目前，我国冠心病、急性心肌梗塞等缺血性心脏病的发病率与死亡率明显上升，严重危害人们健康。溶栓、介入、冠脉搭桥等再灌注疗法是治疗冠心病、急性心肌梗塞等缺血性心脏病最主要的措施，但这些治疗措施在带来有益治疗效果的同时，却会引起潜在的缺血/再灌注(ischemia/reperfusion，I/R)损伤。因此，如何减轻甚至解除心肌I/R损伤受到广大基础和临床研究者的高度关注。针对心肌I/R损伤的保护，人们进行了积极的研究与探讨，从对心肌I/R损伤机制的研究，到心肌缺血预适应保护作用的探索，发展到药理性预适应心肌保护，已取得了许多令人鼓舞的成果，但距临床应用仍有一定的距离。我国中药资源极为丰富，如何从中发掘出更加安全有效的防治心肌I/R损伤的药物，特别是对其活性成分进行药效学及作用机制的研究是当前我国医药界所面临的一大课题。 油茶皂苷(Sasanquasaponin，C₅₈H₉₂O₂₆，SQS)是从油茶好中提取出来的一种有效单体成分。近年来，针对SQS的心肌保护作用我们进行了大量的研究工作，研究证实，SQS预处理具有良好的抗心肌I/R损伤作用，且这种作用与其可有效地抑制I/R损伤时心肌细胞内氯离子浓度升高有关，但SQS通过什么方式来抑制心肌细胞内氯离子浓度升高，目前还不明确。 氯离子(Cl⁻)是机体细胞内最富有生理意义的阴离子，它不仅在调节细胞酸碱平衡，维持静息膜电位和调节动作电位的形成等方面发挥着重要作用，而且在细胞免疫应答、迁移、增殖、分化、凋亡及Ca²⁺稳态中也发挥了一定作用。此外，氯离子在急性心肌I/R损伤中所发挥的作用也逐渐引起人们的注意。Cl⁻在细胞内外的转运，除了经过Cl⁻通道外，还可通过阴离子交换蛋白(anion exchangers， AEs)等进行转运。AEs广泛分布于脊椎动物细胞，主要存在于胞浆膜，其基因家族主要由AE₁、AE₂、AE₃三种基因组成，其中在心脏以AE₃为主，具有Cl⁻/HCO₃⁻可逆性交换及HCO₃⁻依赖性的超氧阴离子(O₂⁻)胞外转运等功能，因而在调节细胞内Cl⁻浓度、活性氧 (reactive oxygen species，ROS)、以及维持细胞内稳定的pH值等方面起重要作用。最近我们研究表明AE₃与心肌缺血预适应保护作用密切相关，且机制与调节和稳定I/R损伤心肌细胞内Cl⁻浓度有关。 综上所述，考虑到AE₃在调节心肌细胞内cr浓度以及ROS系统活性等方面发挥着茁要作用，而SQS可有效地抗脂质过氧化及抑制急性I/R损伤时心肌细胞内Cl⁻浓度升高。因此，我们假设：SQS对I/R所致急性心肌细胞损伤的保护作用可能由AE₃所介导，其机制可能通过AE₃蛋白抑制I/R损伤时细胞内氯离子浓度升高，调节细胞内ROS系统活性而得以实现。 为此，本研究拟从细胞水平，通过心肌细胞缺氧/复氧(anoxia/reoxygenation， A/R)损伤模型模拟在体I/R损伤，应用细胞生物学和基因转染及RNA干扰等分子生物学手段以期阐明SQS预处理所产生的抗心肌细胞A/R损伤作用与AE₃蛋白表达的关系，明确AE₃蛋白介导SQS抗心肌细胞A/R损伤的分子机制，并进一步明确SQS预处理上调AE₃表达的信号通路，从而为进一步开发SQS提供理论和实验依据。 第一部分 油茶皂苷预处理对心肌细胞缺氧/复氧损伤及AE₃蛋白表达的影响 目的： 在观察SQS对原代心肌细胞A/R损伤保护作用的基础上，进一步观察SQS对A/R损伤所诱发的氧化应激、Ca²⁺超载、细胞凋亡等影响；同时观察SQS对A/R损伤心肌细胞内氯离子浓度以及AE₃基因表达的影响，初步探讨AE₃蛋白与SQS抗心肌细胞A/R损伤的相关性。 方法： 1.利用原代分离培养的新生大鼠心肌细胞，随机分为5组：①正常对照(Control)组，②缺氧/复氧组(A/R)，③0.1 μM油茶皂苷预处理组(SQS-0.1)， ④1μM油茶皂苷预处理组(SQS-1)，⑤10 μM油茶皂苷预处理组(SQS-10)。除对照组外，均建立缺氧/复氧损伤模型。 2.MTT法检测各实验组心肌细胞的存活率；按试剂盒说明测定各组心肌细胞内LDH的活性、MDA的含最以及抗氧化物酶SOD、CAT、GSH-Px的活性；流式细胞术检测各实验组心肌细胞内ROS、游离钙以及氯离子浓度的变化； Annexin V-FITC/PI双染法检测各组心肌细胞凋亡率的改变。 3.RT-PCR半定量检测各实验组心肌细胞内AE₃ mRNA的表达变化；Western Blot检测各组心肌细胞中AE₃蛋白的表达。 4.统计学方法：应用SPSS11.0软件对数据进行方差分析和t检验以P<0.05作为判定统计学意义的标准。 结果： 1.心肌细胞A/R处理后，细胞存活率降低、LDH活性增加，与正常组比较有显著差异(P<0.01)；而SQS预处理后可显著提高A/R损伤心肌细胞的存活率、降低LDH的活性，并呈现一定的剂量依赖性。 2.A/R组心肌细胞内ROS及MDA含量增加，而抗氧化物酶SOD、CAT、 GSH-Px的活性降低，与正常组比较均有显著差异(P<0.01)。SQS预处理后可剂量依赖性逆转上述变化。 3.缺氧/复氧损伤导致心肌细胞内游离钙、氯离子浓度以及细胞凋亡率增加，给予SQS可使缺氧/复氧心肌细胞内的游离钙、氯离子浓度以及细胞凋亡率下降。 4.AE₃ mRNA及蛋白在正常心肌细胞内存在基础表达，缺氧/复氧后表达增加，而SQS预处理后可进一步促进缺氧/复氧心肌细胞内AE₃基因的表达上调。 结论： 1.在原代培养的心肌细胞水平上，再次证实SQS预处理对心肌细胞缺氧/复氧损伤具有保护作用，并呈现一定的剂量依赖性；同时SQS剂量依赖性地抑制缺氧/复氧所诱发的心肌细胞氧化应激、钙超载及细胞凋亡。 2.SQS预处理可上调缺氧/复氧心肌细胞AE₃基因表达，同时抑制缺氧/复氧心肌细胞内氯过载。 3.AE₃可能参与SQS抗心肌细胞缺氧/复氧损伤。 关键词：油茶皂苷；AE₃蛋白；缺氧/复氧损伤；氯离子；心肌细胞 第二部分 RNA干扰技术沉默AE₃基因对油茶皂苷抗心肌细胞缺氧/复氧损伤的影响 目的： 构建靶向AE₃的RNAi真核表达载体，建立AE₃低表达的细胞系，观察AE₃低表达对SQS抗心肌细胞缺氧/复氧损伤的影响，反向验证AE₃基因在这一过程中的重要作用及机制。 方法： 1.设计靶向AE₃基因的3条候选RNA干扰序列，利用真核表达载体pSilencer™ 3.1-H1 hygro构建重组质粒转染H9c2心肌样细胞；同时设计并构建靶向GAPDH基因的阳性对照及阴性对照RNA干扰重组载体，用于鉴定pSilencer™ 3.1-H1 hygro质粒介导的RNA干扰体系的有效性。 2.瞬时转染后，RT-PCR和Western Blot检测AE₃基因mRNA和蛋白表达，筛选最有效的RNA干扰靶片段；转染携带绿色荧光蛋白的pEGFP-C1质粒，荧光显微镜监测瞬时转染不同时相的转染效率；将筛选出的抑制AE₃表达最有效的重组载体用于稳定筛选低表达AE₃基因的H9c2细胞，同时筛选稳定转染pSilencer-NC阴性对照载体的细胞作为对照；最后通过RT-PCR及Western Blot测定稳定筛选出的细胞中AE₃基因mRNA和蛋白的表达。 3.SQS分别预处理未转染的H9c2细胞、稳定转染的阴性对照细胞H9c2/pSilencer-NC和AE₃低表达的细胞系H9c2/pSilencer-siAE₃，缺氧/复氧后，通过MTT法检测细胞存活率；化学比色法测定细胞内MDA含量以及抗氧化物酶SOD、CAT、GSH-Px的活性；流式细胞术检测细胞内ROS、游离钙以及氯离子浓度；Annexin V-FITC/PI双染法检测细胞凋亡率；分析AE₃基因沉默前后SQS对上述指标影响的变化。 结果： 1.DNA测序鉴定证实，各重组RNA干扰真核表达载体包括阳性对照pSilencer-GAPDH、阴性对照pSilencer-NC及靶向AE₃基因的3个重组载体pSilencer-AE₃-A、 B、C 均构建成功。 2.转染pEGFP-Cl质粒经荧光显微镜检测发现，H9c2心肌样细胞在转染后48 h达到瞬时转染效率的高峰，此时相用于后续瞬时转染靶基因表达抑制率的分析。RT-PCR 及 Western Blot 检测发现转染pSilencer-GAPDH 的 H9c2 细胞中GAPDH mRNA及蛋白表达抑制率均在75%以上，而转染pSilencer-NC对GAPDH表达无明显影响。此外，RT-PCR和Western Blot检测结果显示pSilencer-AE₃-A重组载体瞬时转染抑制靶基因表达的效果最强，H9c2细胞中AE₃ mRNA和蛋白表达的抑制率分别为83.5%和77.2%。pSilencer-AE₃-A转染稳定筛选建立的AE₃低表达的细胞系H9c2/pSilencer-siAE₃中靶基因AE₃表达能够持续抑制。 3.RNAi封闭AE₃基因表达后，SQS预处理降低缺氧/复氧损伤心肌细胞内[Cl⁻]〓的效应被逆转；同时SQS增加缺氧/复氧损伤心肌细胞的存活率、降低LDH活性的效应也被取消；此外，RNAi封闭AE₃基因表达后，SQS降低缺氧/复氧心肌细胞内MDA含量、增加抗氧化物酶SOD、CAT、GSH-Px活性、降低ROS水平以及抑制钙超载、细胞凋亡等作用均被完成或部分取消。 结论： 1.pSilencer™ 3.1-H hygro质粒介导的RNA干扰体系能够有效抑制H9c2心肌样细胞中靶基因的表达。 2.针对AE₃mRNA序列521～541 bp位置设计并构建的RNA干扰重组载体pSilencer-AE₃-A能够最有效抑制靶基因的表达，抑制率达75%以上。 3.成功构建的低表达AE₃基因的H9c2/pSilencer-siAE₃细胞，可用于AE₃及相关基因功能分析的进一步研究。 4.利用AE₃低表达的细胞系H9c2/pSilencer-siAE₃，反向证实AE₃介导了SQS对心肌细胞缺氧/复氧损伤的保护作用，其机制可能通过抑制缺氧/复氧心肌细胞内氯过载及减少细胞内ROS含量，进而抑制缺氧/复氧所致的氧化应激、钙超载及细胞凋亡，从而对缺氧/复氧损伤心肌细胞产生保护作用。 关键词：RNA干扰；油茶皂苷；AE₃蛋白；缺氧/复氧损伤；氯离子；H9c2细胞 第三部分 油茶皂苷预处理上调缺氧/复氧损伤心肌细胞AE₃表达的信号机制 目的： 利用NO合成酶和ERK1/2抑制剂，从NO和ERK1/2途径初步探讨SQS上调缺氧/复氧损伤心肌细胞AE₃基因表达的信号机制。 方法： 1.利用原代分离培养的新生大鼠心肌细胞，随机分为5组：①正常对照(Control)组，②缺氧/复氧组(A/R)，③10μM油茶皂苷预处理组(SQS-10)， ④NO抑制剂L-NAME处理组(L-NAME)，⑤ERK1/2抑制剂PD98059处理组(PD98059)。除对照组外，均建立缺氧/复氧损伤模型。 2.MTT比色法检测各实验组心肌细胞的存活率；利用全自动生化分析仪测定各组心肌细胞培养液中LDH的活性；硝酸还原酶法测定各组心肌细胞培养液中NO的浓度；Western Blot检测ERK1/2蛋白和磷酸化ERK1/2蛋白的表达水平。 3.RT-PCR半定量检测各实验组心肌细胞内AE₃ mRNA的表达变化；Western Blot检测各组心肌细胞中AE₃蛋白的表达。 4.统计学方法：应用SPSS11.0软件对数据进行方差分析和t检验以P<0.05作为判定统计学意义的标准。 结果： 1.SQS预处理能显著提高缺氧/复氧心肌细胞的存活率，并降低其LDH活性，预先给予NO合酶抑制剂L-NAME或ERK1/2抑制剂PD98059可取消SQS这种作用。 2.SQS预处理能显著增加心肌细胞NO的释放，预先给予L-NAME可取消这种作用，而PD98059不影响NO的水平。 3.SQS预处理能促进缺氧/复氧心肌细胞内ERK1/2蛋白磷酸化，预先给予L-NAME或PD98059均可取消SQS这种作用。 4.SQS预处理能显著上调缺氧/复氧心肌细胞内AE₃ mRNA和蛋白的表达，预先给予L-NAME或PD98059均可取消SQS这种作用。 结论： SQS预处理上调缺氧/复氧心肌细胞内AE₃的表达涉及NO和ERK1/2信号途径。 关键词：油茶皂苷；AE₃蛋白；缺氧/复氧损伤；ERK1/2信号通路；心肌细胞 论文意义： 1.本研究在心肌细胞水平上，进一步验证了SQS可剂量依赖性地对抗心肌细胞缺氧/复氧损伤；同时研究发现SQS可抑制心肌细胞缺氧/复氧损伤所诱发的氧化应激、钙超载和细胞凋亡。 2.本研究率先提出了SQS抗心肌细胞缺氧/复氧损伤的新机制。即SQS可能通过ERK1/2信号途径上调AE₃基因表达从而降低缺氧/复氧损伤心肌细胞内[Cl⁻]〓及ROS水平，进而抑制缺氧/复氧所致的氧化应激、钙超载及细胞凋亡，最终对缺氧/复氧损伤心肌细胞产生保护作用。 3.本研究所建立的AE₃低表达的H9c2细胞系，将为进一步研究AE₃蛋白功能、筛选SQS类似药物、研究SQS类似药物作用机制奠定了实验基础。 4.本研究不仅有助于国内外医药界对SQS心肌保护作用和机制的认识，而且尚可提示其它类似结构的中药有效成份也可能存在心肌保护作用，为寻找心肌保护药物探索了 一条新的思路。 ①*本文受到国家自然科学基金(№：30660209)资助

 At present, in our country, the incidence and lethality rate of coronary heart disease and acute myocardial infarction turn on the increasing trends. Reperfusion therapies such as thrombolytic therapy, interventional therapy, and coronary artery bypass grafting are the most important measures to these ischemic heart diseases which still seriously influence human health. However, these effective measures may cause potential ischemia-reperfusion (I/R) injury. Therefore, how to prevent myocardial I/R injury (MIRI) has attracted considerable attention of basic and clinical workers, and the study in development of new anti-MIRI drugs is still an important but imminent work. The natural resources of Chinese medicinal materials are very rich in China. It is of great significance to search for the more safe and effective herbs for the prevention and treatment of MIRI, particularly for the active components in these herbs and the study of their pharmacodynamics and mechanisms on anti-MIRI, which can help to develop our country's herbal resources. Sasanquasaponin (SQS) is a biologically active ingredient extracted from the Chinese medicinal plant Camellia oleifera Abel and has gained considerable attention due to its wide range of biological and pharmacological properties. Our recent studies have shown that SQS could effectively protect cardiomyocytes against ischemia-reperfusion (I/R) injury by suppressing I/R-induced elevation of intracellular Cl⁻ concentrations ([Cl⁻]〓). However, the precise mechanisms of inhibition by SQS on [Cl⁻]〓 during ischemia and reperfusion remain unclear. Cl⁻ is the primary intracellular anion and participates in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, differentiation, apoptosis, and Ca²⁺ homeostasis. Moreover, cumulated evidence shows that an increase of intracellular Cl⁻ plays an important role in myocardial I/R injury. The regulation of intracellular Cl⁻ has been attributed to several electroneutral carriers or cotransporters. One of them, anion exchangers also is a primary determinant. Anion exchangers are widely distributed in invertebrates, and exist mainly in the cytoplasmic membrane. The AEs family comprises at least 3 members: AE₁, AE₂, and AE₃. AE₃ is mainly expressed in excitable tissues such as brain, heart and retina AEs facilitate the reversible electroneutral exchange of Cl⁻ for HCO₃⁻ across the plasma membrane and thus contribute to regulation of intracellular pH (pH〓), intracellular chloride concentration, reactive oxygen species (ROS) and cell volume. Recently, our research suggested that AE₃ is involved in the cardioprotection of ischemic preconditioning by maintaining intracellular chloride homeostasis. Considering that AE₃ plays important roles in regulating intracellular Cl⁻ and ROS levels, while SQS could effectively inhibit I/R-induced elevation of [Cl⁻]〓 and lipid peroxidation. Therefore, we hypothesized that AE3 could mediate the cardioprotection of SQS against I/R injury through inhibiting I/R-induced elevation of [Cl⁻]〓 and regulating the level of intracellular ROS. Therefore, this study was designed to elucidate the relationships between the cardioprotection of SQS and AE₃ expression and investigate whether AE₃ was involved in the cardioprotection afforded by SQS preconditioning and its potential mechanisms in cultured neonatal rat cardiomyocytes under anoxia/reoxygenation conditions as an in vitro ischemia/reperfusion model through technique of molecular biology and RNA interference. Furthermore, we investigated whether ERK1/2 signaling pathway implicated in the up-regulation of AE₃ protein induced by SQS. The destination is to provide new experimental and theoretical basis for further developing SQS. Part Ⅰ Effects of SQS pretreatment on anoxia/reoxygenation-induced cardiomyocyte injury and AE₃ protein expression Objectives: To investigate effects of SQS on anoxia/reoxygenation-induced oxidative stress, Ca²⁺ overload, and cell apoptosis in a neonatal rat cardiomyocyte model, and to observe effects of SQS on [Cl⁻]〓 and AE₃ expression in cardiomyocytes subjected to anoxia/reoxygenation. To further elucidate the relationships between the cardioprotection of SQS and AE₃ expression. Methods: 1.Primary cardiomyocytes were cultured in vitro. Cardiomyocytes divided randomly into following five groups: control group (Control), A/R injury model group (A/R), 0.1 μM SQS pretreatment group (SQS-0.1), 1 μM SQS pretreatment group (SQS-1), 10 M SQS pretreatment group (SQS-10). A/R injury model was established in each group except control group. 2.Cell viability was detected by Methyl thiazolyl tetrazolium (MTT) method. The content of LDH in cultured conditions, SOD, CAT, GSH-Px and MDA in cardiomyocytes were measured by a colorimetric method. The percentage of apoptosis was measured by annexin V-FITC/PI double staining with flow cytometry. Meanwhile, intracellular ROS, Ca²⁺, and CP levels were also measured by flow cytometry. 3.RT-PCR analysis was used to detect the AE₃ mRNA expression; the expression of AE₃ protein was analyzed by Western Blot. 4.Methods of Statistics: The data were statistically processed analysis of variance (ANOVA) and t test by the software SPSS 11.0, and with the value of P smaller than 0.05 being considered statistically significant. Results: 1.Cardiomyocytes subjected to anoxia/reoxygenation had significant increases of LDH activity and reduction of cell viability as compared with untreated cells (P＜0.01). However, pretreating the cardiomyocytes with SQS significantly suppressed the increase of LDH activity and the decrease of viability that resulted from anoxia/reoxygenation in a dose-dependent manner up to 10 μM. 2.Cardiomyocytes subjected to anoxia/reoxygenation had a rapid and significant increase in intracellular ROS level and concomitantly increase of MDA content and reduction of antioxidant enzymes activity, including SOD, CAT, and GSH-Px. However, pretreating the cardiomyocytes with SQS reversed anoxia/reoxygenationinduced changes in ROS, MDA, SOD, CAT, and GSH-Px in a dose-dependent manner. 3.Cardiomyocytes subjected to anoxia/reoxygenation had significant increase in [Ca²⁺]〓, [Cl⁻]〓, and cell apoposis, compared with that of control group (all P＜0.01). However, treatment with SQS, at 0.1, 1, or 10 μM, inhibited the anoxia/reoxygenation -induced increase in [Ca²⁺]〓, [Cl⁻]〓, and cell apoposis in a dose-dependent manner. 4.The basic expression of AE₃ mRNA and protein was present in untreated cardiomyocytes. However, cardiomyocytes subjected to A/R showed significant increase in AE₃ mRNA and protein expression compared with control group (both P＜0.05). SQS induced further increase in AE₃ mRNA and protein expression compared with A/R group (both P＜0.01). Conclusions: 1.Our data further demonstrate that SQS can exert direct cardioprotection from anoxia/reoxygenation injury in a neonatal rat cardiomyocyte model. Meanwhile, SQS concentration dependently inhibited A/R-induced oxidative stress, Ca²⁺ overload, and cell apoptosis. 2.SQS pretreatment can up-regulate AE₃ expression and inhibit A/R-induced elevation of intracellular Cl⁻ concentrations. 3.AE₃ may be related to the cardioprotection afforded by SQS preconditioning. Key words: Sasanquasaponin; Anion exchanger 3; Anoxia-reoxygenation injury; Chloride ion; Cardiomyocyte. Part Ⅱ Effects of AE₃ gene silence by RNA interference on cardioprotection of SQS against anoxia/reoxygenation injury Objectives: To reversely validate the key status and mechanisms of AE₃ in cardioprotection of SQS, via constructing AE₃ gene-specific small interfering RNA (siRNA) expression vectors, screening AE₃ expression down-regulated cell line, and investigating effects of AE₃ gene silence on cardioprotection of SQS against anoxia/reoxygenation injury. Methods: 1.Three sequences targeting the ORF of AE₃ mRNA were cloned into the RNA interference expression vector pSilencer™ 3.1-H1 hygro, which was used to reconstruct the pSilencer-AE₃ plasmids, the positive control pSilencer-GAPDH and the negative control pSilencer-NC. The construction of the recombinant expression vectors was identified by DNA sequencing. 2.H9c2 cells were transfected with pEGFP-Cl plasmids to observe the lipofectamine transfection efficiency by fluorescence microscope. The expression of GAPDH in H9c2 cells transiently transfected with pSilencer-GAPDH or pSilencer-NC was examined using RT-PCR and Western Blot analyses to examine the efficiency and specificity of pSilencer™ 3.1 system. The mRNA and protein levels of AE₃ in cells transiently transfected with three AE₃-targeting recombinant plasmids were compared using RT-PCR and Western Blot analyses. The AE₃-targeting recombinant plasmid testified being most effective was used to construct cells stably silenced the expression of AE₃ via being selected with hygromycin. Resistant clones were cultured, and the expression of AE₃ was assayed using RT-PCR and Western Blot analyses after 1 month. Others stably transfected with negative control plasmid pSilencer-NC were used as controls. 3.SQS pretreated untransfected cells H9c2, stable screening control cells H9c2/pSilencer-NC, and stable screening cells H9c2/pSilencer-siAE₃, subjected to A/R, respectively. Cell viability was detected by MTT method. The content of LDH in cultured conditions, SOD, CAT, GSH-Px and MDA in cardiomyocytes were measured by a colorimetric method. The percentage of apoptosis was measured by annexin V-FITC/PI double staining with flow cytometry. Meanwhile, intracellular ROS, Ca²⁺, and Cl⁻ levels were also measured by flow cytometry. Results: 1.The constructions of the recombinant expression vectors pSilencer-AE₃-A, B, C, its positive control pSilencer-GAPDH and the negative control pSilencer-NC were successfully confirmed by the results of DNA sequencing. 2.The highest efficiency was in 48 h after transfection in H9c2. pSilencer-GAPDH was able to significantly knockdown GAPDH expression by 75% (P＜0.05), whereas pSilencer-NC was inactive in this assay to show no-selection knockdown was not observed. The inhibition rate of AE₃ gene expression was above 75% 48 h after transient transfection with pSilence-AE₃-A, and that was no significant difference after transfection with pSilencer-NC (P＞0.05). There was specific inhibition of AE₃ in H9c2/pSilencer-siAE₃ (P＜0.01). 3.For untransfected H9c2 cells or stable screening control H9c2/pSilencer-NC cells subjected to anoxia/reoxygenation, SQS pretreatment significantly increased the survival rate of cells, reduced the release of LDH and the generation of MDA, increased the activities of SOD, CAT, and GSH-Px, reduced apoptosis percentage, and reduced the levels of intracellular Ca²⁺, Cl⁻, and ROS (all P＜0.05 vs A/R). However, for H9c2/pSilencer-siAE₃ cells subjected A/R, these protective effects of SQS pretreatment were abolished because AE₃ gene was silenced by RNAi. Conclusions: 1.RNA interference expression vector pSilencer™ 3.1-H1 hygro can downregulate gene expression in H9c2 cells. 2.The recombinant expression vectors pSilence-AE₃-A, which targeted the sequence located in 521-541 bp of AE₃ mRNA, can reduce AE₃ expression by above 75%. 3.H9c2/pSilencer-siAE₃ cell line is quite stable. There is specific inhibition of AE₃ in them. They can help us to have a further understanding of AE₃ gene function. 4.AE₃ is involved in the cardioprotection of SQS against anoxia/reoxygenation injury by inhibiting A/R-induced elevation of [Cl⁻]〓 and ROS, and subsequently inhibiting A/R-induced oxidative stress, Ca²⁺ overload, and cell apoptosis. Key words: RNA interferce; Sasanquasaponin; Anion exchanger 3; Anoxia-reoxygenation injury; Chloride ion; H9c2 cells. Part Ⅲ Signal mechanisms of AE₃ expression up-regulation induced by SQS pretreatment in cardiomyocyte subjected to anoxia/reoxygenation Objective: To explore whether ERK1/2 signal pathway is involved in the up-regulation of AE₃ expression induced by SQS via addition of L-NAME, an NOS inhibitor and PD98059, an inhibitor of ERK1/2. Methods: 1.Primary cardiomyocytes were cultured in vitro. Cardiomyocytes divided randomly into following five groups: control group (Control), A/R injury model group (A/R), 10 μM SQS pretreatment group (SQS-10), L-NAME treatment group (L-NAME), PD98059 treatment group (PD98059). A/R injury model was established in each group except control group. 2.Cell viability was detected by MTT method. LDH activity in the culture medium of cardiomyocytes was analyzed with an automatic biochemical analyzer. The level of NO was measured with the Griess reagent. Meanwhile, the expression of ERK1/2 and phosphorylated ERK1/2 protein was detected by Western Blot. 3.RT-PCR analysis was used to detect the AE₃ mRNA expression; the expression of AE₃ protein was analyzed by Western Blot. 4.Methods of Statistics: The data were statistically processed analysis of variance (ANOVA) and t test by the software SPSS 11.0, and with the value of P smaller than 0.05 being considered statistically significant. Results: 1.SQS preconditioning significantly increased viability of cells and reduced the release of LDH in cardiomyocytes underdoing A/R. The effects of SQS were completely abolished by addition of L-NAME, an NOS inhibitor, and PD98059, an inhibitor of ERK1/2. 2.Pretreating the cardiomyocytes with SQS significantly increased the release of NO, which was significantly attenuated by L-NAME. 3.SQS significantly promoted ERK 1/2 protein phosphorylation in cardiomyo-cytes with anoxia-reoxygeonation. L-NAME and PD98059 abolished the phosphory-lation of ERK 1/2 induced by SQS. 4.SQS significantly up-regulated the expression of AE₃ mRNA and protein in cardiomyocytes subjected to A/R. The effects of SQS were abolished by addition of L-NAME, an NOS inhibitor, and PD98059, an inhibitor of ERK 1/2. Conclusions: The up-regulation of AE₃ expression induced by SQS is related to NO and ERK 1/2 signal passway. Key words: Sasanquasaponin; Anion exchanger 3; Anoxia-reoxygenation injury; ERK1/2 signal passway; Cardiomyocytes. Significance: 1.This study further validated that SQS can protect cardiomyocytes against A/R injury at the cell level. Moreover, this study demonstrated SQS can inhibit A/R-induced elevation of intracellular Cl⁻ concentrations, simultaneously inhibit A/R-induced oxidative stress, Ca²⁺ overload, and cell apoptosis. 2.This study put forward a new mechanism of cardioprotection induced by SQS for the first time, which SQS mediates its cardioprotective effects in part, at least, by up-regulating AE₃ expression via ERK1/2 signaling pathway, subsequently inhibiting A/R-induced oxidative stress, Ca²⁺ overload, and cell apoptosis by inhibiting A/R-induced elevation of [Cl⁻]〓 and ROS. 3.H9c2/pSilencer-siAE₃ stable cell line can help us to have a further understanding of AE₃ gene function. Moreover, this system should be useful for screening SQS-like drugs and studying its mechanisms. 4.This study not only had contribution to understand the cardioprotection of SQS and its mechanisms in medical field, but also suggested that other Chinese medicine ingredients with similar molecular structure may have cardioprotective effects, and consequently offered a new way for searching for the safe and effective medicines for the prevention and treatment of myocardial I/R injury.