【作者】 张韶辉；

【导师】 王嘉陵；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 第一部分二苯乙烯苷对阿霉素心脏毒性的体内外保护作用及机制阿霉素（DOX）是一种高效广谱的葸环类抗肿瘤抗生素，心脏毒性是阿霉素临床应用的主要不良反应之一，因出现剂量依赖性的严重心力衰竭而使它的应用受到限制。DOX所致心脏毒性的机制至今仍未完全阐明，但有许多证据表明其与产生的大量自由基密切相关。2，3，5，4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷（2，3，5，4’-tetrahydroxystilbene-2-O-β-D-glucoside，THSG）是何首乌的活性单体，体外实验研究表明THSG具有较强的抗氧化活性。已有的研究也证实THSG能够降低阿尔茨海默病（Alzheimer’s Disease）模型小鼠和缺血再灌注损伤模型小鼠组织中的过氧化物水平。THSG的化学结构与白藜芦醇相似，后者具有显著的保护受损心肌的作用。所以我们推测THSG对DOX导致的心脏毒性具有保护作用。论文将分别以体外培养的乳鼠心肌细胞和DOX诱导的小鼠急性心脏毒性模型为对象探讨THSG的保护作用及机制。§1 THSG对小鼠急性阿霉素心脏毒性的保护作用及机制1．THSG对急性阿霉素心脏毒性的保护作用方法：一次性腹腔注射DOX 15mg／kg诱导急性心脏毒性模型，给予THSG 0．1g／kg灌胃一天1次，连续7天。观察THSG对小鼠阿霉素心脏毒性的保护作用。结果：①体重和生存率：处理前各组小鼠体重无明显差别。处理第7天，DOX组小鼠体重（21．8±3．6 g）和空白对照组（25．6±2．6 g，P＜0．01）比较明显减轻；给予THSG（0．1g／kg·d）治疗后没有增加造模小鼠的体重（22．5±2．8 g，P＞0．05 compared with DOXalone）。处理7天后，3组小鼠心脏指数（心脏重量与体重的比值）分别为空白对照组：4．5±0．6mg/g；DOX组：4．3±0．7mg/g；THSG组：4．3±0．4 mg/g，统计学分析均无明显差异。对照组20只动物全部存活，DOX处理7天后动物存活70％，给予THSG治疗后动物存活率升高为85％（P＞0．05，log rank test）。②组织学观察：正常对照组小鼠心肌纤维完整，排列整齐，无出血和水肿。DOX模型组可见明显的心肌纤维断裂甚至消失，心肌间质水肿并伴有局灶性心内膜下出血。而THSG治疗组和DOX模型组相比心肌纤维受损程度明显降低。2．THSG对急性阿霉素心脏毒性小鼠血清LDH和CK水平的影响方法：生物化学法测定血清LDH和CK的水平结果：DOX处理后第七天，血清中LDH水平从空白对照组3．87±0．40 U／ml上升到6．00±0．27 U／ml （P＜0．01）。经过THSG（0．1 g／kg·d）治疗后，LDH水平显著降低，为4．86±0．20U／ml（P＜0．05）。血清中CK水平从正常组1．55±0．49 U／ml上升到模型组3．02±0．28U／ml （P＜0．01）。经过THSG（0．1g／kg·d）治疗后CK水平降至1．94±0．4U／ml，与DOX单独处理组比较差异有显著性（P＜0．05）。3．THSG对急性阿霉素心脏毒性小鼠心肌组织中MDA和GSH含量的影响方法：生物化学方法测定心肌组织中MDA和GSH的含量结果：DOX处理后第七天，MDA含量从空白对照组1．15±0．30 nmol／mg protein上升到4．17±0．91 nmol／mg protein（P＜0．01）。THSG（0．1g／kg·d）可以显著抑制MDA的升高，其数值为2．32±0．76 nmol／mg protein（P＜0．01）。DOX处理后第七天，GSH含量从正常组24．43±6．57mg/g protein下降到模型组13．65±3．46 mg/g protein（P＜0．01）。THSG（0．1g／kg·d）可以升高GSH含量，为18．31±3．27 mg/g protein，与DOX组比较差异有显著性（P＜0．01）。§2 THSG对阿霉素损伤心肌细胞的保护作用及机制1．THSG对阿霉素引起的心肌细胞存活减少的影响方法：MTT法分析心肌细胞存活率。结果：空白对照组细胞存活率为100％，THSG300μM作用24h后细胞存活率为101．50±5．84％，与空白对照组比较差异无统计学意义。加入DOX 1μM作用24小时后，细胞存活率下降为72．68±8．52％（P＜0．01）。THSG 3，10，30，100，300μM可剂量依赖性削弱DOX导致的细胞存活率下降，其值分别为76．09±10．24％，81．37±9．84％，83A3±11．47％，91．21±7．54％，95．24±10．87％。除3μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性（P＜0．05，P＜0．01）。2．THSG对阿霉素引起的细胞凋亡的影响方法：TUNEL法检测细胞凋亡。结果：空白对照组凋亡阳性细胞百分率为4．57±+0．52，给予DOX 1μM作用24小时后，细胞凋亡率明显增加为32．55±3．47％（P＜0．01）。THSG10，30，100，300μM可剂量依赖性减少DOX导致的细胞凋亡，其凋亡阳性细胞百分率分别为26．53±3．27％，20．12±2．64％，13．34±8．64％，10．48±1．57％，与DOX单独处理组比较，差异均有统计学意义（P＜0．05，P＜0．01）。3．THSG对阿霉素导致的心肌细胞线粒体膜电位降低的影响方法：采用荧光标记法检测线粒体膜电位。结果：正常对照组细胞伸展良好并呈现出红色或橙色荧光，定量分析显示其红绿荧光比例为14．52±3．86％。给予DOX 1μM作用24小时后，心肌细胞多数固缩为圆形并显示绿色荧光，红绿荧光比例为0．61±0．16％（P＜0．01，compared with control），提示细胞线粒体膜电位降低。THSG10，30，100，300μM可剂量依赖性减轻DOX导致的心肌细胞线粒体膜电位的降低，其荧光比例分别为1．48±0．34％，2．32±0．46％，3．70±0．87％，5．35±1．25％，除10μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性（P＜0．01）。4．THSG对阿霉素引起的活性氧自由基增多的影响方法：采用荧光标记法检测氧自由基。结果：细胞内氧自由基经标记后呈现绿色荧光。定量分析数据用各组细胞绿色荧光面积和正常对照组细胞荧光面积的百分率来表示。正常对照组为100％，给予DOX 1μM作用24小时后荧光比值为337．56±45．56％（P＜0．01，compared with control），提示阿霉素导致心肌细胞氧自由基增多。THSG 30，100，300μM可剂量依赖性减轻DOX导致的氧自由基增多，其荧光比例分别为265，65±23，46％，227．70±2657％，184．17±34．52％，与DOX单独处理组比较，差异均有统计学意义（P＜0．05，P＜0．01）。5．THSG对阿霉素引起的凋亡相关蛋白表达改变的影响方法：用Western blot检测caspase-3，Bcl-2和Bax蛋白表达。结果：①DOX 1μM诱导24h后，caspase-3活化较正常对照组明显增加，且17kDa条带灰度明显深于正常对照组。caspase-3（17kDa）灰度值与内参β-actin灰度值的比值从正常对照组0．08±0．01上升到1．31±0．27，并有显著性差异（P＜0．01）。THSG30，100，300μM可以剂量依赖性抑制DOX导致的caspase-3蛋白的激活，其17kDa条带灰度值与内参β-actin灰度值的比值分别为0．80±0．14、0．42±0．06、0．12±0．13，与DOX单独处理组比较差异均有显著性（P＜0．05，P＜0．01）。②DOX 1μM诱导24h后，Bcl-2蛋白表达量较正常对照组明显增加，Bcl-2蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．42±0．04上升到0．88±0．09，并有显著性差异（P＜0．05）。THSG30，100，300μM可以剂量依赖性地使DOX导致的Bcl-2蛋白表达量的增高更加显著，其灰度比值分别为0．99±0．10，1．37±0．08，1．64±0．12，除30μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性（P＜0．05，P＜0．01）。③DOX 1μM诱导24h后，Bax蛋白表达量较正常对照组明显增加，Bax蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．64±0．07上升到1．09±0．12，差异有显著性意义（P＜0．05）。THSG 30，100，300μM可以剂量依赖性抑制DOX引起的Bax蛋白表达量增加，其灰度比值分别为0．95±0．08，0．75±0．06，0．66±0．05，除30μM THSG组与DOX单独处理组比较差异没有统计学意义外，其余均有显著性（P＜0．05，P＜0．01）。6．THSG对阿霉素导致的心肌细胞胞内钙水平增高的影响方法：加载荧光探针后用激光共聚焦检测细胞胞内钙水平[Ca²⁺]_i。结果：加入1μM DOX后，心肌细胞胞内钙水平快速增高并在5min后持续上升，5min时其荧光强度与基础值的比值为2．33±0．43。THSG 30μM预孵育2h可以显著抑制DOX导致的胞内钙水平增高，并使这种增高在2min内进入平台期，5min时其荧光强度与基础值的比值为1．33±0．07。THSG 100、300μM预孵育2h几乎能够使胞内钙水平在DOX加入后保持不变，5min时其荧光强度与基础值的比值分别为1．09±0．04、1．06±0．02。所有THSG组与DOX单独处理组比较均有统计学差异（P＜0．01）。结论：①THSG对DOX诱导的小鼠急性心脏毒性有保护作用，且作用与抑制DOX引起的氧化损伤有关。②THSG能够减少DOX诱导的细胞凋亡，而且作用与减少细胞线粒体膜电位下降，抑制caspase-3激活，增加Bcl-2及减少Bax的蛋白表达有关。③THSG抑制DOX引起的活性氧自由基增多。④THSG能够抑制DOX诱导的心肌细胞胞内钙浓度增高。第二部分体外培育牛黄镇痛作用的电生理机制研究牛黄是名贵中药，《本草纲目》记载：其“清心解毒、凉肝息风”，主治惊厥抽搐、癫痫发狂、咽喉肿痛等症。以牛黄为主要成分的牛黄千金散、牛黄解毒丸等经典中成药主要用于解热、镇痛，具有良好的临床疗效。已证实，牛黄及培植牛黄均能显著降低醋酸所致的小鼠疼痛反应。关于牛黄镇痛的作用机制，除与抗炎止痛有关外，推测也可能涉及对痛觉信息传入的直接干预作用。天然牛黄药源匮乏、价格昂贵。国家药品监督管理部门自1972年陆续批准了3个牛黄代用品：人工牛黄、培植牛黄、体外培育牛黄。体外培育牛黄（calculus bovis sativus，CBS；曾译名in vitro cultivated calculusbovis，ICCB）是模拟体内胆结石形成的原理和生化过程，在体外牛胆汁内培育的牛胆红素钙结石，其性状、结构、成分、含量、药效及疗效等与天然牛黄一致。批准文号国药准字20010075，已收载入2005年药典（一部）。实验以CBS为对象，以蟾蜍坐骨神经干和三叉神经节细胞为标本，观察CBS对复合动作电位及电压依赖性总钠通道电流(I_Na-T)、TTX不敏感型钠通道电流(I_Na-TTX-r)及电压依赖性钙通道电流(I_Ca)的影响，探讨牛黄镇痛作用的电生理机制，以证实其阻滞痛觉信息传导的推测。1．CBS对蟾蜍坐骨神经干复合动作电位的影响方法：复合动作电位记录法结果：0．4 mg·mL^-1 CBS能轻度抑制蟾蜍坐骨神经干复合动作电位的幅度，30 min时使动作电位由给药前的2．68±0．26 mV下降为给药后的2．55±0．29 mV，抑制率为5．0±2．4％（n=7，P＞0．05）。4 mg·mL^-1 CBS可时间依赖性的显著抑制动作电位，给药后10 min、20 min和30 min时，对动作电位的抑制率分别为21．3±10．3％、36．8±14．2％、42．5±17．6％（n=8，P＜0．01），用任氏液冲洗10～20 min可使动作电位部分恢复。同样条件下，给予0．02％利多卡因10 min、20 min和30 min时，动作电位分别下降26．8±14．9％、48．0±19．1％和60．7±14．2％（n=6，P＜0．05）。溶剂对照组对动作电位无明显影响。2．CBS对大鼠三叉神经元总钠通道电流(I_Na-T)的影响方法：全细胞膜片钳记录结果：溶剂对照对于TRG细胞I_Na-T无明显影响。CBS能够剂量依赖性的抑制TRG细胞的总钠电流幅值，0．2、2、20μg·mL^-1 CBS可使神经元钠电流幅值分别减少25．9±5．7％、45．0±7．6％和55．8±7．8％（n=6，P＜0．01）。20μg·mL^-1 CBS使I_Na由给药前的12．2±0．7 nA减少至给药后的5．4±0．9 nA，但冲洗后电流未见明显恢复（5．6±1．0 nA，P＞0．05），提示药物作用难以逆转。以I_Na幅值对应各测试电位作图，得I_Na I-V曲线。各测试电压下，CBS使I_Na-T幅值减小，给药前后最大激活电位及激活阈值电位无改变。根据所记录到的I_Na，用Boltzmann函数拟合比较激活曲线发现：给药前半数激活电压V_1/2=-19．0±10．6 mV，k=4．3±1．7，给予20μg·mL^-1 CBS后V_1/2=-24．5±10．3 mV，k=4．9±2．5（n=6，P＞0．05），表明20μg·mL^-1 CBS对I_Na-T的激活动力学无明显影响。3．CBS对大鼠三叉神经元TTX不敏感型钠通道电流(I_Na-TTX-r)的影响方法：全细胞膜片钳记录结果：给药前I_Na-TTX-r幅值为10．2±0．2 nA，给予溶剂对照后为9．9±0．4 nA（n=5，P＞0．05），表明溶剂对TRG细胞I_Na-TTX-r无明显影响。CBS可剂量依赖性的抑制TRG细胞I_Na-TTX-r幅值，0．2、2、20μg·mL^-1 CBS可使TRG细胞I_Na-TTX-r幅值分别减少8．1±2．0％、28±5．2％和39．3±5．7％（n=6，P＜0．01）。20μg·mL^-1 CBS使I_Na-TTX-r幅值由给药前的11．4±1．4 nA减少至给药后的6．9±0．6 nA，冲洗后电流同样未见明显恢复（6．8±0．8 nA，P＞0．05）。I_Na-TTX-r给药前后的I-V曲线显示，在各测试电压下CBS可使I_Na-TTX-r幅值减小，对I-V曲线的形状无明显影响。比较给药前后电流的激活曲线发现：给药前半数激活电压V_1/2=-24．1±0．2 mV，k=1．6±0．1，给予20μg·mL^-1 CBS后V_1/2=-23．0±1．4 mV，k=1．6±0．2（n=6，P＞0．05），表明20μg·mL^-1 CBS对TRG细胞I_Na-TTX-r的激活时间过程无明显影响。4．CBS对大鼠三叉神经元细胞电压依赖性钙通道电流(I_Ca)的影响方法：全细胞膜片钳记录结果：0．05％溶剂对照对于TRG细胞I_Ca无明显影响。CBS能够剂量依赖性的抑制TRG细胞电压依赖性钙电流幅值，0．2、2、20μg·mL^-1 CBS可使神经元钙电流幅值分别减少303±4．7％、41．9±3．6％和56．7±6．8％（n=6，P＜0．01）。20μg·mL^-1 CBS使I_Ca由给药前的5．7±0．9 nA减少至给药后的2．5±0．4 nA，但冲洗后电流未见明显恢复（2．4±0．6 nA，P＞0．05），提示药物作用难以逆转。I_Ca I-V曲线显示，各测试电压下，CBS使I_Ca幅值减小，给药前后最大激活电位及激活阈值电位无改变。比较给药前后电流的激活曲线发现：给药前半数激活电压V_1/2=-15．5±3．2m V，k=3．4±0．8，给予20μg·mL^-1 CBS后V_1/2=-12．3±4．6 mV，k=4．5±0．6（n=6，P＞0．05），表明20μg·mL^-1 CBS对I_Ca的激活动力学无明显影响。比较给药前后I_Ca的稳态失活曲线发现：给药前半数失活电压V_1/2=-36．3±4．5 m V，k=21．4±5．4，给予20μg·mL^-1 CBS后V_1/2=-33．5±5．8mV，k=18．5±4．3（n=6，P＞0．05），表明20μg·mL^-1 CBS对I_Ca的失活动力学无明显影响。结论：CBS对电压依赖性钠电流和钙电流的阻滞作用可能是其镇痛作用的机制之一。更多还原

【Abstract】 Part 1Protective effect of THSG on cardiotoxicity induced bydoxorubicin in vitro and in vivoDoxorubicin （also named adriamycin） is an anthracycline antibiotic that has beenused for more than 30 years for the treatment of a wide variety of cancers. However, severecardiomyopathy and heart failure have been observed in DOX-treated cancer patient, whichlimits the clinical chemotheraphy. It has been proposed that doxorubicin-inducedcardiomyopathy is at least partially caused by increased oxidant production in the heart, andthere is a great deal of supportive evidence for this hypothesis.Tetrahydroxystilbene-glucoside（THSG） was the antioxidative component of Polygonummultiflorum Thunb and it showed strong antioxidant activity in vitro. Previous researcheselucidated THSG could diminish peroxidation level of brain in Alzheimer’s Disease modelmice and ischemia-reperfusion model mice. Structurally, THSG belongs to hydroxystilbene,and its structure is similar to that of resveratrol extracted from red wine which hassignificantly protective effects on cardiomyocyte injury. Based on the previous reportsthat THSG protects normal tissues against oxidative stress, we envisioned a possibility thatTHSG might protect heart from DOX injury.In the present study we used neonate rattus cardiomyocytes in vitro and acutemouse model of DOX-induced cardiotoxicity to examine the protective effect of THSG.§1. Protective effects and mechamism of THSG on DOX induced actue cardiotoxicityin mice1. Protective effects of THSG on DOX-induced cardiotoxicity in mice Method: A single DOX （15 mg/kg） was intraperitoneally injected to the animals to inducedthe actue cardiotoxicity, THSG （0.1g/kg·d） were administered with intragastric injection（p.o.） for 7 days continuously. Then it was observed whether THSG have protective effectson DOX-induced cardiotoxity in mice.Results:①Animal body weight and survival: There was no difference on body weightamong 3 groups at day 1. On the 7th day after DOX administration, animals’ body weightswere reduced（21.8±3.6 g） by DOX treatment compared with control（25.6±2.6 g,P＜0.01）. Treatment with THSG did not increase body weights of animals （22.5±2.8 g,P＞0.05 compared with DOX alone）. There was no difference in the heart weight/bodyweight ratio among the 3 groups（4.5±0.6mg/g in control group, 4.3±0.7mg/g in DOXgroup and 4.3±0.4 mg/g in THSG group）. The survival rate of animals in the control groupwas 100%. DOX treated animals had a compromised survival rate （70%）, and there was atrend of increased survival in THSG treated animals （85%） compared with DOX treatedgroup （P＞0.05, log rank test）.②Histological findings : Normal heart histologicalfindings were seen in the control group.On the other hand, there were histological changesin DOX group by the presence of marked interstitial oedema, focal subendocardial bleed,cardiac muscle fibre destruction or loss, which was qualitatively recognized as DOX-inducecardiac damage. The lesions were significantly reduced in the groups treated with THSGcompared to DOX group.2. Effects of THSG on serum LDH and CK in DOX administrated miceMethod: Serum LDH and CK were investigated with biochemical method.Results: On the 7th day after DOX adminstration, serum LDH increased from 3.87±0.40U/ml of normal control group to 6.00±0.27 U/ml of the DOX group （P＜0.01）. However,THSG （0.1g/kg） could prevent the increase of serum LDH induced by DOX, with the value4.86±0.20U/ml （P＜0.05, compared with DOX alone）. Serum CK increased from 1.55±0.49 U/ml of control group to 3.02±0.28U/ml of the DOX alone administrationgroup （P＜0.01）. THSG administration could inhibit the increase of serum CK induced byDOX, with the value of 1.94±0.4U/ml （P＜0.05, compared with DOX alone）。3. Effects of THSG on MDA and GSH in DOX administrated miceMethod: MDA and GSH were investigated with biochemical method.Results: On the 7th day after DOX adminstration, MDA content increased from 1.15±0.30nmol/mg protein of normal control group to 4.17±0.91 nmol/mg protein of the DOX group（P＜0.01）. However, THSG （0.1g/kg） could significantly prevent the increase of MDAinduced by DOX, with the value 2.32±0.76 nmol/mg protein （P＜0.01, compared withDOX alone）. GSH content decreased from 24.43±6.57mg/g protein of control group to13.65±3.46 mg/g protein of the DOX alone administration group（P＜0.01）. THSGadministration could inhibit the decrease of GSH induced by DOX, with the value of18.31±3.27 mg/g protein （P＜0.01, compared with DOX alone）.§2 Protective effects of THSG on DOX induced cardiomyocyte lesion1. Effects of THSG on viability of DOX treated cellsMethod: Cell viability was determinated by MTT assay.Results: Cell viability of control group was 100%. DOX markedly affected cell survival asthe rate of cell survival after exposure to 1μM DOX for 24h was 72.68±8.52%（P＜0.01）,while in the presence of THSG（3,10,30,100,300μM）, cell viability was significantlyenhanced in a concentration-dependent manner with the values of 76.09±10.24 %, 81.37±9.84 %, 83.43±11.47 %, 91.21±7.54 %, 95.24±10.87 % respectively. Except 3μMTHSG group, 10-300μM THSG groups had significant difference compared with the DOXalone group （P＜0.05, P＜0.01）. 2. Effects of THSG on DOX-induced apoptosisMethod: Cell apoptosis was investigated by TUNEL assay.Results: The rate of apoptotic cells of control group was 4.57±0.52%. DOX markedlyincreased the rate of positive cells after exposure to 1μM DOX for 24h with the value was32.55±3.47% （P＜0.01）, while in the presence of THSG（10,30,100,300μM）, the rate ofapoptotic cells was significantly decreased in a concentration-dependent manner with thevalues 26.53±3.27%,20.12±2.64 %,13.34±8.64 %,10.48±1.57 % respectively. Allgroups had significant difference compared with the DOX alone group （P＜0.05, P＜0.01）.3. Effects of THSG on DOX-induced loss of mitochondrial membrane potentialMethod: Mitochondrial membrane potential was investigated by fluorescence probe.Results: Nearly all cells were well spread and exhibited red or orange fluorescence in anuntreated culture, Quantitative analysis showed ratio of JC-1 aggregates/monomericwas14.52±3.86 %. In contrast, a culture treated with DOX for 24h demonstrated multiplerounded cells, a majority of which fluoresce green exclusively with the value of 0.61±0.16% （P＜0.01, compared with control）, indicating loss of mitochondrial membrane potential.A culture treated with DOX in the presence of THSG（10,30,100,300μM） appeared similarto untreated control, with spread cells exhibiting red or orange fluorescence with thevaluesl.48±0.34%, 2.32±0.46 %,3.70±0.87%,5.35±1.25% respectively. Except 10μMTHSG group, 30-300μM THSG groups had significant difference compared with DOXgroup （P＜0.01）.4. Effects of THSG on DOX-mediated ROS generationMethod: ROS were investigated by fluorescence probe.Results: Quantitative results （green fluorescence area） are expressed as % of values foundin cells untreated. The value was increased from 100% of control group to 337.56±45.56 % after exposure of 1μM DOX for 24h （P＜0.01）, indicated that DOX stimulatedsignificant increase of ROS level. A culture treated with DOX in the presence of THSG（30,100,300μM） could inhibited DOX-mediated ROS generation with the values 265.65±23.46 %, 227.70±26.57%, 184.17±34.52%. All groups had significant differencecompared with the DOX alone group （P＜0.05, P＜0.01）.5. Effects of THSG on apoptosis-related proteins expressionMethod: Protein expressions were investigated by Western blot.Results:①Treatment of the cells with 1μM DOX for 24h induced the cleavage ofprocaspase-3（32 kDa） to its 17 kDa subunits. Caspase-3（17 kDa）/β-actin density ratio wassignificantly increased from normal control group 0.08±0.01 to DOX group 1.31±0.27（P＜0.01）. THSG （30,100,300μM） could inhibit caspase-3 protein activation indose-dependent manner, and density ratio were 0.80±0.14,0.42±0.06,0.12±0.13. Allthese THSG groups had significant difference from DOX alone group （P＜0.05, P＜0.01） .②DOX treated for 24h markly increased Bcl-2 protein expression. Bcl-2/β-actin densityratio was significantly increased from normal control group 0.42±0.04 to DOX group0.88±0.09 （P＜0.05）. THSG （30,100,300μM） could dose-dependently make this potentiallyadaptive response more outstanding, and density ratio were 0.99±0.10, 1.37±0.08,1.64±0.12. Except 30μM THSG group, 100 and 300μM THSG groups had significantdifference from DOX alone group （P＜0.05, P＜0.01）.③DOX treated for 24h marklyincreased Bax protein expression. Bax/β-actin density ratio was increased from normalcontrol group 0.64±0.07 to DOX group 1.09±0.12 （P＜0.05）. THSG （30,100,300μM） coulddose-dependently inhibit the increase of Bax protein expression, and density ratio were0.95±0.08, 0.75±0.06, 0.66±0.05. Except 30μM THSG group, 100 and 300μM THSGgroups had significant difference from DOX alone group （P＜0.05, P＜0.01）. 6. Effects of THSG on DOX-induced increase of [Ca²⁺]_iMethod: [Ca²⁺]_i was investigated by fluorescence probe.Results: Treatment of cardiomyocytes with DOX greatly increased level of [Ca²⁺]_i. TheCa²⁺ increase was very quick and did not reach plateau at 5 min. Quantitative analysisshowed fluorescence intensity after treatment of DOX /basal value was 2.33±0.43.Pretreatment of THSG inhibited DOX-induced increase of [Ca²⁺]_i. It reached plateau atapproximately 2 min with pretreatment of 30μM THSG and the ratio was 1.33±0.07.Treatment of 100 and 300μM THSG nearly maintained basal [Ca²⁺]_i after DOX was addedwith the value 1.09±0.04 and 1.06±0.02. All these THSG groups had siginificant differencefrom DOX group （P＜0.01）.Conclusions:1. THSG had protective effects on acute cardiotoxicity induced by DOX in mice, and itseffects may be associated with inhibiting oxidative damage by DOX.2. THSG could inhibit DOX-induced apoptosis and it may be involved that it preventedDOX-mediated loss of mitochondrial membrane potential, caspase-3 activation andchanges of Bax and Bcl-2 protein expression.3. THSG could inhibit DOX-mediated ROS generation.4. THSG could inhibit the increase of [Ca²⁺]_i induced by DOX. Part 2The electrophysiological mechanism of calculus bovissativus on analgesic effectCalculus bovis has been used for thousand years as the Chinese traditional medicine.The classical Chinese patent medicine NiuHuang JieDu Wan and NiuHuang QianJing Shanwhose major ingredient were calculus bovis had antipyretic and analgesic effect andapproved for clinical use. Previous reports had verified that calculus bovis and culturalcalculus bovis could relieve pain induced by acid in mice. We presumed that themechanism of calculus bovis on analgesic effect was not only involved itsanti-inflammatory effect but also the directly intervention of afferent pain sense. In order toresolve the shortage raw material of calulus bovis, the national management of drugsurveilance had validated in succession three substitutes since 1972: artificial bezoar,cultural calulus boris and calulus bovis stativus （CBS）.CBS is prepared in ox bile bysimulating the biochemical process of bilirubin calcium formation and by employingbioengineering techniques. It has same quality as that of natural calculus bovis in terms ofproperties, structure, component and content.In the present study we used sciatic nerve trunk of toads and cultured adult rattrigeminal ganglion neurons to examine the effect of CBS on action potential, currents ofvoltage-dependent sodium channel and voltage-dependent calcium channel.1. The effect of CBS on action potential of sciatic nerve trunk of toadsMethod: The action potential of sciatic nerve trunk of toads was recorded by RM6240BDpolygraph.Results: 0.4 mg·mL^-1 CBS could solfly decrease the action potential of satatic nerve trunkof toads. The amplitude of action potential was decreased from 2.68±0.26 mV to 2.55±0.29 mV of CBS treatment for 30min. The inhibitory rate was 5.0±2.4% （n=7, P＞0.05）. 4mg·mL^-1 CBS could significantly decrease the action potential of satatic nerve trunk oftoads in time-dependent manner. The inhibitory rates were 21.3±10.3%, 36.8±14.2%,42.5±17.6% respectively when CBS treated for 10, 20 and 30min （n=8, P＜0.01）, washed byRinger solution for 30 min could recovery the action potential partly. Under the samecondition, treated satatic nerve trunk of toads with 0.02% lidocaine for10, 20 and 30min,the inhibitory rates of action potential were 26.8±14.9 %, 48.0±19.1%, 60.7±14.2 %respectively （n=6, P＜0.05）. Dissolvant control had no effect on action potential.2. The effect of CBS on total currents of voltage-dependent sodium channel (I_Na-T).Method: whole cell patch clamp record.Results: The amplitude of total currents before treatment was 12.1±0.5 nA and the valuewas 12.1±0.6 nA （n=5, P＞0.05） after 0.05% dissolvent treatment, indicated that thedissolvent had no effect on I_Na-T. CBS could dose-dependently inhibit the total currents ofvoltage-dependent sodium channel on trigeminal ganglion neurons （TRG）. The inhibitoryrates of 0.2, 2 and 20μg·mL^-1 CBS on I_Na-T were 25.9±5.7%, 45.0±7.6%, 55.8±7.8%respectively （n=6, P＜0.01）. Treatment with 20μg·mL^-1 CBS decreased the currents from12.2±0.7 nA to 5.4±0.9 nA and washing could not make it recovery（5.6±1.0 nA,P＞0.05）, indicated that the drug action was inreversible. Current-voltage （I-V） relation ofI_Na-T under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage （G-V） relation showedno significant change after 20μg·mL^-1 CBS treatment (control: V_1/2=-19.0±10.6 mV,k=4.3±1.7; 20μg·mL^-1 CBS:V_1/2=-24.5±10.3 mV, k=4.9±2.5 （n=6, P＞0.05）), indicatedthat CBS had no effect on activation kinetics of I_Na-T. 3. The effect of CBS on TTX-resistant currents of voltage-dependent sodium channel(I_Na-TTX-r).Method: whole cell patch clamp record.Results: The dissolvent had no effect on I_Na-TTX-r. CBS could dose-dependently inhibitthe TTX-resistant currents of voltage-dependent sodium channel on TRG. The inhibitoryrate of 0.2, 2 and 20μg·mL^-1 CBS on I_Na-TTX-r were 8.1±2.0%, 28±5.2%, 39.3±5.7%respectively（n=6, P＜0.01）. Treatment with 20μg·mL^-1 CBS decreased the currents from11.4±1.4 nA to 6.9±0.6 nA and washing could not make it recovery（6.8±0.8 nA,P＞0.05）, indicated that the drug action was inreversible. Current-voltage （I-V） relation ofI_Na-TTX-r under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage （G-V） relation showedno significant change after 20μg·mL^-1 CBS treatment.( control: V_1/2= -24.1±0.2 mV,k=1.6±0.1; 20μg·mL^-1 CBS: V_1/2= -23.0±1.4 mV, k=1.6±0.2 （n=6, P＞0.05）), indicatedthat CBS had no effect on activation kinetics of I_Na-TTX-r.4. The effect of CBS on the currents of voltage-dependent calcium channel (I_Ca).Method: whole cell patch clamp record.Results: The amplitude of the currents before treatment was 5.3±0.8 nA and the value was5.1±0.6 nA （n=6, P＞0.05） after treated with 0.05% dissolvent, indicated that the dissolventhad no effect on I_Ca. CBS could dose-dependently inhibit the currents of voltage-dependentcalcium channel on TRG. The inhibitory rate of 0.2, 2, 20μg·mL^-1 CBS on I_Ca were30.3±4.7%, 41.9±3.6% and 56.7±6.8% respectively （n=6, P＜0.01）. Treatment with 20μg·mL^-1 CBS decreased the currents from 5.7±0.9 nA to 2.5±0.4 nA and washing couldnot make it recovery（2.4±0.6 nA, P＞0.05）. Current-voltage （I-V） relation of I_Ca underdifferent concentration showed CBS had no effect on maximum activation potential andthreshold of activation potential. Conductance-voltage （G-V） relation showed no significantchange after 20μg·mL^-1 CBS treatment( control:V_1/2=-15.5±3.2 mV, k=3.4±0.8; 20 μg·mL^-1 CBS:V_1/2= -12.3±4.6 mV, k=4.5±0.6 （n=6, P＞0.05）), indicated that CBS had noeffect on activation kinetics of I_Ca. Steady-state inactivation curves showed no significantchange after 20μg·mL^-1 CBS treatment( control:V_1/2= -36.3±4.5 mV, k=21.4±5.4; 20μg·mL^-1 CBS: V_1/2= -33.5±5.8 mV, k=18.5±4.3 （n=6, P＞0.05）), indicated that CBS hadno effect on inactivation kinetics of I_Ca.Conclusion:The inhibitory effect of CBS on the voltage-dependent sodium and calcium currents maycontribute to its relieving pain.更多还原