银杏叶制剂对哮喘T淋巴细胞及其与PKC关系影响的研究
|
摘要
前言
在众多的发病机制中，有T淋巴细胞参与的慢性气道炎症是公认的哮喘重要的发病机制之一。T淋巴细胞(主要是Th2细胞)的活化、增殖和分泌大量的细胞因子(如：IL-4、IL-5等)在哮喘的发生、发展中起着重要作用。而在T淋巴细胞活化、增殖和分泌细胞因子促进炎性介质释放的过程中，细胞信号转导通路的作用十分重要，其中尤以蛋白激酶C(Protein Kinase C，PKC)信号转导通路的作用最引人注目。PKC是一个具有广泛生物学活性的酶，先前的研究表明PKC家族的重要成员PKCα活性的变化在T淋巴细胞活化、增殖和分泌细胞因子的过程中起着重要作用。糖皮质激素是目前治疗哮喘气道炎症的最强大最有效的药物，但临床应用显示其也有缺点和局限性，寻找新的有效治疗气道炎症的药物对提高哮喘的总体治疗效果具有一定的临床意义。银杏叶提取物的化学成分复杂，其主要活性成分银杏黄酮和萜内酯现已广泛应用于心脑血管系统疾病的治疗；在呼吸系统方面的治疗作用，特别是在哮喘的治疗方面早有记载。迄今为止，较多的临床研究证实银杏制剂对哮喘有确切的疗效，其对哮喘患者T淋巴细胞功能及其与PKCα关系的影响如何目前尚无报道。因此，将银杏叶制剂对哮喘T淋巴细胞功能及其与PKCα关系的影响进行系统地的研究，对银杏叶制剂治疗哮喘的临床效果和部分作用机理进行深入的探讨，明确其对哮喘的疗效及部分可能的作用机理，对于改善哮喘疗效和预防复发有一定的临床意义。
第一部分银杏叶制剂对哮喘外周血T淋巴细胞PKC表达及Th1／Th2类细胞因子分泌影响的研究
分题一银杏叶制剂对哮喘大鼠T淋巴细胞体外增殖和凋亡的影响
目的检测不同银杏叶制剂在体外对T淋巴细胞增殖和凋亡的影响，探讨银杏叶制剂治疗哮喘的部分机理。
方法将14只大鼠分成2组(健康对照组7只；哮喘组7只)，分别采集外周血，并分离出T淋巴细胞，体外分组培养(分空白组、银杏叶制剂BN-52021组、银杏叶制剂EGb761组)，各组分别以不同浓度的BN-52021和EGb761干预不同时间。之后，用MTT法分别测定各组细胞的增殖情况；用Annexin V／PI双染法流式细胞仪测定BN-52021不同浓度组的细胞凋亡情况。
结果与空白组比较，EGb761在低浓度时促进T淋巴细胞增殖，而高浓度时则起抑制作用(均P＜0.05)；BN-52021对体外培养的哮喘和健康大鼠T淋巴细胞的增殖均有抑制作用，其作用强度(在一定的范围内)随剂量的增加和时间的延长而加强(组间比较均P＜0.05)；但对哮喘大鼠的抑制作用要明显强于健康大鼠(P＜0.05)；T淋巴细胞的凋亡率随BN-52021浓度的增加而增加(P＜0.01)。
结论银杏叶制剂因其成分和干预浓度的不同而对体外培养的大鼠T淋巴细胞增殖活性的影响不同，对哮喘与健康大鼠T淋巴细胞的作用也有差异，银杏内酯B可能是抑制T淋巴细胞增殖的主要活性成分，其不仅可抑制T淋巴细胞的体外增殖，而且还可增加T淋巴细胞的凋亡率。
分题二哮喘大鼠外周血T淋巴细胞Th1／Th2细胞因子和PKCα的表达及银杏叶制剂对它们的影响
目的探讨银杏叶制剂对在哮喘大鼠免疫学发病机制中起关键作用的T淋巴细胞部分生物学功能的影响情况。
方法将14只SD大鼠随机分成哮喘和正常两组，每组7只。从每只大鼠外周血分离出T淋巴细胞，分组培养72小时后，分别用逆转录—聚合酶链式反应(Reverse Transcriptase-Polymerase Chain Reaction，RT-PCR)检测各组IL-2、IL-4、IL-5 mRNA的表达量和Western Blot检测各组细胞膜与细胞浆PKCα的表达比率。
结果IL-2、4、5 mRNA表达量在哮喘组均明显高于正常组，各因子两组间比较差异有显著性(P＜0.01)；哮喘组T淋巴细胞给予BN-52021干预后IL-2、4、5mRNA的表达量明显下降；正常组IL-2／IL-4mRNA的比值较哮喘组高，两组比较有显著差异(P＜0.01)，其中哮喘组给予BN-52021干预后比值有所增大，与未干预组比较有明显变化(P＜0.01)。哮喘PMA+BN-52021干预组IL-2、4、5mRNA表达量，明显低于哮喘PMA干预组(P＜0.05)，但高于PMA+Ro31-8220组(P＜0.05)，而PMA+Ro31-8220组与PMA+Ro31-8220+BN-52021干预组比较无明显差异(P＞0.05)。哮喘空白组PKCα在T淋巴细胞胞膜与胞浆的表达量比率显著高于正常对照组(P＜0.01)，BN-52021干预后哮喘组比率较未干预组明显下降(P＜0.05)，PMA+BN-52021干预组PKCα比率为较PMA干预组1.28±0.28低(P＜0.05)；哮喘各组T淋巴细胞胞膜与胞浆PKCα表达量的比率与IL-4mRNA表达量的相关性分析显示呈明显正相关(n=42，r=0.845，P＜0.01)。
结论银杏叶制剂对体外培养的哮喘大鼠T淋巴细胞因子IL-2、4、5的分泌均具有抑制作用，而对IL-2分泌的抑制作用相对较弱；银杏叶制剂对哮喘大鼠T淋巴细胞胞膜与胞浆PKCα的表达量比率具有明显的下调作用；其对T淋巴细胞因子及Th1／Th2细胞因子平衡的影响，可能是通过影响PKC信号转导途径起作用。
分题三PKCα对哮喘患者外周血T淋巴细胞IL-2、IL-4、IL-5mRNA表达的调节以及银杏叶提取物对其的影响
目的了解银杏叶制剂对哮喘患者免疫学发病机制中起关键作用的T淋巴细胞部分生物学功能的影响情况，探讨其治疗哮喘的可能机制。
方法从12位哮喘患者和10位健康对照者静脉采取外周血10ml，分离出T淋巴细胞并进行分组培养(A组：空白组，B组：PMA干预组，C组：BN-52021干预组，D组：PMA+BN-52021干预组，E组：PMA+PKC抑制剂(Ro31-8220)干预组，F组：PMA+Ro31-8220+BN-52021干预组)。体外培养72小时后分别用Western Blot检测各组细胞膜与细胞浆PKCα表达比率和RT-PCR检测各组细胞IL-2、4、5mRNA的表达量。
结果哮喘组IL-2、4、5mRNA表达量明显高于正常组，各因子两组间比较差异有显著性(P＜0.01)；哮喘组T淋巴细胞给予BN-52021干预后IL-2、4、5mRNA的表达量明显下降(均P＜0.05)；正常组IL-2／IL-4mRNA的比值高于哮喘组，两组比较有显著差异(P＜0.01)，给予BN-52021干预后哮喘组比值有所增大，与干预前比较有明显变化(P＜0.01)。哮喘PMA+BN-52021组IL-2、4、5mRNA表达量明显低于PMA干预组(P＜0.05)，高于PMA+Ro31-8220组(P＜0.05)，而PMA+Ro31-8220组与PMA+Ro31-8220+BN-52021干预组比较无明显差异(P＞0.05)。哮喘空白组PKCα在T淋巴细胞胞膜与胞浆表达量的比率显著高于正常对照组(P＜0.01)，BN-52021干预后哮喘组比率较干预前明显下降(P＜0.05)，PMA+BN-52021干预组PKCα比率较PMA干预组低(P＜0.05)；哮喘组T淋巴细胞胞膜与胞浆PKCα表达量的比率与IL-4、5mRNA表达量的相关性分析呈明显正相关(分别r=0.885；r=0.938，均n=132，P＜0.01)。
结论银杏叶制剂对体外培养的哮喘患者T淋巴细胞因子IL-2、4、5的分泌均具有抑制作用，而对IL-2分泌的抑制作用相对较弱；银杏叶制剂对哮喘患者T淋巴细胞胞膜与胞浆PKCα表达比率具有明显的下调作用；其可能通过影响PKC信号转导途径而进一步影响IL-4、5等Th2细胞因子的分泌及Th1／Th2细胞因子的平衡。
第二部分银杏叶制剂对PKC在哮喘气道炎性细胞中的表达及其对气道中部分Th2类细胞因子浓度影响的研究
分题一银杏叶制剂对哮喘大鼠气道炎性细胞蛋白激酶Cα表达及白细胞介素-4分泌的影响
目的探讨银杏叶提取物(Extract of Ginkgo biloba，EGb)对哮喘大鼠气道炎症的治疗效果及可能机制。
方法48只SD大鼠按数字随机法均分成8组：正常对照组、哮喘组(此组再分为：未处理组、激素1周组、激素2周组、激素4周组、EGb1周组、EGb2周组、EGb4周组)。采用Wright's染色法分类计数肺泡灌洗液中各类炎性细胞的相对数，免疫组化(SP法)测定PKCα在各组大鼠肺泡灌洗液炎性细胞中的表达情况，ELISA法测定肺泡灌洗液上清中IL-4的含量。
结果哮喘未处理组大鼠肺泡灌洗液中嗜酸性粒细胞、淋巴细胞相对计数百分比、PKCα在淋巴细胞和总的细胞中的阳性表达率以及上清液中IL-4含量均显著高于正常对照组(P＜0.05)。激素处理不同时间组与未处理组间比较前述各指标明显下降(均P＜0.05)。EGb处理1、2周组相应指标明显高于激素处理组(P＜0.05)；EGb处理4周组与激素处理组比较无明显差异。EGb处理1周组与哮喘未处理组比较相应检测指标无明显差异(P＜0.05)，EGb处理2周组相关检测指标明显低于EGb处理1周组(P＜0.05)，但明显高于EGb处理4周组(P＜0.05)。EGb处理组炎性细胞PKCα阳性表达率与上清液中IL-4、嗜酸性粒细胞及淋巴细胞的相对计数百分比明显相关(n=18，r值分别为0.641、0.699、0.625，均P＜0.01)。
结论EGb可减轻气道EOS和淋巴细胞等炎性细胞的浸润以及气道炎性细胞PKCα的阳性表达率和IL-4的分泌量，其作用弱于糖皮质激素，但随着药物治疗时间的延长其药理作用逐渐明显。其治疗哮喘的作用可能与其减少炎性细胞PKCα的表达进而降低EOS和淋巴细胞在气道的浸润以及IL-4的浓度有关。
分题二吸入激素对哮喘患者诱导痰炎性细胞中PKCα表达及IL-5浓度的影响
目的了解哮喘患者气道炎性细胞中PKCα的表达及TL-5的分泌情况，探讨它们与气道炎症的关系以及吸入激素对它们的影响。
方法29例哮喘患者分2组：激素2周组(14例)、激素4周组(15例)，治疗并随访，在治疗前后行诱导痰和肺功能检查，另选14名健康者为对照组。采用免疫组化(SP法)测定PKCα在诱导痰炎性细胞中的表达，ELISA法测定诱导痰上清中IL-5的含量。
结果治疗前哮喘患者诱导痰中嗜酸性粒细胞和淋巴细胞相对计数、炎性细胞中PKCα阳性表达率和IL-5含量均高于健康组(P＜0.05)，治疗后同组比较均明显下降(P＜0.01)，FEV1占预计值的百分比的变化与它们的变化呈明显负相关(P＜0.05)；嗜酸性粒细胞和淋巴细胞的相对百分比与IL-5和炎性细胞PKCα阳性表达率显著相关(P＜0.01)。
结论吸入激素可明显降低气道IL-5的含量，其机制可能是通过抑制PKCα的表达来发挥作用。
分题三银杏叶提取物对哮喘患者诱导痰炎性细胞中PKCα表达及IL-5浓度的影响
目的探讨银杏叶提取物对哮喘患者的疗效及可能机制。
方法75例哮喘患者随机分4组：激素2周组20例、激素4周组20例、激素+银杏叶制剂2周组18例、激素+银杏叶制剂4周组17例，治疗并随访，在治疗前后行诱导痰及肺功能检查，另选15名健康者为对照。采用瑞氏染色法对诱导痰中细胞进行分类计数，SP法测定PKCα在诱导痰炎性细胞中的表达，ELISA法测定诱导痰上清中IL-5的含量。
结果治疗前哮喘患者诱导痰中EOS和淋巴细胞相对计数百分比、炎性细胞PKCα总阳性表达率和IL-5含量均显著高于健康组，FEV1占预计值的百分比显著低于健康对照组(均P＜0.05)。治疗后除FEV1占预计值的百分比较治疗前增加外，哮喘组其它相应指标较治疗前明显下降(P＜0.05)，但仍高于健康对照组(P＜0.05)。与激素治疗2周组比较，银杏叶制剂+激素治疗2周组EOS、淋巴细胞、诱导痰上清中IL-5浓度及各类炎性细胞PKCα阳性表达率均无明显降低(P＞0.05)；但银杏叶制剂+激素治疗4周组与激素治疗2、4周组比较相应指标有明显降低(P＜0.05)。IL-5与诱导痰中炎性细胞PKCα总阳性表达率和EOS的相对百分比明显正相关(分别r=0.83，r=0.76，均n=150，P＜0.01)；FEV1占预计值的百分比与诱导痰炎性细胞PKCα总阳性表达率及IL-5的含量呈明显负相关(分别r=-0.77，r=-0.64，均n=150，P＜0.01)。
结论银杏叶制剂可明显降低气道EOS、淋巴细胞等炎性细胞在气道的浸润，对哮喘气道炎症具有明显的改善作用；其部分机制可能通过影响气道炎性细胞PKCα的活性进而降低气道IL-5等细胞因子的浓度而发挥作用；其药理作用与糖皮质激素可能具有一定的互补性，但作用效果相对缓慢。
Introduction
Among the masses factors of mechanism of asthma, it was widely acknowledged that T-lymphocytes were one of the most important factors to chronic airway inflammation of asthma. The exacerbation and development of asthma was closely associated with T-lymphocytes (it is mainly Th2 cells) while it was activating, prolifering and producing amount of cytokines such as interleukin 4 (IL-4), IL-5 and so on. PKC is one of the major regulatory enzymes involved in the control of wide variety of physiological processes including T lymphocytes differentiation, proliferation, and activation. The role of PKC in T lymphocytes differentiation and activation has been extensively investigated. The evidence indicated that the differentiation and activation of T lymphocytes, when triggered through the T cell receptor, is dependent on PKC activity. PKC alpha is one of twelve diverse isotypes of PKC gene family. More and more investigation indicated that PKC alpha involves and play a main role in the T lymphocyte differentiation and activation. In severe asthma, glucocorticosteroids was the most effectively drugs to inhibit the airway inflammation. However, it has much adverse effects when it is used systemic. To minimize the side effects or avoid the use of steroids, other less toxic immunotherapeutic agents including Ginkgo biloba extracts based on an understanding of the pathogenesis of asthma have been proposed. Ginkgo biloba, an ancient plant, its standardized extract of leaves has been extensively used in disease of cardiovascular system and cerebrovascular system. Ginkgo biloba extract has been mentioned in the traditional Chinese pharmacopoeia, and Chinese has used the ginkgo leaf for the treatment of bronchial asthma and bronchitis for many centuries. However, so far, the effects of Ginkgo biloba extract on the activation and function of T-lymphocytes, an important population among immune effector cells in
inflammatory responses of airway inflammation of asthma, and PKC singnals conducts in that, has been very limited studies. Therefore, to acquire more information of effect of EGb on treatment asthma and the mechanism, it is necensary to systemicly investigating the effect of EGb on T-lymphocytes function and the relationship between T-lymphocytes and PKCa.The results will be benefit for the treatment and prevention of asthma.
Part 1
The Effect of Ginkgo biloba Extract on Production of cytokines of Thl/Th2 and Protein Kinase Ca Expression in Peripheral Blood T Lymphocytes of Asthma
Chapter 1
Effects of Ginkgo Biloba Extract on Proliferation and Apoptosis of T Lymphocytes Cultured in vitro of the Rat Model for Asthma
Objective: to study the partial therapeutic mechanism of Ginkgo Biloba extract in asthmatic patient treatment, we detected the effects of Ginkgo Biloba extract on proliferation of T lymphocytes cultured in vitro of the rat model for asthma.
Methods: 14 Wistar rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) of the rats, and were cultured in vitro with Ginkgolide B (BN-52021 group) or Ginkgo Biloba extract 761 (EGb761 group) in different concentration or without any of them (control group). After different period of time culture, the effect of Ginkgo Biloba extract on T lymphocytes viability was measured using the MTT proliferation assay, and the effect of BN-52021 on T lymphocytes apoptosis was analysis by Flow Cytometry.
Results: Compared with the control group, BN-52021 could significantly suppress the proliferation of T lymphocytes in vitro of healthy and sensitized rat (P<0.05). The effects of suppression were enhanced by the concentration of
BN-52021 increasing and the time prolonging. To the effects of the BN-52021's suppression, there is a significant difference between the healthy and the asthmatic model rat (P<0.05). However, EGb761 had a contrary pharmic effect on the in vitro culture T lymphocytes' proliferation as the concentration varied from low to high level, it promote T lymphocytes growth at low level and suppressed it at high level. Compared with the control group there were a significant difference (P<0.05). With the concentration of BN-52021 increasing, the rate of T lymphocytes apoptosis is increasing. There is a significant changes of rate among the different concentration levels (P<0.01).
Conclusions: Ginkgolide Biloba Extract had a different effect on the T lymphocytes cultured in vitro because it had different ingredients. They had a different effect on T lymphocytes proliferation between healthy and asthmatic rat. Ginkgolide B was the mainly for suppressing the T lymphocyte proliferation among the ingredients, and it probably could increasing the rate of apoptosis of T lymphocytes while they were cultured with them in vitro.
Chapter 2 The Effect of Ginkgo biloba Extract on Production of Interleukin-2,4,5 and
Protein Kinase Co Expression in Peripheral Blood T Lymphocytes Culture in
Vitro of Asthmatic Rats
Objective To investigate therapeutic effect of Ginkgo biloba extract on bronchial asthma.
Methods Fourteen SD rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells of rats, and were cultured in different conditions, blank group, group treated with Phorbol Myristate Acetate (PMA), group treated with Ginkgolide B (BN-52021), group treated with PMA + BN-52021, group treated with PMA+ Ro31-8220 (an inhibitor of PKC ), group treated with PMA+ Ro31-8220 + BN-52021. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chain
reaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western blotting.
Results The quantity of IL-2, 4 and 5 mRNA in blank group of asthmatic models (respectively 0.58 ± 0.086, 1.03 ± 0.12 and 0.48 ± 0.08) were significantly increased as compared to those in blank group of normal rats (respectively 0.45 ± 0.03, 0.35 ± 0.08 and 0.15 ± 0.05) (PO.05). The quantity of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes of asthmatic model rats were cultured with BN-52021 (respectively 0.49± 0.05, 0.55± 0.09 and 0.27±0.05) (P<0.05). The ratio of IL-2/IL-4 mRNA in normal rats was 1.27±0.20, significantly higher than that in asthmatic model rats (0.60±0.18) (PO.01), and BN-52021 could significantly increase the ratio when the T lymphocytes of asthmatic model rats were cultured with it (PO.01). BN-52021 could significantly counteract the levels of IL-2, 4 and 5 mRNA that increased by PMA (P<0.05), but the decreasing effect was less than the Ro31-8220, a specific inhibitor of PKCα (P<0.05). And BN-52021 couldn't further counteract significantly the levels of IL-2, 4 and 5 mRNA that increased by PMA which had significantly decreased by the Ro31-8220. The expression ratio of PKCα in membrane and cytoplasm of T lymphocytes of asthmatic model rats was significantly higher than that of normal rats (P<0.01), and BN-52021 could significantly decrease the ratio when the T lymphocytes were cultured with it (PO.05). Furthermore, it also can significantly counteract the expression ratio of PKCα in membrane and cytoplasm which increased by PMA (P<0.05). The expression ratio of PKCα in membrane and cytoplasm was significantly positively correlated with the level of IL-4 mRNA in T lymphocytes cultured in vitro in asthmatic model rats group (n=42, r=0.845, P<0.01).
Conclusion Ginkgo biloba extract could depress the expression of IL2, 4 and 5 mRNA of asthmatic model rat T lymphocytes in vitro. However, the inhibition effects were distinct in degree, so the balance of IL-2/IL-4 was changed according to the relative variability. Ginkgo biloba extract also could decrease the expression ratio of PKCα in membrane and cytoplasm. It is possible that Ginkgo biloba extract
influences the production of interleukin and the balance of Thl/Th2 in the asthmatic model rats through its influence to the PKC signal pathway.
Chapter 3 The Effects of the Ginkgo Biloba Extract on the Expression of Interleukin-2,
Interleukin-4, and Interleukin-5 Regulated by Protein Kinase Cot in Peripheral Blood T Lymphocytes Cultured in Vitro from Asthmatic Patients
Objective To investigate whether the secretion of T helper (Th) interleukin(IL), such as IL-2, IL-4 and IL-5, are regulated by protein kinase Cα (PKCα) in asthmatic T lymphocytes cultured in vitro, and the probably mechanism of the effects of Ginkgo biloba Extract (EGb) to asthma.
Methods Peripheral blood T lymphocytes were isolated from 12 asthmatic patients and 10 health volunteers, and were cultured in vitro in different groups: blank group, group cultured with Phorbol Myristate Acetate (PMA), group cultured with Ginkgolide B (BN 52021), group cultured with PMA + BN 52021, group cultured with PMA+ Ro31-8220(a specific inhibitor of PKCα), and group cultured with PMA+ Ro31-8220 + BN 52021, respectively. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chain reaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western Blot.
Results The levels of IL-2, 4 and 5 mRNA in blank group of asthmatic patients (0.68 ± 0.103, 1.14 ± 0.12 and 0.56 ± 0.06, respectively) were significantly increased than those in blank group of healthy control (0.55 ± 0.05, 0.41 ± 0.07 and 0.24 ± 0.04, respectively, P<0.05). The levels of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes from asthmatic patients were cultured with BN-52021(0.59±0.06, 0.67±0.09 and 0.36±0.05, respectively, P<0.05). The ratio of IL-2/IL-4 mRNA in T lymphocytes from normal controls was significantly higher than that from asthmatic patients (1.36±0.21 and 0.71±0.18, respectively, P<0.01), and BN52021 could significantly increased the ratio when the T lymphocytes from asthmatic patients were cultured with it (P<0.01). BN52021 could significantly
decreased the levels of IL-2, 4 and 5 mRNA that increased by the PMA (P<0.05), but its effects were less than Ro31-8220(P<0.05). The BN52021 couldn't further significantly decreased the levels of IL-2, 4 and 5 mRNA which had significantly decreased by the Ro31-8220 in the group cultured with PMA+ Ro31-8220 + BN52021. The activity of PKCα in T lymphocytes from asthmatic patients were significantly higher than that of healthy controls (P<0.01), and BN52021 could significantly decreased the ratio when T lymphocytes were cultured with it (P<0.05). Furthermore, it also can significantly decrease the activity of PKCα which increased by the PMA (P<0.05). The activity of PKCα were significantly correlated with the level of mRNA of IL-4 and IL-5 in T lymphocytes cultured in vitro (r=0.885 and r=0.938, respectively, P<0.01).
Conclusions EGb could depress the level of IL2, IL-4 and IL-5 mRNA in T lymphocytes from asthmatic patients cultured in vitro. However its ability for inhibition was difference in degree among different interleukins, and therefore it could recover the imbalance of IL-2/IL-4 in asthmatic T lymphocytes. EGb also could decrease the activity of PKCα in asthmatic T lymphocytes. We concluded that EGb influenced on the production of interleukin and the balance of Thl/Th2 in the asthmatic patients probable partly through its influence on the PKC signal pathway.
Part 2
The Investigation of the Effect of Extract of Ginkgo Biloba on the Expression of PKC in airway Inflammation Cells and the level of Th2-cytokines in Airway
Secretion of Asthma
Chapter 1
The Effect of Extract of Ginkgo Biloba on the Expression of PKCα in the Inflammation Cells and the level of IL-4 in Alveolar lavage of Asthmatic Rat
Objectives To investigate the effect and possible mechanism of the Extract of Ginkgo biloba (EGb) in the asthma therapy.
Methods Forty-eight rats were classified randomly 8 groups; these were normal control group, asthmatic control group, asthmatic rats treated with glucocorticosteroid 1, 2 and 4 weeks groups, and asthmatic rats treated with EGb 1, 2 and 4 weeks groups. The expression of PKCα in the alveolar lavage cells was detected using immunocytochemistry and positive staining cells in different cells were counted. Interleukin-4 (IL-4) in alveolar lavage supernatants was detected using enzyme-linked immunosorbent assay.
Results The percentage of eosinophils (EOS), lymphocytes in alveolar lavage cells, the ratio of PKCα positive expressing in lymphocytes and total cells of alveolar lavage and the concentration of IL-4 in alveolar lavage supernatants, all that in rat asthmatic models were higher than that of the controls (P<0.05). And glucocorticosteroid could decrease significantly the amount of eosinophils and lymphocytes in airway of the asthmatic rats (P<0.05), and it could suppress significantly the expressing of PKCα in lymphocytes and total cells of alveolar lavage (P<0.05), and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats (P<0.05). There were significantly higher in the groups of EGb treatment 1, 2 weeks than the groups treatment with glucocorticosteroid in the same items were detected (respectively P<0.05). There was no significantly different between group treated with EGb 4 weeks and group treated with glucocorticosteroid in the same items were detected (P>0.05). There was no significantly different between group treated with EGb 1 week and asthmatic control group in the same items were detected (P>0.05). The same items were detected in group treated with EGb 2 weeks were significantly lower than that in group treated with EGb 1 week (P<0.05), but were significantly higher than that in group treated with EGb 4 weeks (P<0.05). The ratio of PKCα positive expressing in total cells of alveolar lavage was significantly correlated to the percentage of EOS , lymphocytes in alveolar lavage cells, and the concentration of IL-4 in alveolar lavage supernatants (n=18, respectively r=0.641、 r=0.69、 r=0.625, respectively P<0.01).
Conclusions EGb could decrease the amount of eosinophils and lymphocytes in airway of the asthmatic rats, and it could suppress the expressing of PKCα in lymphocytes and total cells of alveolar lavage, and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats. However, the effect of that was less than that of glucocorticosteroid, but the effect would improve with the time prolonged. The probably mechanism of the EGb in the asthma therapy is that it suppress the expression of PKCα in airway inflammation cells and thus then depress the secretion of IL-4, decrease the amount of eosinophils and lymphocytes in airway of asthmatic rats.
Chapter 2
The Effect of Inhaled Glucocorticosteroid on Expression of PKCα in the Inflammation Cells and Producing of IL-5 in Induced Sputum of Asthma
Patients
Objectives To investigate the Expression of PKCα in the Inflammation Cells and the producing of interleukin-5(IL-5) in induced sputum in asthma patients and the effect of inhaled glucocorticosteroid on them.
Methods 29 asthma patients were classified 2 groups, 14 patients were treated with fluticasone propionate 2 weeks and the others were treated with fluticasone propionate 4 weeks. All individuals needed followed up and a method to induce sputum with inhaled hypertonic saline(4%～5%) was respectively used in them. 14 health volunters were as control. The expression of PKCα in the cells was detected using immunocytochemistry and positive percentage in different cells were counted. Interleukin-5(IL-5) in sputum supernatants was detected using enzyme-linked immunosorbent assay.
Results The percentage of eosinophils(11.1%±4.3%) , lymphocytes(4.3%±2.6%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in asthmas were higher than the eosinophils(0.9%± 0.9%) , lymphocytes(1.2% ± 0.8%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in the controls(P<0.05). The concentration of IL-5 is correlated to the percentage positive of PKCα in inflammation cells (P<0.01). The
positives percentage of PKCα and the concentration of IL-5 in all groups were lower than treated before(P<0.05), and they are correlated to the forced expiratory volume in first second (FEV1).
Conclusions PKCα in the inflammation cells and IL-5 may play an important part in the airway inflammation., and the signal transduction of the PKCα may be one of the mechanisms of the airway inflammation in asthma patients. It is powerful to the glucocorticosteroid in asthma therapy. The probably mechanism is that glucocorticosteroid suppress the expression of PKCα in airway inflammation cells and then decrease the producing of IL-5 in the airway.
Chapter 3
The Effect of Ginkgo Biloba Extract on the Expression of PKCα in the
Inflammation Cells and the Level of IL-5 in Induced Sputum of Asthma
Patients
Objectives To investigate the effect of the EGb on the therapy of asthma and the probable mechnisms.
Methods 75 asthma patients were classified into 4 groups, treated with fluticasone propionate 2 weeks or 4 weeks, or treated with fluticasone propionate and EGb 2 weeks or 4 weeks. 15 health volunteers were as normal control. All individuals needed followed up. Induced sputum after inhaling hypertonic saline (4%～5%) was performed respectively to them. Lung ventilatory function and forced expiratory volume in one second (FEV1) were measured to all individuals. The numbers of different cells in induced sputum were calculated after cells stained with Wright's staining. The expression of PKCα in the cells was detected with immunocytochemistry and positive percentage in different cells were counted and calculated. Interleukin-5 (IL-5) in sputum supernatants was detected with enzyme-linked immunosorbent assay.
Results The percentage of eosinophils (14.3±2.7) %, lymphocytes (7.9±2.6) %, PKCα positive inflammation cells (80.7±9.1) % and the concentration of IL-5 (56.8±10.8) ng/L in asthmatic patients are higher than that percentage of the
eosinophils (1.1±0.4) % , lymphocytes (2.4±0.7)%, PKCα positive inflammation cells (13.6±3.4) % and the concentration of IL-5 (9.7±4.3) ng/L in the controls (P<0.05), and it was significantly lower than that after they were treated with fluticasone propionate or fluticasone propionate and EGb. However, it was still significantly higher than that of the controls. Compared to the group treatment with glucocorticosteroid 2 weeks, there was no significantly decreasing in the percentage of eosinophils, lymphocytes, PKCα positive inflammation cells and the induced sputum supernatant levels of IL-5. Compared to the group treatment with glucocorticosteroid 2 or 4 weeks, There was a significantly decrease in the same items in the group treated with fluticasone propionate and EGb 4 weeks. The levels of IL-5 in supernatant of induced sputum was significantly correlated positive to the percentage of PKCα positive inflammation cells and the percentage of eosinophils in the induced sputum in groups of asthma patients (respectively: n=150, r= 0.83, P<0.01; n=150, r=0.76, P<0.01). The forced expiratory volume in first second are significantly correlated negative to the percentage of PKCα positive inflammation cells and the levels of IL-5 in supernatant of induced sputum in groups of asthma patients (respectively: n=150, i= -0.77, P<0.01; n=150, r= -0.64, P<0.01).
Conclusions EGb could significantly decrease the infiltration of inflammation cells such as eosinophils and lymphocytes in airway of bronchial asthma. It could relieve the airway inflammation in some degree. Part mechanism of that probably is that EGb could decrease the activation of the PKCα in the inflammation cells and then decrease the induced sputum supernatant levels of IL-5. EGb was probably as glucocorticosteroid complement in therapy of asthma, but it took much longer time to display the effect of treatment of asthma than glucocorticosteroid.
在众多的发病机制中，有T淋巴细胞参与的慢性气道炎症是公认的哮喘重要的发病机制之一。T淋巴细胞(主要是Th2细胞)的活化、增殖和分泌大量的细胞因子(如：IL-4、IL-5等)在哮喘的发生、发展中起着重要作用。而在T淋巴细胞活化、增殖和分泌细胞因子促进炎性介质释放的过程中，细胞信号转导通路的作用十分重要，其中尤以蛋白激酶C(Protein Kinase C，PKC)信号转导通路的作用最引人注目。PKC是一个具有广泛生物学活性的酶，先前的研究表明PKC家族的重要成员PKCα活性的变化在T淋巴细胞活化、增殖和分泌细胞因子的过程中起着重要作用。糖皮质激素是目前治疗哮喘气道炎症的最强大最有效的药物，但临床应用显示其也有缺点和局限性，寻找新的有效治疗气道炎症的药物对提高哮喘的总体治疗效果具有一定的临床意义。银杏叶提取物的化学成分复杂，其主要活性成分银杏黄酮和萜内酯现已广泛应用于心脑血管系统疾病的治疗；在呼吸系统方面的治疗作用，特别是在哮喘的治疗方面早有记载。迄今为止，较多的临床研究证实银杏制剂对哮喘有确切的疗效，其对哮喘患者T淋巴细胞功能及其与PKCα关系的影响如何目前尚无报道。因此，将银杏叶制剂对哮喘T淋巴细胞功能及其与PKCα关系的影响进行系统地的研究，对银杏叶制剂治疗哮喘的临床效果和部分作用机理进行深入的探讨，明确其对哮喘的疗效及部分可能的作用机理，对于改善哮喘疗效和预防复发有一定的临床意义。
第一部分银杏叶制剂对哮喘外周血T淋巴细胞PKC表达及Th1／Th2类细胞因子分泌影响的研究
分题一银杏叶制剂对哮喘大鼠T淋巴细胞体外增殖和凋亡的影响
目的检测不同银杏叶制剂在体外对T淋巴细胞增殖和凋亡的影响，探讨银杏叶制剂治疗哮喘的部分机理。
方法将14只大鼠分成2组(健康对照组7只；哮喘组7只)，分别采集外周血，并分离出T淋巴细胞，体外分组培养(分空白组、银杏叶制剂BN-52021组、银杏叶制剂EGb761组)，各组分别以不同浓度的BN-52021和EGb761干预不同时间。之后，用MTT法分别测定各组细胞的增殖情况；用Annexin V／PI双染法流式细胞仪测定BN-52021不同浓度组的细胞凋亡情况。
结果与空白组比较，EGb761在低浓度时促进T淋巴细胞增殖，而高浓度时则起抑制作用(均P＜0.05)；BN-52021对体外培养的哮喘和健康大鼠T淋巴细胞的增殖均有抑制作用，其作用强度(在一定的范围内)随剂量的增加和时间的延长而加强(组间比较均P＜0.05)；但对哮喘大鼠的抑制作用要明显强于健康大鼠(P＜0.05)；T淋巴细胞的凋亡率随BN-52021浓度的增加而增加(P＜0.01)。
结论银杏叶制剂因其成分和干预浓度的不同而对体外培养的大鼠T淋巴细胞增殖活性的影响不同，对哮喘与健康大鼠T淋巴细胞的作用也有差异，银杏内酯B可能是抑制T淋巴细胞增殖的主要活性成分，其不仅可抑制T淋巴细胞的体外增殖，而且还可增加T淋巴细胞的凋亡率。
分题二哮喘大鼠外周血T淋巴细胞Th1／Th2细胞因子和PKCα的表达及银杏叶制剂对它们的影响
目的探讨银杏叶制剂对在哮喘大鼠免疫学发病机制中起关键作用的T淋巴细胞部分生物学功能的影响情况。
方法将14只SD大鼠随机分成哮喘和正常两组，每组7只。从每只大鼠外周血分离出T淋巴细胞，分组培养72小时后，分别用逆转录—聚合酶链式反应(Reverse Transcriptase-Polymerase Chain Reaction，RT-PCR)检测各组IL-2、IL-4、IL-5 mRNA的表达量和Western Blot检测各组细胞膜与细胞浆PKCα的表达比率。
结果IL-2、4、5 mRNA表达量在哮喘组均明显高于正常组，各因子两组间比较差异有显著性(P＜0.01)；哮喘组T淋巴细胞给予BN-52021干预后IL-2、4、5mRNA的表达量明显下降；正常组IL-2／IL-4mRNA的比值较哮喘组高，两组比较有显著差异(P＜0.01)，其中哮喘组给予BN-52021干预后比值有所增大，与未干预组比较有明显变化(P＜0.01)。哮喘PMA+BN-52021干预组IL-2、4、5mRNA表达量，明显低于哮喘PMA干预组(P＜0.05)，但高于PMA+Ro31-8220组(P＜0.05)，而PMA+Ro31-8220组与PMA+Ro31-8220+BN-52021干预组比较无明显差异(P＞0.05)。哮喘空白组PKCα在T淋巴细胞胞膜与胞浆的表达量比率显著高于正常对照组(P＜0.01)，BN-52021干预后哮喘组比率较未干预组明显下降(P＜0.05)，PMA+BN-52021干预组PKCα比率为较PMA干预组1.28±0.28低(P＜0.05)；哮喘各组T淋巴细胞胞膜与胞浆PKCα表达量的比率与IL-4mRNA表达量的相关性分析显示呈明显正相关(n=42，r=0.845，P＜0.01)。
结论银杏叶制剂对体外培养的哮喘大鼠T淋巴细胞因子IL-2、4、5的分泌均具有抑制作用，而对IL-2分泌的抑制作用相对较弱；银杏叶制剂对哮喘大鼠T淋巴细胞胞膜与胞浆PKCα的表达量比率具有明显的下调作用；其对T淋巴细胞因子及Th1／Th2细胞因子平衡的影响，可能是通过影响PKC信号转导途径起作用。
分题三PKCα对哮喘患者外周血T淋巴细胞IL-2、IL-4、IL-5mRNA表达的调节以及银杏叶提取物对其的影响
目的了解银杏叶制剂对哮喘患者免疫学发病机制中起关键作用的T淋巴细胞部分生物学功能的影响情况，探讨其治疗哮喘的可能机制。
方法从12位哮喘患者和10位健康对照者静脉采取外周血10ml，分离出T淋巴细胞并进行分组培养(A组：空白组，B组：PMA干预组，C组：BN-52021干预组，D组：PMA+BN-52021干预组，E组：PMA+PKC抑制剂(Ro31-8220)干预组，F组：PMA+Ro31-8220+BN-52021干预组)。体外培养72小时后分别用Western Blot检测各组细胞膜与细胞浆PKCα表达比率和RT-PCR检测各组细胞IL-2、4、5mRNA的表达量。
结果哮喘组IL-2、4、5mRNA表达量明显高于正常组，各因子两组间比较差异有显著性(P＜0.01)；哮喘组T淋巴细胞给予BN-52021干预后IL-2、4、5mRNA的表达量明显下降(均P＜0.05)；正常组IL-2／IL-4mRNA的比值高于哮喘组，两组比较有显著差异(P＜0.01)，给予BN-52021干预后哮喘组比值有所增大，与干预前比较有明显变化(P＜0.01)。哮喘PMA+BN-52021组IL-2、4、5mRNA表达量明显低于PMA干预组(P＜0.05)，高于PMA+Ro31-8220组(P＜0.05)，而PMA+Ro31-8220组与PMA+Ro31-8220+BN-52021干预组比较无明显差异(P＞0.05)。哮喘空白组PKCα在T淋巴细胞胞膜与胞浆表达量的比率显著高于正常对照组(P＜0.01)，BN-52021干预后哮喘组比率较干预前明显下降(P＜0.05)，PMA+BN-52021干预组PKCα比率较PMA干预组低(P＜0.05)；哮喘组T淋巴细胞胞膜与胞浆PKCα表达量的比率与IL-4、5mRNA表达量的相关性分析呈明显正相关(分别r=0.885；r=0.938，均n=132，P＜0.01)。
结论银杏叶制剂对体外培养的哮喘患者T淋巴细胞因子IL-2、4、5的分泌均具有抑制作用，而对IL-2分泌的抑制作用相对较弱；银杏叶制剂对哮喘患者T淋巴细胞胞膜与胞浆PKCα表达比率具有明显的下调作用；其可能通过影响PKC信号转导途径而进一步影响IL-4、5等Th2细胞因子的分泌及Th1／Th2细胞因子的平衡。
第二部分银杏叶制剂对PKC在哮喘气道炎性细胞中的表达及其对气道中部分Th2类细胞因子浓度影响的研究
分题一银杏叶制剂对哮喘大鼠气道炎性细胞蛋白激酶Cα表达及白细胞介素-4分泌的影响
目的探讨银杏叶提取物(Extract of Ginkgo biloba，EGb)对哮喘大鼠气道炎症的治疗效果及可能机制。
方法48只SD大鼠按数字随机法均分成8组：正常对照组、哮喘组(此组再分为：未处理组、激素1周组、激素2周组、激素4周组、EGb1周组、EGb2周组、EGb4周组)。采用Wright's染色法分类计数肺泡灌洗液中各类炎性细胞的相对数，免疫组化(SP法)测定PKCα在各组大鼠肺泡灌洗液炎性细胞中的表达情况，ELISA法测定肺泡灌洗液上清中IL-4的含量。
结果哮喘未处理组大鼠肺泡灌洗液中嗜酸性粒细胞、淋巴细胞相对计数百分比、PKCα在淋巴细胞和总的细胞中的阳性表达率以及上清液中IL-4含量均显著高于正常对照组(P＜0.05)。激素处理不同时间组与未处理组间比较前述各指标明显下降(均P＜0.05)。EGb处理1、2周组相应指标明显高于激素处理组(P＜0.05)；EGb处理4周组与激素处理组比较无明显差异。EGb处理1周组与哮喘未处理组比较相应检测指标无明显差异(P＜0.05)，EGb处理2周组相关检测指标明显低于EGb处理1周组(P＜0.05)，但明显高于EGb处理4周组(P＜0.05)。EGb处理组炎性细胞PKCα阳性表达率与上清液中IL-4、嗜酸性粒细胞及淋巴细胞的相对计数百分比明显相关(n=18，r值分别为0.641、0.699、0.625，均P＜0.01)。
结论EGb可减轻气道EOS和淋巴细胞等炎性细胞的浸润以及气道炎性细胞PKCα的阳性表达率和IL-4的分泌量，其作用弱于糖皮质激素，但随着药物治疗时间的延长其药理作用逐渐明显。其治疗哮喘的作用可能与其减少炎性细胞PKCα的表达进而降低EOS和淋巴细胞在气道的浸润以及IL-4的浓度有关。
分题二吸入激素对哮喘患者诱导痰炎性细胞中PKCα表达及IL-5浓度的影响
目的了解哮喘患者气道炎性细胞中PKCα的表达及TL-5的分泌情况，探讨它们与气道炎症的关系以及吸入激素对它们的影响。
方法29例哮喘患者分2组：激素2周组(14例)、激素4周组(15例)，治疗并随访，在治疗前后行诱导痰和肺功能检查，另选14名健康者为对照组。采用免疫组化(SP法)测定PKCα在诱导痰炎性细胞中的表达，ELISA法测定诱导痰上清中IL-5的含量。
结果治疗前哮喘患者诱导痰中嗜酸性粒细胞和淋巴细胞相对计数、炎性细胞中PKCα阳性表达率和IL-5含量均高于健康组(P＜0.05)，治疗后同组比较均明显下降(P＜0.01)，FEV1占预计值的百分比的变化与它们的变化呈明显负相关(P＜0.05)；嗜酸性粒细胞和淋巴细胞的相对百分比与IL-5和炎性细胞PKCα阳性表达率显著相关(P＜0.01)。
结论吸入激素可明显降低气道IL-5的含量，其机制可能是通过抑制PKCα的表达来发挥作用。
分题三银杏叶提取物对哮喘患者诱导痰炎性细胞中PKCα表达及IL-5浓度的影响
目的探讨银杏叶提取物对哮喘患者的疗效及可能机制。
方法75例哮喘患者随机分4组：激素2周组20例、激素4周组20例、激素+银杏叶制剂2周组18例、激素+银杏叶制剂4周组17例，治疗并随访，在治疗前后行诱导痰及肺功能检查，另选15名健康者为对照。采用瑞氏染色法对诱导痰中细胞进行分类计数，SP法测定PKCα在诱导痰炎性细胞中的表达，ELISA法测定诱导痰上清中IL-5的含量。
结果治疗前哮喘患者诱导痰中EOS和淋巴细胞相对计数百分比、炎性细胞PKCα总阳性表达率和IL-5含量均显著高于健康组，FEV1占预计值的百分比显著低于健康对照组(均P＜0.05)。治疗后除FEV1占预计值的百分比较治疗前增加外，哮喘组其它相应指标较治疗前明显下降(P＜0.05)，但仍高于健康对照组(P＜0.05)。与激素治疗2周组比较，银杏叶制剂+激素治疗2周组EOS、淋巴细胞、诱导痰上清中IL-5浓度及各类炎性细胞PKCα阳性表达率均无明显降低(P＞0.05)；但银杏叶制剂+激素治疗4周组与激素治疗2、4周组比较相应指标有明显降低(P＜0.05)。IL-5与诱导痰中炎性细胞PKCα总阳性表达率和EOS的相对百分比明显正相关(分别r=0.83，r=0.76，均n=150，P＜0.01)；FEV1占预计值的百分比与诱导痰炎性细胞PKCα总阳性表达率及IL-5的含量呈明显负相关(分别r=-0.77，r=-0.64，均n=150，P＜0.01)。
结论银杏叶制剂可明显降低气道EOS、淋巴细胞等炎性细胞在气道的浸润，对哮喘气道炎症具有明显的改善作用；其部分机制可能通过影响气道炎性细胞PKCα的活性进而降低气道IL-5等细胞因子的浓度而发挥作用；其药理作用与糖皮质激素可能具有一定的互补性，但作用效果相对缓慢。
Introduction
Among the masses factors of mechanism of asthma, it was widely acknowledged that T-lymphocytes were one of the most important factors to chronic airway inflammation of asthma. The exacerbation and development of asthma was closely associated with T-lymphocytes (it is mainly Th2 cells) while it was activating, prolifering and producing amount of cytokines such as interleukin 4 (IL-4), IL-5 and so on. PKC is one of the major regulatory enzymes involved in the control of wide variety of physiological processes including T lymphocytes differentiation, proliferation, and activation. The role of PKC in T lymphocytes differentiation and activation has been extensively investigated. The evidence indicated that the differentiation and activation of T lymphocytes, when triggered through the T cell receptor, is dependent on PKC activity. PKC alpha is one of twelve diverse isotypes of PKC gene family. More and more investigation indicated that PKC alpha involves and play a main role in the T lymphocyte differentiation and activation. In severe asthma, glucocorticosteroids was the most effectively drugs to inhibit the airway inflammation. However, it has much adverse effects when it is used systemic. To minimize the side effects or avoid the use of steroids, other less toxic immunotherapeutic agents including Ginkgo biloba extracts based on an understanding of the pathogenesis of asthma have been proposed. Ginkgo biloba, an ancient plant, its standardized extract of leaves has been extensively used in disease of cardiovascular system and cerebrovascular system. Ginkgo biloba extract has been mentioned in the traditional Chinese pharmacopoeia, and Chinese has used the ginkgo leaf for the treatment of bronchial asthma and bronchitis for many centuries. However, so far, the effects of Ginkgo biloba extract on the activation and function of T-lymphocytes, an important population among immune effector cells in
inflammatory responses of airway inflammation of asthma, and PKC singnals conducts in that, has been very limited studies. Therefore, to acquire more information of effect of EGb on treatment asthma and the mechanism, it is necensary to systemicly investigating the effect of EGb on T-lymphocytes function and the relationship between T-lymphocytes and PKCa.The results will be benefit for the treatment and prevention of asthma.
Part 1
The Effect of Ginkgo biloba Extract on Production of cytokines of Thl/Th2 and Protein Kinase Ca Expression in Peripheral Blood T Lymphocytes of Asthma
Chapter 1
Effects of Ginkgo Biloba Extract on Proliferation and Apoptosis of T Lymphocytes Cultured in vitro of the Rat Model for Asthma
Objective: to study the partial therapeutic mechanism of Ginkgo Biloba extract in asthmatic patient treatment, we detected the effects of Ginkgo Biloba extract on proliferation of T lymphocytes cultured in vitro of the rat model for asthma.
Methods: 14 Wistar rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) of the rats, and were cultured in vitro with Ginkgolide B (BN-52021 group) or Ginkgo Biloba extract 761 (EGb761 group) in different concentration or without any of them (control group). After different period of time culture, the effect of Ginkgo Biloba extract on T lymphocytes viability was measured using the MTT proliferation assay, and the effect of BN-52021 on T lymphocytes apoptosis was analysis by Flow Cytometry.
Results: Compared with the control group, BN-52021 could significantly suppress the proliferation of T lymphocytes in vitro of healthy and sensitized rat (P<0.05). The effects of suppression were enhanced by the concentration of
BN-52021 increasing and the time prolonging. To the effects of the BN-52021's suppression, there is a significant difference between the healthy and the asthmatic model rat (P<0.05). However, EGb761 had a contrary pharmic effect on the in vitro culture T lymphocytes' proliferation as the concentration varied from low to high level, it promote T lymphocytes growth at low level and suppressed it at high level. Compared with the control group there were a significant difference (P<0.05). With the concentration of BN-52021 increasing, the rate of T lymphocytes apoptosis is increasing. There is a significant changes of rate among the different concentration levels (P<0.01).
Conclusions: Ginkgolide Biloba Extract had a different effect on the T lymphocytes cultured in vitro because it had different ingredients. They had a different effect on T lymphocytes proliferation between healthy and asthmatic rat. Ginkgolide B was the mainly for suppressing the T lymphocyte proliferation among the ingredients, and it probably could increasing the rate of apoptosis of T lymphocytes while they were cultured with them in vitro.
Chapter 2 The Effect of Ginkgo biloba Extract on Production of Interleukin-2,4,5 and
Protein Kinase Co Expression in Peripheral Blood T Lymphocytes Culture in
Vitro of Asthmatic Rats
Objective To investigate therapeutic effect of Ginkgo biloba extract on bronchial asthma.
Methods Fourteen SD rats were randomly divided into two groups, 7 rats were sensitized as asthmatic model and the others as healthy controls. T lymphocytes were isolated from peripheral blood mononuclear cells of rats, and were cultured in different conditions, blank group, group treated with Phorbol Myristate Acetate (PMA), group treated with Ginkgolide B (BN-52021), group treated with PMA + BN-52021, group treated with PMA+ Ro31-8220 (an inhibitor of PKC ), group treated with PMA+ Ro31-8220 + BN-52021. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chain
reaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western blotting.
Results The quantity of IL-2, 4 and 5 mRNA in blank group of asthmatic models (respectively 0.58 ± 0.086, 1.03 ± 0.12 and 0.48 ± 0.08) were significantly increased as compared to those in blank group of normal rats (respectively 0.45 ± 0.03, 0.35 ± 0.08 and 0.15 ± 0.05) (PO.05). The quantity of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes of asthmatic model rats were cultured with BN-52021 (respectively 0.49± 0.05, 0.55± 0.09 and 0.27±0.05) (P<0.05). The ratio of IL-2/IL-4 mRNA in normal rats was 1.27±0.20, significantly higher than that in asthmatic model rats (0.60±0.18) (PO.01), and BN-52021 could significantly increase the ratio when the T lymphocytes of asthmatic model rats were cultured with it (PO.01). BN-52021 could significantly counteract the levels of IL-2, 4 and 5 mRNA that increased by PMA (P<0.05), but the decreasing effect was less than the Ro31-8220, a specific inhibitor of PKCα (P<0.05). And BN-52021 couldn't further counteract significantly the levels of IL-2, 4 and 5 mRNA that increased by PMA which had significantly decreased by the Ro31-8220. The expression ratio of PKCα in membrane and cytoplasm of T lymphocytes of asthmatic model rats was significantly higher than that of normal rats (P<0.01), and BN-52021 could significantly decrease the ratio when the T lymphocytes were cultured with it (PO.05). Furthermore, it also can significantly counteract the expression ratio of PKCα in membrane and cytoplasm which increased by PMA (P<0.05). The expression ratio of PKCα in membrane and cytoplasm was significantly positively correlated with the level of IL-4 mRNA in T lymphocytes cultured in vitro in asthmatic model rats group (n=42, r=0.845, P<0.01).
Conclusion Ginkgo biloba extract could depress the expression of IL2, 4 and 5 mRNA of asthmatic model rat T lymphocytes in vitro. However, the inhibition effects were distinct in degree, so the balance of IL-2/IL-4 was changed according to the relative variability. Ginkgo biloba extract also could decrease the expression ratio of PKCα in membrane and cytoplasm. It is possible that Ginkgo biloba extract
influences the production of interleukin and the balance of Thl/Th2 in the asthmatic model rats through its influence to the PKC signal pathway.
Chapter 3 The Effects of the Ginkgo Biloba Extract on the Expression of Interleukin-2,
Interleukin-4, and Interleukin-5 Regulated by Protein Kinase Cot in Peripheral Blood T Lymphocytes Cultured in Vitro from Asthmatic Patients
Objective To investigate whether the secretion of T helper (Th) interleukin(IL), such as IL-2, IL-4 and IL-5, are regulated by protein kinase Cα (PKCα) in asthmatic T lymphocytes cultured in vitro, and the probably mechanism of the effects of Ginkgo biloba Extract (EGb) to asthma.
Methods Peripheral blood T lymphocytes were isolated from 12 asthmatic patients and 10 health volunteers, and were cultured in vitro in different groups: blank group, group cultured with Phorbol Myristate Acetate (PMA), group cultured with Ginkgolide B (BN 52021), group cultured with PMA + BN 52021, group cultured with PMA+ Ro31-8220(a specific inhibitor of PKCα), and group cultured with PMA+ Ro31-8220 + BN 52021, respectively. After 72 h, we detected the levels of IL-2, 4, 5 mRNA in each groups with reverse transcriptase polymerase chain reaction (RT-PCR), and detected the activity of PKCα by checking the ratio of PKCα in membrane to cytoplasm in T lymphocytes with Western Blot.
Results The levels of IL-2, 4 and 5 mRNA in blank group of asthmatic patients (0.68 ± 0.103, 1.14 ± 0.12 and 0.56 ± 0.06, respectively) were significantly increased than those in blank group of healthy control (0.55 ± 0.05, 0.41 ± 0.07 and 0.24 ± 0.04, respectively, P<0.05). The levels of IL-2, 4 and 5 mRNA decreased significantly when the T lymphocytes from asthmatic patients were cultured with BN-52021(0.59±0.06, 0.67±0.09 and 0.36±0.05, respectively, P<0.05). The ratio of IL-2/IL-4 mRNA in T lymphocytes from normal controls was significantly higher than that from asthmatic patients (1.36±0.21 and 0.71±0.18, respectively, P<0.01), and BN52021 could significantly increased the ratio when the T lymphocytes from asthmatic patients were cultured with it (P<0.01). BN52021 could significantly
decreased the levels of IL-2, 4 and 5 mRNA that increased by the PMA (P<0.05), but its effects were less than Ro31-8220(P<0.05). The BN52021 couldn't further significantly decreased the levels of IL-2, 4 and 5 mRNA which had significantly decreased by the Ro31-8220 in the group cultured with PMA+ Ro31-8220 + BN52021. The activity of PKCα in T lymphocytes from asthmatic patients were significantly higher than that of healthy controls (P<0.01), and BN52021 could significantly decreased the ratio when T lymphocytes were cultured with it (P<0.05). Furthermore, it also can significantly decrease the activity of PKCα which increased by the PMA (P<0.05). The activity of PKCα were significantly correlated with the level of mRNA of IL-4 and IL-5 in T lymphocytes cultured in vitro (r=0.885 and r=0.938, respectively, P<0.01).
Conclusions EGb could depress the level of IL2, IL-4 and IL-5 mRNA in T lymphocytes from asthmatic patients cultured in vitro. However its ability for inhibition was difference in degree among different interleukins, and therefore it could recover the imbalance of IL-2/IL-4 in asthmatic T lymphocytes. EGb also could decrease the activity of PKCα in asthmatic T lymphocytes. We concluded that EGb influenced on the production of interleukin and the balance of Thl/Th2 in the asthmatic patients probable partly through its influence on the PKC signal pathway.
Part 2
The Investigation of the Effect of Extract of Ginkgo Biloba on the Expression of PKC in airway Inflammation Cells and the level of Th2-cytokines in Airway
Secretion of Asthma
Chapter 1
The Effect of Extract of Ginkgo Biloba on the Expression of PKCα in the Inflammation Cells and the level of IL-4 in Alveolar lavage of Asthmatic Rat
Objectives To investigate the effect and possible mechanism of the Extract of Ginkgo biloba (EGb) in the asthma therapy.
Methods Forty-eight rats were classified randomly 8 groups; these were normal control group, asthmatic control group, asthmatic rats treated with glucocorticosteroid 1, 2 and 4 weeks groups, and asthmatic rats treated with EGb 1, 2 and 4 weeks groups. The expression of PKCα in the alveolar lavage cells was detected using immunocytochemistry and positive staining cells in different cells were counted. Interleukin-4 (IL-4) in alveolar lavage supernatants was detected using enzyme-linked immunosorbent assay.
Results The percentage of eosinophils (EOS), lymphocytes in alveolar lavage cells, the ratio of PKCα positive expressing in lymphocytes and total cells of alveolar lavage and the concentration of IL-4 in alveolar lavage supernatants, all that in rat asthmatic models were higher than that of the controls (P<0.05). And glucocorticosteroid could decrease significantly the amount of eosinophils and lymphocytes in airway of the asthmatic rats (P<0.05), and it could suppress significantly the expressing of PKCα in lymphocytes and total cells of alveolar lavage (P<0.05), and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats (P<0.05). There were significantly higher in the groups of EGb treatment 1, 2 weeks than the groups treatment with glucocorticosteroid in the same items were detected (respectively P<0.05). There was no significantly different between group treated with EGb 4 weeks and group treated with glucocorticosteroid in the same items were detected (P>0.05). There was no significantly different between group treated with EGb 1 week and asthmatic control group in the same items were detected (P>0.05). The same items were detected in group treated with EGb 2 weeks were significantly lower than that in group treated with EGb 1 week (P<0.05), but were significantly higher than that in group treated with EGb 4 weeks (P<0.05). The ratio of PKCα positive expressing in total cells of alveolar lavage was significantly correlated to the percentage of EOS , lymphocytes in alveolar lavage cells, and the concentration of IL-4 in alveolar lavage supernatants (n=18, respectively r=0.641、 r=0.69、 r=0.625, respectively P<0.01).
Conclusions EGb could decrease the amount of eosinophils and lymphocytes in airway of the asthmatic rats, and it could suppress the expressing of PKCα in lymphocytes and total cells of alveolar lavage, and it also could depress significantly the secretion of IL-4 in airway inflammation cells of asthmatic rats. However, the effect of that was less than that of glucocorticosteroid, but the effect would improve with the time prolonged. The probably mechanism of the EGb in the asthma therapy is that it suppress the expression of PKCα in airway inflammation cells and thus then depress the secretion of IL-4, decrease the amount of eosinophils and lymphocytes in airway of asthmatic rats.
Chapter 2
The Effect of Inhaled Glucocorticosteroid on Expression of PKCα in the Inflammation Cells and Producing of IL-5 in Induced Sputum of Asthma
Patients
Objectives To investigate the Expression of PKCα in the Inflammation Cells and the producing of interleukin-5(IL-5) in induced sputum in asthma patients and the effect of inhaled glucocorticosteroid on them.
Methods 29 asthma patients were classified 2 groups, 14 patients were treated with fluticasone propionate 2 weeks and the others were treated with fluticasone propionate 4 weeks. All individuals needed followed up and a method to induce sputum with inhaled hypertonic saline(4%～5%) was respectively used in them. 14 health volunters were as control. The expression of PKCα in the cells was detected using immunocytochemistry and positive percentage in different cells were counted. Interleukin-5(IL-5) in sputum supernatants was detected using enzyme-linked immunosorbent assay.
Results The percentage of eosinophils(11.1%±4.3%) , lymphocytes(4.3%±2.6%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in asthmas were higher than the eosinophils(0.9%± 0.9%) , lymphocytes(1.2% ± 0.8%) and the concentration of IL-5(64.9 ng/L±46.0ng/L) in the controls(P<0.05). The concentration of IL-5 is correlated to the percentage positive of PKCα in inflammation cells (P<0.01). The
positives percentage of PKCα and the concentration of IL-5 in all groups were lower than treated before(P<0.05), and they are correlated to the forced expiratory volume in first second (FEV1).
Conclusions PKCα in the inflammation cells and IL-5 may play an important part in the airway inflammation., and the signal transduction of the PKCα may be one of the mechanisms of the airway inflammation in asthma patients. It is powerful to the glucocorticosteroid in asthma therapy. The probably mechanism is that glucocorticosteroid suppress the expression of PKCα in airway inflammation cells and then decrease the producing of IL-5 in the airway.
Chapter 3
The Effect of Ginkgo Biloba Extract on the Expression of PKCα in the
Inflammation Cells and the Level of IL-5 in Induced Sputum of Asthma
Patients
Objectives To investigate the effect of the EGb on the therapy of asthma and the probable mechnisms.
Methods 75 asthma patients were classified into 4 groups, treated with fluticasone propionate 2 weeks or 4 weeks, or treated with fluticasone propionate and EGb 2 weeks or 4 weeks. 15 health volunteers were as normal control. All individuals needed followed up. Induced sputum after inhaling hypertonic saline (4%～5%) was performed respectively to them. Lung ventilatory function and forced expiratory volume in one second (FEV1) were measured to all individuals. The numbers of different cells in induced sputum were calculated after cells stained with Wright's staining. The expression of PKCα in the cells was detected with immunocytochemistry and positive percentage in different cells were counted and calculated. Interleukin-5 (IL-5) in sputum supernatants was detected with enzyme-linked immunosorbent assay.
Results The percentage of eosinophils (14.3±2.7) %, lymphocytes (7.9±2.6) %, PKCα positive inflammation cells (80.7±9.1) % and the concentration of IL-5 (56.8±10.8) ng/L in asthmatic patients are higher than that percentage of the
eosinophils (1.1±0.4) % , lymphocytes (2.4±0.7)%, PKCα positive inflammation cells (13.6±3.4) % and the concentration of IL-5 (9.7±4.3) ng/L in the controls (P<0.05), and it was significantly lower than that after they were treated with fluticasone propionate or fluticasone propionate and EGb. However, it was still significantly higher than that of the controls. Compared to the group treatment with glucocorticosteroid 2 weeks, there was no significantly decreasing in the percentage of eosinophils, lymphocytes, PKCα positive inflammation cells and the induced sputum supernatant levels of IL-5. Compared to the group treatment with glucocorticosteroid 2 or 4 weeks, There was a significantly decrease in the same items in the group treated with fluticasone propionate and EGb 4 weeks. The levels of IL-5 in supernatant of induced sputum was significantly correlated positive to the percentage of PKCα positive inflammation cells and the percentage of eosinophils in the induced sputum in groups of asthma patients (respectively: n=150, r= 0.83, P<0.01; n=150, r=0.76, P<0.01). The forced expiratory volume in first second are significantly correlated negative to the percentage of PKCα positive inflammation cells and the levels of IL-5 in supernatant of induced sputum in groups of asthma patients (respectively: n=150, i= -0.77, P<0.01; n=150, r= -0.64, P<0.01).
Conclusions EGb could significantly decrease the infiltration of inflammation cells such as eosinophils and lymphocytes in airway of bronchial asthma. It could relieve the airway inflammation in some degree. Part mechanism of that probably is that EGb could decrease the activation of the PKCα in the inflammation cells and then decrease the induced sputum supernatant levels of IL-5. EGb was probably as glucocorticosteroid complement in therapy of asthma, but it took much longer time to display the effect of treatment of asthma than glucocorticosteroid.
引文
1. Cohn L, Elias JA, Chupp GL. Asthma: mechanism of disease persistence and progression. Annu Rev Immunol. 2004; 22: 789-815.
2. Shirai T, Suxuki K, Inui N, et al. Th1/Th2 profile in peripheral blood in atopic cough and atopic asthma. Clin Exp Allergy, 2003, 33: 84-89.
3. Berry N, Nishizuka Y. Protein kinase C and T cell activation. Eur J Biochem. 1990; 189: 205-214.
4 刘先胜，徐永健，张珍祥，等．哮喘患者外周血淋巴细胞蛋白激酶C活性变化的研究．中华结核和呼吸杂志，1999，22：461-465．
5 Badou A, Savignac M, Moreau M, et al. HgC12-induced interleukin-4 gene expression in T cells involves a protein kinase C-dependent calcium influx through L-type calcium channels. J Biol Chem, 1997, 272:32411-32418.
6 McKerma D J, Jones K, Hughes K. Efficacy, safety, and use of Ginkgo biloba in clinical and preclinical applications. Altern Ther Health Med 2001; 7: 88-90
7 Watanabe CM, Wolffram S, Ader P, et al. The in vivo neuromodulatory effects of the herbal medicine Ginkgo biloba. Proc Natl Acad Sci USA. 2001; 98: 6577-6580.
8 Sheffer AL: International consensus report on diagnosis and management of asthma. Clin Exp Allergy. 1992; 22S: 1-72.
9 Stanciu LA, Shute J, Holgate ST, et al. Production of IL-8 and IL-4 by positively and negatively selected CD4+ and CD8+ human T cells following a four-step cell separation method including magnetic cell sorting (MACS). J Immunol Methods. 1996; 189: 107-115.
10 Fikri Y, Nyabenda J, Denis M, et al. Purification and characterization of bovine WC1+ gamma delta T lymphocytes from peripheral blood. Vet Res. 2000; 31: 229-239.
11 Braun DC, Garfield SH, Blumberg PM. Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase C delta in the intact cell. J Biol Chem. 2005; 280: 8164-8171.
12 Aoshiba K, Nagai A. Differences in airway remodeling between asthma and chronic obstructive pulmonary disease. Clin Rev Allergy Immunol. 2004; 27: 35-43.
13 Umetsu DT, Akbari O, Dekruyff RH. Regulatory T cells control the development of allergic disease and asthma. J Allergy Clin Immunol. 2003; 112: 480-487.
14.Packard KA, Khan MM. Effects of histamine on Thl/Th2 cytokine balance. Int Immunopharmacol, 2003; 3:909 -920.
15. Ying S, Durham SR, Corrigan CJ, et al. Phenotype of cells expressing mRNA for Th2-type (interleukin 4 and interleukin 5) and Thl-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell Mol Biol, 1995,12:477-487.
16. Park CS, Lee SM, Chung SW, et al. Interleukin-2 and soluble interleukin-2 receptor in bronchoalveolar lavage fluid from patients with bronchial asthma. Chest,1994, 106:400-406
17. Bansal SK, Jha A, Jaismal AS, et al. Increased levels of proteinkinase C in lymphocytes in asthma: possible mechanism of regulation. Eur Respir J, 1997, 10: 308-313.
18. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995, 9:484-496.
1．李明华，张洪亮等．浓缩银杏叶口服液治疗支气管哮喘临床观察．中国中西医结合杂志，1997；17：216-218．
2．薛建敏，徐永健，张珍祥，等．雷公腾内酯醇对致敏大鼠淋巴细胞凋亡的影响．中华微生物学和免疫学杂志，2001；21：130—133．
3. Shi H, Qin S, Huang G, et al. Infiltration of eosinophils into the asthmatic airways caused by interleukin-5. Am J Respir Cell Mol Biol, 1997, 16(3): 220-224.
4. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. J Allergy Clin Immunol, 1994; 94(6): 1195-1202.
5. Renauld JC. New insights into the role of cytokines in asthma. J Clin Pathol, 2001; 54:577-589.
6．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用．国外医学呼吸分册，1996，154：308-317．
7. Jang AS, Choi IS, Koh YI, et al. Effects of prednisolone on eosinophils,IL-5, eosinophil cationic protein, EG2+ eosinophils, and nitric oxide metabolies in the sputum of patients with exacebated asthma. J Korean Med Sci. 2000; 15(5): 521-528.
8. Jaracz S, Stromgaard K, Nakanishi K. Ginkgolide: selective acetylations, translactonization, and biological evalution. J Org Chem. 2002; 67(13): 4623-4626.
1．中华医学会呼吸病学分会哮喘学组．支气管哮喘防治指南(支气管哮喘的定义、诊断、治疗、疗效判断标准及教育和管理方案)．中华结核和呼吸杂志，1997，20：261-267．
2. Pavord I D, Pizzichini MMM, Pizzichini E, et al. The use of induced sputum to investigate airway inflammation. Thorax, 1997, 52:498-50
3. Fahy JV, Wong H, Liu J, et al. Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects. Am J Respir Crit Care Med, 1995, 152:53-58.
4. Ronchi MC, Piragino C, Rosi E, et al. Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax, 1996, 51:1000-1004.
5. Greenfeder S, Umland SP, Cuss FM, et al. Th2 cytokines and asthma-the role of interleukin-5 in allergic eosinophilic disease. Respir Res, 2001, 2(2): 71-79.
6. Hogan SP, Koskonen A, Matthaei K I, et al. Interleukin 5 producing CD4+ T cells play a pivotal role in aeroallergen induced eosinophilia, bronchial hyperactivity, and lung damage in mice. Am J Respir Critcare Med, 1998, 157:210-218.
7．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用。国外医学呼吸分册，1996，154：308-317．
1. Agrawal DK, Bharadwaj A. Allergic airway inflammation. Curr Allergy Asthma Rep, 2005; 5:142-148.
2. Smith PF, Maclennan K, Darlington CL.The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activation factor (PAF). J Ethnopharmacol, 1996; 50:131-139.
3．董竞成，倪健，宫兆华．中药单体复方防治支气管哮喘的实验研究．中国中西医结合杂志，2004；24：717-722．
4．李明华，张洪亮，杨本英，等．浓缩银杏叶口服液治疗支气管哮喘临床观察．中国中西医结合杂志，1997；17：216-218．
5．中华医学会呼吸病学分会哮喘学组．支气管哮喘防治指南(支气管哮喘的定义、诊断、治疗、疗效判断标准及教育和管理方案)．中华结核和呼吸杂志，2003：26：132-138．
6. Pavord I D, Pizzichini MMM, Pizzichini E, et al. The use of induced sputum to investigate airway inflammation. Thorax, 1997; 52:498-50
7. Louis R, Lau LC, Bron AO, et al. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med, 2000; 161:9-16.
8. Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med, 2000; 161:64-72.
9. Ronchi MC, Piragino C, Rosi E, et al. Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax, 1996; 51:1000-1004.
10. Greenfeder S, Umland SP, Cuss FM, et al. Th2 cytokines and asthma-The role of interleukin-5 in allergic eosinophilic disease. Respir Res, 2001; 2:71-79.
11．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用．国外医学呼吸分册，1996；154：308-317．
12．熊维宁，徐永健，张珍祥，等．核因子KB和蛋白激酶C对哮喘Th2类细胞因子表达的调控．中华结核和呼吸杂志，2001；24：355-359．
13. Jang A S, Chai I S,et al. Effects of Prednisolone on Eosinophils,IL-5, Eosinophil Cationic Protein, EG2+ Eosinophils, and Nitric Oxide Metabolies in the Sputum of Patients with Exacebated Asthma. J Korean Med Sci 2000; 15:521-528.
14. Jaracz S, Stromgaard K, Nakanishi K. Ginkgolide: Selective Acetylations, Translactonization, and Biological Evalution. J Org Chem. 2002; 67: 4623-4626.
1. Stiche, O. Quality of Ginkgo Preparations [J]. Planta Med, 1993, 59(1): 2.
2．孙福红，王慧力，高习文。达浓缩银杏叶口服液治疗支气管哮喘的临床疗效观察[J]。药学实践杂志，1998，16(3)：142-144。
3．李昆，侯杰。银杏叶片对哮喘炎性细胞浸润的影响及疗效观察[J]。中华结核和呼吸杂志，2000，23(3)：171。
4．李明华，刘颖慧，杨本英，等。辅舒酮吸入配合金纳康治疗中度哮喘患者的临床观察[J]。中国中西医结合杂志，2001，21(11)：819-821。
5．董竞成，李明，吴金桐，等。银杏内酯拮抗血小板激活因子对豚鼠肺条作用的实验研究。中国中西医结合杂志。1997；17(8)：481—483。
6．施庆忠，胡文彬，钱伟靖，等。银杏制剂治疗慢性喘息性支气管炎急性发作[J]。中华结核和呼吸杂志，1998，21(10)：630。
7．苏百龄。银杏叶片对肺心病治疗使用的研究[J]。上海医药，2001，32(4)：163-164。
8．邢国宏，孙晓明，周士发，等。银杏叶提取物在治疗慢性肺心病中的抗血小板作用[J]。中国临床药学杂志，2000，9(4)：209-211。
9．李海明，张华茹。银杏叶片治疗慢性阻塞性肺疾病稳定期疗效观察。现代中国中西医结合杂志。2004；13(2)：146—147。
10. Braquet P, Touqui L, Shen TY, et al. Perspectives in platelet—ativating factor research [J]. Pharmacol Rev, 1987, 39(2):97.
11．张煜，黄芸，焦青萍，等。银杏叶提取物预防离体大鼠心肌缺血再灌注损伤。中国临床药学杂志，2000；9(3)：163—165。
12．李麟仙。三七根总皂甙对家兔急缺血的保护作用[J]。中华神经精神病杂志，1987，20(2)：109-110。
13．崔艳英，孔祥军，王淑仙。银杏叶提取物对急性脑缺血的保护作用[J]。中草药，1996；27(1)：22-24。
14. Heiss D, Zeiler K. The influence of drugs on cerebral blood flow [J]. Pharm ako therapie, 1987, 2: 137.
15. Chandrasekaran K, Mehrabian Z, Spinnewyn B, et al. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb761), in gerbil global brain ischemia. Brain Res, 2001, 20; 922(2): 282-292.
16. Krieglstein J, Ausmeier F, E1-Abhar H, et al. Neuroprotective effects of Ginkgo biloba constituents [J]. Eur J Pharma Sci, 1995, 3: 39-48.
17. Petkov VD, Kehayov R, Belcheva S, et al. Memory effects of standardized extracts of Panax ginseng (G115), Ginkgo biloba (GK 501) and their combination Gincosan (PHL-00701). Planta Med. 1993, 59: 106-114.
18．常发伟。银杏叶片治疗慢性精神分裂症临床研究[J]。临床心身疾病杂志，2003，9(2)：117-118。
19．解旭东，冀风云。银杏叶提取物辅助治疗癫痫临床观察[J]。脑与神经疾病杂志，2000，8(3)：172—173。
20．尹浩军，郭志义。银杏叶提取物辅助治疗癫痫33例[J]。医药导报，2001，20(2)：98。
21．罗和春，刘平，赵安全。舒血宁合并阿米替林治疗抑郁症多中心双盲对照研究[J]。中国心理卫生杂志，1999，13(3)：167—169。
22．严光，孙梦雯，刘美云。银杏叶提取物对老年糖耐量减退高胰岛素血症及血糖代谢的影响[J]。安徽中医学院学报，2000，19(4)：14。
23. Loffier T, Lee SK, Noldner M, et al. Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocin-damaged rat brain. J Neural Transm, 2001; 108 (12): 1457-1474.
24．李薇，戴庆棠，刘作恩。复方银杏叶冲剂治疗慢性乙型肝炎早期纤维化的初步观察[J]。中国中西医结合杂志，1995，15(10)：593—595。
25．吴东方，罗顺德，冯小东。银杏叶黄酮对肝脏MDA生成的影响[J]。中国中药杂志，1997，22(1)：51—52。
26．彭巍，向开础，刘斌，等。银杏叶片治疗慢性肾功能衰竭30例临床观察[J]。湖南中医杂志，1998，14(8)：5—6。
27. Janssens D, Remacle J, Drieu K, et al. Protection of mitochondrial respiration activity by bilobalide [J]. Biochem Pharmacol, 1999, 58:109-119.
28. Seif-E1-Nasr M, E1-Fattah AA. Lipid peroxide, phospholipids, glutathione levels and superoxide dismutase activity in rat brain after ischaemia: effect of Ginkgo biloba extract [J]. Pharmacol Res, 1995, 32:273-278.
29. Du ZY, Li XY. Inhibitory effects of ginkgolides on nitric oxide production in neonatal rat microglia in vitro [J]. Acta Pharmacol Sin, 1998, 19 (5): 467-470.
30. Kobuchi H, Droy-Lefaix MT, Christen Y, et al. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7 [J]. Bio chem. Pharmacol, 1997, 53: 897-903.
31. Cheung F, Siow YL, Chen WZ, et al. Inhibitory effect of Ginkgo biloba extract on the expression of inducible nitrc oxide synthase in endothelial cells [J]. Bio chem. Pharmacol, 1999, 58: 1665-1673.
32．周君富。天保宁(银杏叶片)在治疗冠心病中的抗自由基作用[J]。首都医药，1998，5(1)：41。
33．张依宁，张健，黄桂秋，等。银杏叶提取物对溶血卵磷脂胆碱致血管内皮细胞损伤的保护作用[J]。药学学报，1997，32(10)：735—739。
34．吴东方，周本宏，罗顺德，等。银杏叶总黄酮对化学性肝损伤的影响[J]。中草药，1997，28(6)：348—350。
35. Zhu L, Wu J, Liao H, et al. Antagonistic effects of extract from leaves of Ginkgo biloba on glutuanate neurotoxicity [J]. Zhongguo Yao Li Xue Bao, 1997, 18: 344-347.
36. Porsolt RD, Martin P, Lenegre A, et al. Effects of an Extract of Ginkgo Biloba (EGb761) on 'Learned Helplessness' and Other Models of Stress in Rodents [J]. Pharmacol Biochem Behav, 1990, 36: 963-971.
37．吴春福，游松，刘雯，等。银杏内酯和银杏提取物对纹状体和边缘系统多巴胺及其代谢产物含量的影响[J]。中草药，1995，26(5)：253—254。
38. Cheng SM, Yang SP, Ho LJ, et al. Down-regulation of c-jun N-terminal kinase-activator protein-1 signaling pathway by Ginkgo biloba extract in human peripheral blood T cells. Biochem Pharmacol, 2003, 66: 679-689.
1. Joken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol, 1996, 8: 168-173.
2. Hoffman J. The potential for isoemzym-selective modulation of protein kinase C. FASEB J, 1997,11:649-669.
3. Monick MM, Carter AB, Gudmundsson G, et al. Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes. Am J Physiol, 1998; 275: 389-397.
4. Szamel M, Resch K. T-cell antigen receptor-induced signal-transduction pathways— activation and function of protein kinases C in T lymphocytes. Eur J Biochem, 1995; 228: 1-15.
5. Mann J, Farrukh IS, Michael. Mechanisms by which endothelin 1 induces pulmonary vasoconstriction in the rabbit. J Appl Physiol, 1991; 71: 410-416
6. Zulueta JJ, Yu FS, Hertig IA, et al. Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. Am J Respir Cell Mol Bio, 1995; 12: 41-49
7. Weissmann N, Voswinckel R, Hardebusch T, et al. Evidence for a role of protein kinase C in hypoxic pulmonary vasoconstriction. Am J Physiol, 1999; 276; L90-L95
8. Fleming I, MacKenzie SJ, Vernon RG, et al. Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation. Biochem J, 1998; 333: 712-727
9. Frey MR, Saxon ML, Zhao X, et al. Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21 (waf1/cip1) and p27 (kip 1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J Biol chem, 1997, 272: 9424-9435
10. Jaken S, Leach K, Klauck T, et al. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol, 1989, 109: 697-704
11. Throckmorton DC, Packer CS, Brophy CM, et al. Protein kinase C activation during Ca~(2+)-independent vascular smooth muscle contraction. J Surg Res, 1998; 78:48-53.
12. Church M K, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol, 1997,99: 155-160.
13.Fecktistov I, Biaggioni I. Adenosine A_2b receptors evoke interleukin-8 secretionin human mast cells, An enprofylline-sensitive mechanism with implications for asthma. J Clin Invest, 1995, 96:1979-1986.
14. Hide I, Toriu N, Nuike T, et al. Supression of TNF-α secretion by azelastine in a rat mast (RBL-2H3) cell line. J Immunol, 1997,159:2932-2940.
15.Bahattacharyya SP, Drucker I, Roshef T, et al. Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact. J Leukocyte Biol, 1998, 63: 337-341.
16. Chang EY, Szallasi Z, Acs P, et al. Functional effects of over expression of protein kinase C -α, -β, -δ, -ε and -η in the mast cell line RBL-2H_3. J Immunol, 1997,159:2624-2632.
17. Vasquez NJ, Pettiford SM, Maffield SA, et al. Identification of a small molecule that selectively inhibits IL-4, but not gamma interfereon production in human T cell lines. J Allergy Clin Immunol, 1997, 99 Suppl:300.
18.Snyder JT, Finn OJ. The role of protein kinase C - beta in T-lymphocyte activation and tolerance induction. J Allergy Clin Immunol, 1997, 99 Suppl: 307.
19. Monks CRF, Kupfer H, Tamie I, et al. Selective modulation of protein kinase C-H during T cell activation. Nature, 1997, 385: 83-86.
20. Liu D, Sy MS. Phorbol myristate acetate stimulates the dimerization of CD44 involving acysteine in the transmembrane domain. J Immunol, 1997, 159: 2702-2711.
21.Kucik DF, Dustin ML, Miller JM, et al. Adhesion activating phorbol ester increase the mobility of leukocyte intergin LFA-1 in cultured lymphocytes. J Clin Invest, 1996, 97: 2139-2144.
22. Bansal SK, Jha A, Jaismal AS, et al. Increased levels of protein kinase C in lymphocytes in asthma: possible mechanism of regulation. Eur Respir J, 1997, 10: 308-313.
23.Kroegel C, Matthys H. Platelet-activating factor-induced human eosinophil activation, Generation and release of cyclo-ocygenase metabolites in human blood eosinophils from asthmatic. Immunol, 1993, 78: 279-285.
24. Sakakibara H, Takamet SU, Said SL. Eosinophil-mediated injury of culture bronchial epithelial cells: attenuation by vasoactive intestinal peptide (VIP). Am J Rev Respir Dis, 1991,143:44.
25. Bates ME, Bertics PJ, Calhoun WJ, et al. Increased protein kinase C activity in low density eosinophils. J Immunol, 1993,150: 4486-4493.
26. Frangova V, Sacco O, Silvestri M, et al. BA neutrophilia in asthmatic patients. Chest 1996, 110:1236-1242.
27. Ogino T, Kobuchi H, Sen CK, et al Monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and T cell interleukin-2 receptor expression by inhibiting inducible protein kinase C activity. J Biol Chem, 1997,272: 26247-26252.
28. Sergeant S, McPhailL C. Opsonized zymosa stimulates the redistribution of protein kinase isoforms in human neutrophils. J Immunol 1997,159: 2877- 2885.
29. Powell WS, Gravel S, Hawani F, et al. Effect of 5-oxo-6, 8, 11, 14- eicosatet-raenoic acid on expression of CD_(11)b, actin polymerization, and adherence in human neutropils. J Immunol. 1997, 159: 2952-2959.
30. Vachier I, Chanz P, Radeau T, et al. Cellular protein kinase C activity in asthma. Am Respir Crit Care Med, 1997, 155: 1211-1216.
31. Majori M, Vachier I, Godard P, et al. Superoxide abion production by monocytes corticosteroid - treated asthmatic patients. Eu Respir J, 1998,11: 133-138.
32. Amon U, Detz KR. Protein kinase C: a target of anti-inflammatory therapy. Arch Dermat Res, 1992,284: 8-10.
33.Guo CB, Liu MC, Galis SJ, et al. Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils. Am J Respir Cell Mol Biol, 1994,10: 384-390.
34. Schroeder JT, Mac Glasham DW. New concepts: the basophil. J Allergy Clin Immunol, 1997, 99: 429- 433.
35. Rossetti M, Savineau JP, Crevel H et al. Role of protein kinase C in nonsensitized and passively sensitized human isolated bronchial smooth muscle. Am J Physio 1, 1995,268 :L996-971.
36.Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995; 9: 484-496.
37. Yang KX, Black JL. The involvement of protein kinase C in the contraction of human airway smooth muscle. Eur J Pharmacol, 1995, 275: 283-289.
38. Barnes PJ, Haddad EB, Rousell J et al. Regulation of muscarinic M2 receptors. Life Sci, 1997,60:1015-1021.
39. Zaagsma J , Roffel AF, Meurs H, et al. Muscarinic control of airway function. Life Sci, 1997,60:1061-1068.
40. Knox AJ. Airway re-modelling in asthma: role of airway smooth muscle. Clin Sci, 1994,86:647-652.
41. Black JL. Role of airway smooth muscle. Am J Respir Crit Care Med, 1996, 153: S2- S4.
42. Stewart AG, Tomlison PR, Wilson J, et al. Airway wall remodeling in asthma: a novel target for the development of anti-asthma drugs. Trends Pharmacol Sci, 1993,14:275-279.
2. Shirai T, Suxuki K, Inui N, et al. Th1/Th2 profile in peripheral blood in atopic cough and atopic asthma. Clin Exp Allergy, 2003, 33: 84-89.
3. Berry N, Nishizuka Y. Protein kinase C and T cell activation. Eur J Biochem. 1990; 189: 205-214.
4 刘先胜，徐永健，张珍祥，等．哮喘患者外周血淋巴细胞蛋白激酶C活性变化的研究．中华结核和呼吸杂志，1999，22：461-465．
5 Badou A, Savignac M, Moreau M, et al. HgC12-induced interleukin-4 gene expression in T cells involves a protein kinase C-dependent calcium influx through L-type calcium channels. J Biol Chem, 1997, 272:32411-32418.
6 McKerma D J, Jones K, Hughes K. Efficacy, safety, and use of Ginkgo biloba in clinical and preclinical applications. Altern Ther Health Med 2001; 7: 88-90
7 Watanabe CM, Wolffram S, Ader P, et al. The in vivo neuromodulatory effects of the herbal medicine Ginkgo biloba. Proc Natl Acad Sci USA. 2001; 98: 6577-6580.
8 Sheffer AL: International consensus report on diagnosis and management of asthma. Clin Exp Allergy. 1992; 22S: 1-72.
9 Stanciu LA, Shute J, Holgate ST, et al. Production of IL-8 and IL-4 by positively and negatively selected CD4+ and CD8+ human T cells following a four-step cell separation method including magnetic cell sorting (MACS). J Immunol Methods. 1996; 189: 107-115.
10 Fikri Y, Nyabenda J, Denis M, et al. Purification and characterization of bovine WC1+ gamma delta T lymphocytes from peripheral blood. Vet Res. 2000; 31: 229-239.
11 Braun DC, Garfield SH, Blumberg PM. Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase C delta in the intact cell. J Biol Chem. 2005; 280: 8164-8171.
12 Aoshiba K, Nagai A. Differences in airway remodeling between asthma and chronic obstructive pulmonary disease. Clin Rev Allergy Immunol. 2004; 27: 35-43.
13 Umetsu DT, Akbari O, Dekruyff RH. Regulatory T cells control the development of allergic disease and asthma. J Allergy Clin Immunol. 2003; 112: 480-487.
14.Packard KA, Khan MM. Effects of histamine on Thl/Th2 cytokine balance. Int Immunopharmacol, 2003; 3:909 -920.
15. Ying S, Durham SR, Corrigan CJ, et al. Phenotype of cells expressing mRNA for Th2-type (interleukin 4 and interleukin 5) and Thl-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell Mol Biol, 1995,12:477-487.
16. Park CS, Lee SM, Chung SW, et al. Interleukin-2 and soluble interleukin-2 receptor in bronchoalveolar lavage fluid from patients with bronchial asthma. Chest,1994, 106:400-406
17. Bansal SK, Jha A, Jaismal AS, et al. Increased levels of proteinkinase C in lymphocytes in asthma: possible mechanism of regulation. Eur Respir J, 1997, 10: 308-313.
18. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995, 9:484-496.
1．李明华，张洪亮等．浓缩银杏叶口服液治疗支气管哮喘临床观察．中国中西医结合杂志，1997；17：216-218．
2．薛建敏，徐永健，张珍祥，等．雷公腾内酯醇对致敏大鼠淋巴细胞凋亡的影响．中华微生物学和免疫学杂志，2001；21：130—133．
3. Shi H, Qin S, Huang G, et al. Infiltration of eosinophils into the asthmatic airways caused by interleukin-5. Am J Respir Cell Mol Biol, 1997, 16(3): 220-224.
4. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. J Allergy Clin Immunol, 1994; 94(6): 1195-1202.
5. Renauld JC. New insights into the role of cytokines in asthma. J Clin Pathol, 2001; 54:577-589.
6．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用．国外医学呼吸分册，1996，154：308-317．
7. Jang AS, Choi IS, Koh YI, et al. Effects of prednisolone on eosinophils,IL-5, eosinophil cationic protein, EG2+ eosinophils, and nitric oxide metabolies in the sputum of patients with exacebated asthma. J Korean Med Sci. 2000; 15(5): 521-528.
8. Jaracz S, Stromgaard K, Nakanishi K. Ginkgolide: selective acetylations, translactonization, and biological evalution. J Org Chem. 2002; 67(13): 4623-4626.
1．中华医学会呼吸病学分会哮喘学组．支气管哮喘防治指南(支气管哮喘的定义、诊断、治疗、疗效判断标准及教育和管理方案)．中华结核和呼吸杂志，1997，20：261-267．
2. Pavord I D, Pizzichini MMM, Pizzichini E, et al. The use of induced sputum to investigate airway inflammation. Thorax, 1997, 52:498-50
3. Fahy JV, Wong H, Liu J, et al. Comparison of samples collected by sputum induction and bronchoscopy from asthmatic and healthy subjects. Am J Respir Crit Care Med, 1995, 152:53-58.
4. Ronchi MC, Piragino C, Rosi E, et al. Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax, 1996, 51:1000-1004.
5. Greenfeder S, Umland SP, Cuss FM, et al. Th2 cytokines and asthma-the role of interleukin-5 in allergic eosinophilic disease. Respir Res, 2001, 2(2): 71-79.
6. Hogan SP, Koskonen A, Matthaei K I, et al. Interleukin 5 producing CD4+ T cells play a pivotal role in aeroallergen induced eosinophilia, bronchial hyperactivity, and lung damage in mice. Am J Respir Critcare Med, 1998, 157:210-218.
7．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用。国外医学呼吸分册，1996，154：308-317．
1. Agrawal DK, Bharadwaj A. Allergic airway inflammation. Curr Allergy Asthma Rep, 2005; 5:142-148.
2. Smith PF, Maclennan K, Darlington CL.The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activation factor (PAF). J Ethnopharmacol, 1996; 50:131-139.
3．董竞成，倪健，宫兆华．中药单体复方防治支气管哮喘的实验研究．中国中西医结合杂志，2004；24：717-722．
4．李明华，张洪亮，杨本英，等．浓缩银杏叶口服液治疗支气管哮喘临床观察．中国中西医结合杂志，1997；17：216-218．
5．中华医学会呼吸病学分会哮喘学组．支气管哮喘防治指南(支气管哮喘的定义、诊断、治疗、疗效判断标准及教育和管理方案)．中华结核和呼吸杂志，2003：26：132-138．
6. Pavord I D, Pizzichini MMM, Pizzichini E, et al. The use of induced sputum to investigate airway inflammation. Thorax, 1997; 52:498-50
7. Louis R, Lau LC, Bron AO, et al. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med, 2000; 161:9-16.
8. Jatakanon A, Lim S, Barnes PJ. Changes in sputum eosinophils predict loss of asthma control. Am J Respir Crit Care Med, 2000; 161:64-72.
9. Ronchi MC, Piragino C, Rosi E, et al. Role of sputum differential cell count in detecting airway inflammation in patients with chronic bronchial asthma or COPD. Thorax, 1996; 51:1000-1004.
10. Greenfeder S, Umland SP, Cuss FM, et al. Th2 cytokines and asthma-The role of interleukin-5 in allergic eosinophilic disease. Respir Res, 2001; 2:71-79.
11．徐永健，张珍祥．蛋白激酶C信息通道在支气管哮喘发病机制中的作用．国外医学呼吸分册，1996；154：308-317．
12．熊维宁，徐永健，张珍祥，等．核因子KB和蛋白激酶C对哮喘Th2类细胞因子表达的调控．中华结核和呼吸杂志，2001；24：355-359．
13. Jang A S, Chai I S,et al. Effects of Prednisolone on Eosinophils,IL-5, Eosinophil Cationic Protein, EG2+ Eosinophils, and Nitric Oxide Metabolies in the Sputum of Patients with Exacebated Asthma. J Korean Med Sci 2000; 15:521-528.
14. Jaracz S, Stromgaard K, Nakanishi K. Ginkgolide: Selective Acetylations, Translactonization, and Biological Evalution. J Org Chem. 2002; 67: 4623-4626.
1. Stiche, O. Quality of Ginkgo Preparations [J]. Planta Med, 1993, 59(1): 2.
2．孙福红，王慧力，高习文。达浓缩银杏叶口服液治疗支气管哮喘的临床疗效观察[J]。药学实践杂志，1998，16(3)：142-144。
3．李昆，侯杰。银杏叶片对哮喘炎性细胞浸润的影响及疗效观察[J]。中华结核和呼吸杂志，2000，23(3)：171。
4．李明华，刘颖慧，杨本英，等。辅舒酮吸入配合金纳康治疗中度哮喘患者的临床观察[J]。中国中西医结合杂志，2001，21(11)：819-821。
5．董竞成，李明，吴金桐，等。银杏内酯拮抗血小板激活因子对豚鼠肺条作用的实验研究。中国中西医结合杂志。1997；17(8)：481—483。
6．施庆忠，胡文彬，钱伟靖，等。银杏制剂治疗慢性喘息性支气管炎急性发作[J]。中华结核和呼吸杂志，1998，21(10)：630。
7．苏百龄。银杏叶片对肺心病治疗使用的研究[J]。上海医药，2001，32(4)：163-164。
8．邢国宏，孙晓明，周士发，等。银杏叶提取物在治疗慢性肺心病中的抗血小板作用[J]。中国临床药学杂志，2000，9(4)：209-211。
9．李海明，张华茹。银杏叶片治疗慢性阻塞性肺疾病稳定期疗效观察。现代中国中西医结合杂志。2004；13(2)：146—147。
10. Braquet P, Touqui L, Shen TY, et al. Perspectives in platelet—ativating factor research [J]. Pharmacol Rev, 1987, 39(2):97.
11．张煜，黄芸，焦青萍，等。银杏叶提取物预防离体大鼠心肌缺血再灌注损伤。中国临床药学杂志，2000；9(3)：163—165。
12．李麟仙。三七根总皂甙对家兔急缺血的保护作用[J]。中华神经精神病杂志，1987，20(2)：109-110。
13．崔艳英，孔祥军，王淑仙。银杏叶提取物对急性脑缺血的保护作用[J]。中草药，1996；27(1)：22-24。
14. Heiss D, Zeiler K. The influence of drugs on cerebral blood flow [J]. Pharm ako therapie, 1987, 2: 137.
15. Chandrasekaran K, Mehrabian Z, Spinnewyn B, et al. Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb761), in gerbil global brain ischemia. Brain Res, 2001, 20; 922(2): 282-292.
16. Krieglstein J, Ausmeier F, E1-Abhar H, et al. Neuroprotective effects of Ginkgo biloba constituents [J]. Eur J Pharma Sci, 1995, 3: 39-48.
17. Petkov VD, Kehayov R, Belcheva S, et al. Memory effects of standardized extracts of Panax ginseng (G115), Ginkgo biloba (GK 501) and their combination Gincosan (PHL-00701). Planta Med. 1993, 59: 106-114.
18．常发伟。银杏叶片治疗慢性精神分裂症临床研究[J]。临床心身疾病杂志，2003，9(2)：117-118。
19．解旭东，冀风云。银杏叶提取物辅助治疗癫痫临床观察[J]。脑与神经疾病杂志，2000，8(3)：172—173。
20．尹浩军，郭志义。银杏叶提取物辅助治疗癫痫33例[J]。医药导报，2001，20(2)：98。
21．罗和春，刘平，赵安全。舒血宁合并阿米替林治疗抑郁症多中心双盲对照研究[J]。中国心理卫生杂志，1999，13(3)：167—169。
22．严光，孙梦雯，刘美云。银杏叶提取物对老年糖耐量减退高胰岛素血症及血糖代谢的影响[J]。安徽中医学院学报，2000，19(4)：14。
23. Loffier T, Lee SK, Noldner M, et al. Effect of Ginkgo biloba extract (EGb761) on glucose metabolism-related markers in streptozotocin-damaged rat brain. J Neural Transm, 2001; 108 (12): 1457-1474.
24．李薇，戴庆棠，刘作恩。复方银杏叶冲剂治疗慢性乙型肝炎早期纤维化的初步观察[J]。中国中西医结合杂志，1995，15(10)：593—595。
25．吴东方，罗顺德，冯小东。银杏叶黄酮对肝脏MDA生成的影响[J]。中国中药杂志，1997，22(1)：51—52。
26．彭巍，向开础，刘斌，等。银杏叶片治疗慢性肾功能衰竭30例临床观察[J]。湖南中医杂志，1998，14(8)：5—6。
27. Janssens D, Remacle J, Drieu K, et al. Protection of mitochondrial respiration activity by bilobalide [J]. Biochem Pharmacol, 1999, 58:109-119.
28. Seif-E1-Nasr M, E1-Fattah AA. Lipid peroxide, phospholipids, glutathione levels and superoxide dismutase activity in rat brain after ischaemia: effect of Ginkgo biloba extract [J]. Pharmacol Res, 1995, 32:273-278.
29. Du ZY, Li XY. Inhibitory effects of ginkgolides on nitric oxide production in neonatal rat microglia in vitro [J]. Acta Pharmacol Sin, 1998, 19 (5): 467-470.
30. Kobuchi H, Droy-Lefaix MT, Christen Y, et al. Ginkgo biloba extract (EGb761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7 [J]. Bio chem. Pharmacol, 1997, 53: 897-903.
31. Cheung F, Siow YL, Chen WZ, et al. Inhibitory effect of Ginkgo biloba extract on the expression of inducible nitrc oxide synthase in endothelial cells [J]. Bio chem. Pharmacol, 1999, 58: 1665-1673.
32．周君富。天保宁(银杏叶片)在治疗冠心病中的抗自由基作用[J]。首都医药，1998，5(1)：41。
33．张依宁，张健，黄桂秋，等。银杏叶提取物对溶血卵磷脂胆碱致血管内皮细胞损伤的保护作用[J]。药学学报，1997，32(10)：735—739。
34．吴东方，周本宏，罗顺德，等。银杏叶总黄酮对化学性肝损伤的影响[J]。中草药，1997，28(6)：348—350。
35. Zhu L, Wu J, Liao H, et al. Antagonistic effects of extract from leaves of Ginkgo biloba on glutuanate neurotoxicity [J]. Zhongguo Yao Li Xue Bao, 1997, 18: 344-347.
36. Porsolt RD, Martin P, Lenegre A, et al. Effects of an Extract of Ginkgo Biloba (EGb761) on 'Learned Helplessness' and Other Models of Stress in Rodents [J]. Pharmacol Biochem Behav, 1990, 36: 963-971.
37．吴春福，游松，刘雯，等。银杏内酯和银杏提取物对纹状体和边缘系统多巴胺及其代谢产物含量的影响[J]。中草药，1995，26(5)：253—254。
38. Cheng SM, Yang SP, Ho LJ, et al. Down-regulation of c-jun N-terminal kinase-activator protein-1 signaling pathway by Ginkgo biloba extract in human peripheral blood T cells. Biochem Pharmacol, 2003, 66: 679-689.
1. Joken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol, 1996, 8: 168-173.
2. Hoffman J. The potential for isoemzym-selective modulation of protein kinase C. FASEB J, 1997,11:649-669.
3. Monick MM, Carter AB, Gudmundsson G, et al. Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes. Am J Physiol, 1998; 275: 389-397.
4. Szamel M, Resch K. T-cell antigen receptor-induced signal-transduction pathways— activation and function of protein kinases C in T lymphocytes. Eur J Biochem, 1995; 228: 1-15.
5. Mann J, Farrukh IS, Michael. Mechanisms by which endothelin 1 induces pulmonary vasoconstriction in the rabbit. J Appl Physiol, 1991; 71: 410-416
6. Zulueta JJ, Yu FS, Hertig IA, et al. Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane. Am J Respir Cell Mol Bio, 1995; 12: 41-49
7. Weissmann N, Voswinckel R, Hardebusch T, et al. Evidence for a role of protein kinase C in hypoxic pulmonary vasoconstriction. Am J Physiol, 1999; 276; L90-L95
8. Fleming I, MacKenzie SJ, Vernon RG, et al. Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation. Biochem J, 1998; 333: 712-727
9. Frey MR, Saxon ML, Zhao X, et al. Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21 (waf1/cip1) and p27 (kip 1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J Biol chem, 1997, 272: 9424-9435
10. Jaken S, Leach K, Klauck T, et al. Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol, 1989, 109: 697-704
11. Throckmorton DC, Packer CS, Brophy CM, et al. Protein kinase C activation during Ca~(2+)-independent vascular smooth muscle contraction. J Surg Res, 1998; 78:48-53.
12. Church M K, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol, 1997,99: 155-160.
13.Fecktistov I, Biaggioni I. Adenosine A_2b receptors evoke interleukin-8 secretionin human mast cells, An enprofylline-sensitive mechanism with implications for asthma. J Clin Invest, 1995, 96:1979-1986.
14. Hide I, Toriu N, Nuike T, et al. Supression of TNF-α secretion by azelastine in a rat mast (RBL-2H3) cell line. J Immunol, 1997,159:2932-2940.
15.Bahattacharyya SP, Drucker I, Roshef T, et al. Activated T lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact. J Leukocyte Biol, 1998, 63: 337-341.
16. Chang EY, Szallasi Z, Acs P, et al. Functional effects of over expression of protein kinase C -α, -β, -δ, -ε and -η in the mast cell line RBL-2H_3. J Immunol, 1997,159:2624-2632.
17. Vasquez NJ, Pettiford SM, Maffield SA, et al. Identification of a small molecule that selectively inhibits IL-4, but not gamma interfereon production in human T cell lines. J Allergy Clin Immunol, 1997, 99 Suppl:300.
18.Snyder JT, Finn OJ. The role of protein kinase C - beta in T-lymphocyte activation and tolerance induction. J Allergy Clin Immunol, 1997, 99 Suppl: 307.
19. Monks CRF, Kupfer H, Tamie I, et al. Selective modulation of protein kinase C-H during T cell activation. Nature, 1997, 385: 83-86.
20. Liu D, Sy MS. Phorbol myristate acetate stimulates the dimerization of CD44 involving acysteine in the transmembrane domain. J Immunol, 1997, 159: 2702-2711.
21.Kucik DF, Dustin ML, Miller JM, et al. Adhesion activating phorbol ester increase the mobility of leukocyte intergin LFA-1 in cultured lymphocytes. J Clin Invest, 1996, 97: 2139-2144.
22. Bansal SK, Jha A, Jaismal AS, et al. Increased levels of protein kinase C in lymphocytes in asthma: possible mechanism of regulation. Eur Respir J, 1997, 10: 308-313.
23.Kroegel C, Matthys H. Platelet-activating factor-induced human eosinophil activation, Generation and release of cyclo-ocygenase metabolites in human blood eosinophils from asthmatic. Immunol, 1993, 78: 279-285.
24. Sakakibara H, Takamet SU, Said SL. Eosinophil-mediated injury of culture bronchial epithelial cells: attenuation by vasoactive intestinal peptide (VIP). Am J Rev Respir Dis, 1991,143:44.
25. Bates ME, Bertics PJ, Calhoun WJ, et al. Increased protein kinase C activity in low density eosinophils. J Immunol, 1993,150: 4486-4493.
26. Frangova V, Sacco O, Silvestri M, et al. BA neutrophilia in asthmatic patients. Chest 1996, 110:1236-1242.
27. Ogino T, Kobuchi H, Sen CK, et al Monochloramine inhibits phorbol ester-inducible neutrophil respiratory burst activation and T cell interleukin-2 receptor expression by inhibiting inducible protein kinase C activity. J Biol Chem, 1997,272: 26247-26252.
28. Sergeant S, McPhailL C. Opsonized zymosa stimulates the redistribution of protein kinase isoforms in human neutrophils. J Immunol 1997,159: 2877- 2885.
29. Powell WS, Gravel S, Hawani F, et al. Effect of 5-oxo-6, 8, 11, 14- eicosatet-raenoic acid on expression of CD_(11)b, actin polymerization, and adherence in human neutropils. J Immunol. 1997, 159: 2952-2959.
30. Vachier I, Chanz P, Radeau T, et al. Cellular protein kinase C activity in asthma. Am Respir Crit Care Med, 1997, 155: 1211-1216.
31. Majori M, Vachier I, Godard P, et al. Superoxide abion production by monocytes corticosteroid - treated asthmatic patients. Eu Respir J, 1998,11: 133-138.
32. Amon U, Detz KR. Protein kinase C: a target of anti-inflammatory therapy. Arch Dermat Res, 1992,284: 8-10.
33.Guo CB, Liu MC, Galis SJ, et al. Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils. Am J Respir Cell Mol Biol, 1994,10: 384-390.
34. Schroeder JT, Mac Glasham DW. New concepts: the basophil. J Allergy Clin Immunol, 1997, 99: 429- 433.
35. Rossetti M, Savineau JP, Crevel H et al. Role of protein kinase C in nonsensitized and passively sensitized human isolated bronchial smooth muscle. Am J Physio 1, 1995,268 :L996-971.
36.Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J, 1995; 9: 484-496.
37. Yang KX, Black JL. The involvement of protein kinase C in the contraction of human airway smooth muscle. Eur J Pharmacol, 1995, 275: 283-289.
38. Barnes PJ, Haddad EB, Rousell J et al. Regulation of muscarinic M2 receptors. Life Sci, 1997,60:1015-1021.
39. Zaagsma J , Roffel AF, Meurs H, et al. Muscarinic control of airway function. Life Sci, 1997,60:1061-1068.
40. Knox AJ. Airway re-modelling in asthma: role of airway smooth muscle. Clin Sci, 1994,86:647-652.
41. Black JL. Role of airway smooth muscle. Am J Respir Crit Care Med, 1996, 153: S2- S4.
42. Stewart AG, Tomlison PR, Wilson J, et al. Airway wall remodeling in asthma: a novel target for the development of anti-asthma drugs. Trends Pharmacol Sci, 1993,14:275-279.