量知母总皂苷能显著升高PC12细胞活力(P<0.05);和正常组比较(0.476±0.072,0.14±0.02),模型组抗凋亡基因Bcl-2的mRNA(0.316±0.026)和蛋白(0.08±0.01)表达水平降低(P<0.01);和模型组比较,高、中、低剂量知母总皂苷能显著性升高抗凋亡基因Bcl-2的mRNA(0.447±0.016,0.465±0.043,0.472±0.023)和蛋白(0.17±0.01,0.19±0.02,0.13±0.01)的表达水平(P<0.05)。结论: 知母总皂苷能升高Aβ_25-35诱导的PC12细胞的活力降低,其机制可能是升高Bcl-2的表达水平。     

表没食子儿茶素没食子酸酯对乳胞素诱导PC12细胞损伤的影响

目的 探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对乳胞素(lactacystin) 诱导的PC12细胞损伤的影响。     

川芎嗪对t-BHP致PC12细胞损伤的保护及机制

目的:研究川芎嗪(TMP)对叔丁基过氧化氢(t-BHP)诱导的PC12细胞损伤的保护作用及对磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(m TOR)信号通路的影响。方法:培养PC12细胞,孵育不同浓度的川芎嗪(10,25,50,100,200μmol·L~(-1)),12 h后采用细胞增殖毒性检测试剂盒(CCK-8)确定川芎嗪的保护浓度。随后将PC12细胞分为空白组,t-BHP组,TMP组。空白组加入培养基,t-BHP组加入t-BHP(200μmol·L~(-1)),TMP组加入TMP(25,50,100μmol·L~(-1))预处理12 h后加入t-BHP(200μmol·L~(-1))。共作用6 h后采用酶联免疫吸附测定(ELISA)检测乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、活性氧(ROS)含量和谷胱甘肽过氧化物酶(GSH-Px)活性。采用蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),Akt,磷酸化Akt(p-Akt)及m TOR,p-mTOR的表达情况。结果:与空白组比较,t-BHP组细胞生存率降低(P0.01),与t-BHP组比较,25,50,100μmol·L~(-1)的川芎嗪促进细胞增殖(P0.05,P0.01),其中50μmol·L~(-1)组的细胞增殖率最高(P0.01);与空白组比较,t-BHP组LDH的释放量,ROS和MDA含量显著增加(P0.01),SOD和GSH-Px的活性显著降低(P0.01),与t-BHP组比较,TMP组LDH的释放量,ROS和MDA含量显著降低(P0.05,P0.01),SOD和GSH-Px的活性显著升高(P0.05,P0.01);Western blot表明与空白组比较,t-BHP组中p-Akt,p-mTOR蛋白表达水平明显下降(P0.01),Bcl-2/Bax值明显降低(P0.01),与t-BHP组比较,TMP(50μmol·L~(-1))组中p-Akt,p-mTOR蛋白表达水平明显上升(P0.05),Bcl-2/Bax值明显上升(P0.05)。另外LY294002(PI3K蛋白抑制剂)处理减弱了这些变化。结论:川芎嗪通过激活PI3K/Akt/m TOR信号通路保护t-BHP诱导的PC12细胞损伤。