阿魏酸钠对氧化损伤大鼠晶状体上皮细胞凋亡的防护

目的　探讨阿魏酸钠 (SF)对实验性氧化损伤大鼠晶状体上皮细胞 (LEC)凋亡有无抑制作用。方法　采用无菌操作摘取SD大鼠双眼 ,并在手术显微镜下分离晶状体 ,随机分成空白对照组、过氧化氢组 (H2 O2 )、吡诺克辛 (PS)组和SF组。使晶状体孵育在 30 0 μmol·L- 1H2 O2 培养液中复制LEC凋亡模型 ,同时加入终浓度为 5mmol·L- 1的SF ,置CO2 培养箱共同孵育 2 4h。取晶状体前囊膜 ,采用TUNEL法检测LEC凋亡及凋亡率、透射电子显微镜观察LEC超微结构改变和凋亡小体形成。结果　H2 O2组LEC凋亡率 (92 .0± 2 .6 ) %显著高于空白对照组(3.5± 1.8) %;SF组LEC凋亡率 (2 0 .8± 3.0 ) %显著低于H2 O2 组 ,且与PS组 (5 6 .0± 9.9) %有非常显著的差异。超微结构研究显示 ,H2 O2 组绝大多数LEC发生凋亡 ,并呈凋亡各期严重改变的全过程 ;SF组仅少数LEC发生凋亡 ,并多为早期或中期的轻微改变。结论　SF可明显抑制实验性氧化损伤大鼠LEC凋亡 ,且明显优于PS滴眼液。     

补骨脂素和异补骨脂素的急性毒性和相互作用

目的 探究补骨脂素、异补骨脂素的急性毒性及两者（1∶1）混合使用后的相互作用。方法 补骨脂素（1 125、843、633、475 mg/kg）、异补骨脂素（475、404、343、292 mg/kg）以及两者1∶1的混合物（633、538、457、389、330 mg/kg）1次性ig给予小鼠,连续观察并记录14 d小鼠的毒性反应和死亡情况,用SPSS计算补骨脂素、异补骨脂素以及两者混合使用后的半数致死量（LD₅₀）,用等效线图解法判断两者的相互作用。结果 给药组小鼠均出现僵直、腹部贴地、活动力减弱,甚至抽搐、口眼周有分泌物,心率减慢直至死亡的现象,与助溶剂组比较,体质量呈降低趋势;补骨脂素LD₅₀为638.69 mg/kg,95%可信限为526.91～785.78mg/kg;异补骨脂素LD₅₀为351.72 mg/kg,95%可信限为248.17～394.57 mg/kg;两者1∶1混合给药的LD₅₀为454.66 mg/kg,95%可信限为422.58～489.59;两者合用的LD₅₀在补骨脂素和异补骨脂素相加等效线上。结论 补骨脂素和异补骨脂素大剂量给予小鼠时,引起药物急性毒性反应,1∶1混合给药具有相加作用。     

HPLC法测定肺气肿片中补骨脂素和异补骨脂素的含量

摘要：目的 建立肺气肿片中补骨脂素和异补骨脂素的含量测定方法。方法 采用HPLC法，用Kromasil5-C18（4.6mm×250mm, 5μm）色谱柱，以甲醇-0.05mol/l磷酸二氢钾溶液（50:50）为流动相，检测波长246nm。结果 方法线性良好，平均加样回收率：补骨脂素为97.8%，RSD0.20%（n=5）异补骨脂素为98.8%，RSD0.13%。结论 本法简便快速，结果准确，可作为控制肺气肿片质量的方法。     

毛细管气相色谱法测定固本咳喘片中补骨脂素和异补骨脂素的含量

目的：建立超声提取毛细管气相色普（CGC）法分离测定中成药大本咳喘片中有效成分补骨脂素和异补骨脂素的含量测定方法。方法：采用HP－5毛细管柱，氢火焰离子检测器，以超声法提取样品，用蒽作内标物，内标二点法定量。结果：CGC对补骨脂素和异补骨脂素能达到完全分离。结论：该法是固本咳喘片质量控制的简便易行的方法之一。     

HPLC法测定肺气肿片中补骨脂素和异补骨脂素的含量

目的建立肺气肿片中补骨脂素和异补骨脂素的含量测定方法。方法采用HPLC法,用Kromasil C18(4.6 mm×250 mm,5μm)色谱柱;以甲醇-0.05 mol.L-1磷酸二氢钾溶液(50∶50)为流动相;检测波长246 nm。结果方法线性良好,平均加样回收率:补骨脂素为97.8%,RSD为0.20%(n=5);异补骨脂素为98.8%,RSD为0.13%。结论本法简便快速,结果准确,可作为控制肺气肿片质量的方法。     

HPLC法测定不同来源补骨脂中补骨脂素和异补骨脂素的含量

目的：建立HPLC法测定不同来源补骨脂中补骨脂素和异补骨脂素含量的方法。方法：选用Hypersil C18柱（250mm×4．6mm,5μm）,以甲醇：0．015％磷酸溶液（40：60）为流动相,流速1．0ml·min^-1,检测波长246／1／11,柱温25℃。结果：补骨脂素在4．16～14．56μg·ml^-1间呈良好的线性关系（r=0．9997）,平均加样回收率为99．9％（RSD=0．90％,n=9）;异补骨脂素在4．16～14．56μg·ml^-1间呈良好的线性关系（r=0．9996）,平均加样回收率为98．0％（RSD=0．78％,n=9）。结论：该方法快速、准确,可用于补骨脂的质量控制。     

HPLC法测定肠胃宁片中补骨脂素、异补骨脂素的含量

目的建立HPLC法测定肠胃宁片中补骨脂素、异补骨脂素含量的方法。方法采用Agilent Extend-C18色谱柱,以乙腈-0.1%磷酸溶液(18∶82)为流动相,流速为1.0mL.min-1,检测波长为246nm,柱温为30℃。结果补骨脂素在0.0542～0.8124μg范围内线性良好,r=1.0000;异补骨脂素在0.0530～0.7956μg范围内有较好的线性关系,r=1.0000;补骨脂素的平均回收率为97.82%,RSD=1.17%;异补骨脂素平均回收率为97.26%,RSD=1.00%。结论所建立的方法简便、准确,可用于肠胃宁片的质量控制。     

补骨脂酊中补骨脂素、异补骨脂素的含量测定

目的建立补骨脂酊中补骨脂素、异补骨脂素的含量测定方法。方法采用高效液相色谱法,色谱柱为Agilent Eclipse XDB-C18柱（150mm×4．6mm,5μm）．流动相为甲醇-水（40：60）．流速为1．0mL·min^-1;检测波长为246nm。结果补骨脂素在0．022～0．44μg（r=0．9999）,异补骨脂素在0．02388～0．4776μg（r=0．9999）具有良好线性关系。补骨脂素平均回收率为98．10％（n=9．RSD=1．20%）,异补骨脂素平均回收率为97．74％（n=9,RSD=1．10％）。结论本方法简便、准确、可靠,可用于补骨脂酊中补骨脂素、异补骨脂素的含量测定。     

微小 RNA-595在人晶状体上皮细胞凋亡中的调控作用

目的 探讨微小RNA-595(miR-595)在年龄相关性白内障晶状体组织中的表达情况及其对人晶状体上皮细胞凋亡的影响与机制.方法 收集年龄相关性白内障病人晶状体前囊膜组织(白内障组)及正常供体眼球晶状体前囊膜组织(健康组)标本,以H2O2诱导构建人晶状体上皮细胞SRA01/04凋亡模型(H2O2组),并以正常培养的SRA01/04细胞作为对照(对照组),采用实时荧光定量PCR检测miR-595的表达改变.将体外培养的SRA01/04细胞分为mimics-NC组、miR-595 mimics组、inhibitor-NC组和miR-595 inhibitor组,将miR-595 mimics/inhibitor及其阴性对照mimics/inhibitor-NC转染至SRA01/04细胞后,采用实时荧光定量PCR检测检测各组细胞中miR-595的表达情况;转染48 h后暴露于200μmol/L H2O21 h,采用流式细胞仪检测各组细胞凋亡率,免疫印迹法检测细胞中B细胞淋巴瘤-2(Bcl-2)蛋白的表达水平;采用双荧光素酶报告基因实验检测miR-595和Bcl-2的靶向关系.结果 与健康组相比,白内障组中miR-595表达水平明显升高[(5.52±1.64)比(1.00±0.00),P<0.05];同时,H2O2组细胞中miR-595表达水平明显高于对照组[(3.86±1.12)比(1.00±0.00),P<0.05].与mimics-NC组比较,miR-595 mimics组细胞中miR-595表达水平[(4.16±0.78)比(1.00±0.00)]、细胞凋亡率[(17.52±3.05)％比(8.86±1.21)％]明显升高,而Bcl-2蛋白的表达水平明显降低(P<0.05);但miR-595 inhibitor组细胞中miR-595表达水平、细胞凋亡率明显低于inhibitor-NC组,而Bcl-2蛋白的表达水平明显高于inhibitor-NC组(P<0.05).Bcl-2是miR-595的靶基因.结论 miR-595在年龄相关性白内障晶状体组织中高表达,并靶向调控Bcl-2表达促进人晶状体上皮细胞凋亡.     

白藜芦醇抑制晶体上皮细胞生长与诱导细胞凋亡的实验研究

目的 探讨白藜芦醇(Res)对晶体上皮细胞体外增殖的影响,观察Res诱导晶体上皮细胞凋亡的作用.方法 用不同浓度的Res处理晶体上皮细胞,用四甲基偶氮唑蓝(MTT)比色法检测白藜芦醇对晶体上皮细胞增殖的影响,通过HE染色、电子显微镜、原位末端标记法(TUNEL)和流式细胞仪Annexin V荧光染色,观察细胞的形态学改变,并定量检测细胞凋亡.结果:Res抑制了晶体上皮细胞的生长与增殖(P＜0.01),呈浓度依赖性反应;Res能明显诱导晶体上皮细胞凋亡,对照组与100μmol/L、200μmol/L Res处理24h后的细胞凋亡率分别为4.67%、17.31%、32.77%.结论 Res能通过诱导晶体上皮细胞凋亡而抑制其生长与增殖,该研究为进一步探讨Res在白内障术后后囊膜混浊发生的防治提供了一定的实验依据.     

Cadmium-induced induction of cell death in human lens epithelial cells: Implications to smoking associated cataractogenesis

Cadmium is reported to accumulate in human eye tissues suggesting its implication in diverse ocular pathology. Using an in vitro cell culture model we investigated the effects of cadmium on human lens epithelial cells (HLECs) (HLE-B3). We observed cadmium-induced dose- as well as time-dependent decline in HLECs viability which was exacerbated significantly upon reduction of intracellular glutathione levels by buthionine sulfoximine (BSO). There was a dose-dependent significant increase in lactate dehydrogenase (LDH) release from HLECs suggesting cadmium-induced alteration of membrane integrity as well as necrotic cell death. The decline in cell viability was also due to apoptosis of the HLECs as determined by quantifying % apoptotic cells as well as PARP cleavage. Moreover, release of apoptosis inducing factor (AIF) into the cytosol was also detected. Cadmium was also observed to increase oxidative stress, lipid peroxidation and activation of MAPK pathway in HLECs. Antioxidants like N-acetylcysteine (NAC) and α-Tocopherol significantly prevented cadmium-induced toxicity in HLECs. Our findings suggest that cadmium-induced elevated oxidative stress as well as activation of MAPK signaling cascade eventually led to cell death of HLECs through apoptosis as well as necrosis. The loss of HLECs by cadmium could possibly explain its implication in cataract development particularly associated with smoking.     

Oxyresveratrol protects human lens epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis by activation of Akt/HO-1 pathway

Oxidative stress induced by hydrogen peroxide (H₂O₂) triggers human lens epithelial cell (HLEC) apoptosis and initiates cataract formation. Oxyresveratrol (Oxy) was reported to possess antioxidant and free radical scavenging activities. Herein, we investigated the effects of Oxy on H₂O₂-induced oxidative stress and apoptosis in HLECs and the associated mechanisms. Cell viability was detected by MTT assay. The oxidative damage was assessed by measuring the activities of superoxide dismutases-1 (SOD-1), catalase (CAT), glutathione reductase (GSH), and malondialdehyde (MDA). Apoptosis was analyzed by flow cytometry analysis. The changed expressions of heme oxygenase-1 (HO-1) and protein kinase B (Akt) pathways were evaluated by qRT-PCR and western blot. We found that exposure to H₂O₂ dose-dependently reduced cell viability, and induced oxidative stress and apoptosis in HLECs, which were reversed by pretreatment with Oxy. Oxy increased p-Akt and HO-1 expressions in H₂O₂-stimulated HLECs. Akt and HO-1 expressions form a regulatory axis and Oxy activated the Akt/HO-1 pathway in H₂O₂-stimulated HLECs. Inhibition of the Akt/HO-1 pathway by LY294002 or ZnPP attenuated the effects of Oxy on oxidative stress and apoptosis in H₂O₂-stimulated HLECs. In conclusion, Oxy protected H₂O₂-induced oxidative stress and apoptosis through activating the Akt/HO-1 pathway, suggesting the protective effect of Oxy against H₂O₂-induced cataract.     

HPLC法测定生血颗粒中补骨脂素、异补骨脂素的含量

目的:建立HPLC法测定生血颗粒中补骨脂素和异补骨素的含量。方法:色谱柱为SB-C_(18)柱(250mm×4.6mm,10μm);柱温:40℃;流动相为甲醇-水(50:50);流速为1.0ml·min~(-1);检测波长为246nm。结果:补骨脂素在1.135～45.400μg·ml~(-1)范围内、异补骨脂素在1.035～41.400μg·ml~(-1)范围内呈良好的线性关系(相关系数分别为r=0.999 9和r=1.000 0),平均回收率分别为97.05%和96.36%(RSD分别为0.78%和0.86%,n=6)。结论:所建立的方法稳定性可靠、灵敏,结果准确,可用于生血颗粒的质量控制。     

Allethrin toxicity on human corneal epithelial cells involves mitochondrial pathway mediated apoptosis

Pyrethroids including allethrin are the most common commercial household insecticides. The detrimental effects caused by pyrethroids on humans are gaining considerable attention. The present study was aimed to elucidate the effects of allethrin on the human corneal epithelial (HCE) cells. Allethrin inhibited the proliferation of HCE cells in a dose-dependent manner. In the presence of allethrin, cells showed membrane blebbing and nuclear fragmentation along with significant decrease in mitochondrial membrane potential resulting in increased cytochrome c (Cyt c) release into the cytosol. Further, flow cytometry analysis demonstrated a marked increase in sub G₀–G₁ cells, characteristic of apoptosis. Increased expression of pro-apoptotic protein, Bax, a simultaneous decrease of anti-apoptotic protein, Bcl-2, and activation of Caspase 3 was evident in the treated cells. In addition, extracellular matrix digesting metalloproteinase 9 (MMP-9) was also stimulated. Furthermore, significant increase in the gene expression of inflammatory cytokines, TNF-α and IL-1β was observed. Taken together, these findings suggest that allethrin (IC₅₀ ≈ 85 μM) is toxic to HCE cells causing death through mitochondrial pathway.     

HPLC法测定腰痛丸中补骨脂素和异补骨脂素的含量

目的：建立腰痛丸中补骨脂素和异补骨脂素含量测定HPLC方法。色谱柱：ZORBAX SB—C18（250mm×4．6mm,5μm）,流动相：甲醇-水（55：45）,流速1．0ml·min^-1,检测波长：246nm。结果：补骨脂素和畀补骨脂素分别在0．05～0．29μg（r=0．9999）和0．05～0．27μg（r=0．9998）范围内线性关系良好,回收率分别为96．0％（RSD=1．5％）和98．6％（RSD=1．6％,n=6）。结论：方法简便、快速,可用于腰痛丸的含量测定。     

HPLC测定旱莲胶囊中补骨脂素和异补骨脂素的含量

目的建立测定旱莲胶囊中补骨脂素和异补骨脂素含量的高效液相色谱方法.方法色谱柱为Termo C18柱(250mm×4.6mm,5μm),流动相为甲醇-水(50∶50),流速为0.6 ml·min-1,检测波长为295nm.结果补骨脂素的线性范围为0.2～0.6μg,r=0.9998(n=5),平均回收率为99.2%,RSD为1.01%;异补骨脂素的线性范围为0.2～0.6μg,r=0.9999(n=5),平均回收率为100.3%,RSD为2.22%.结论该方法操作简便,准确,精密度高,可用于控制旱莲胶囊的质量.     

RP-HPLC法测定鱼鳔补肾丸中补骨脂素和异补骨素的含量

目的：建立RP—HPLC法测定鱼鳔补肾丸中补骨脂素和异补骨脂素的含量。方法：采用ZORBOAX Eclipse XDB C18柱,以乙腈-0．74％磷酸溶液（三乙胺调节pH至3．5）（33：67）为流动相,检测波长为245nm。结果：补骨脂素和异补骨脂素分别在0．1038～1．0380μg（r=0．99995）及0．0819—0．8190μg（r=0．99993）范围内线性关系良好,平均加样回收率（n=6）分别为94．1％及95．9％,RSD分别为2．13％及1．67％。结论：本方法操作简便,重现性好,可作为该产品质量控制的方法。