【作者】 高永刚；

【导师】 楚立；

【作者基本信息】 河北医科大学 ， 药理学， 2014， 博士

【摘要】 铁是人体必需的微量元素之一，它不仅参与细胞血红素的构成，还参与细胞的增殖和分化，并且通过催化氧化-还原反应参与机体的能量代谢、解毒等重要生理过程，同时它也参与调节NOS、PKC-β等与细胞生长功能有关的基因表达。但是当体内铁超载时，则引起器官的广泛受损。心脏、肝脏等是铁超载受损的主要靶器官。自1981年Swanson提出机体铁不断增加是冠心病的危险因素以来，铁超载致心脏损伤一直成为铁超载研究的热点。另外，肝脏是机体铁贮存和代谢的重要脏器，体外培养的肝细胞中加入非转铁蛋白结合铁，可导致肝细胞形态发生变化。研究发现，铁超载可使机体内有氧化活性游离铁大量增加，从而促进OH·的产生，目前认为OH·是体内最具有损伤性的自由基，它能攻击线粒体双层磷脂中不饱和脂肪酸，产生过量的脂质过氧化物，从而导致线粒体膜功能的损伤如物质转运、信号传导等，这会进一步影响整个细胞的结构和功能，最终导致细胞死亡和组织坏死，这可能是铁超载致器官损伤的重要机制之一。目前，临床铁超载疾病的治疗药物主要是铁的螯合剂，如去铁胺（Deferoxamine, DFO）、去铁酮（Deferiprone, DFP）、去铁斯若（Deferasirox,DFS）等，其中DFO是目前广泛应用治疗铁超载疾病的药物。DFO具有减少自由基形成、抑制脂质过氧化等作用，但DFO也有很多副作用，可引起低血压、心悸等不良反应，长期静脉注射治疗会引起病人耐受性差、生活质量下降等问题。近年有研究发现钙通道阻滞剂如维拉帕米可以抑制机体脂质过氧化、逆转铁超载，但是其作用机制尚未完全明确。传统中医药治疗铁超载的研究很少，国内有学者从黄芩中提取黄芩苷、从硕苞蔷薇中提取儿茶素和漆黄素来治疗铁超载所致脏器损伤，发现黄芩苷、儿茶素和漆黄素具有抗氧化和铁螯合剂双重作用，但缺点是黄芩苷由于在黄芩中含量低难以提取。因此，研究开发一种新的药物治疗铁超载疾病有着非常重要的意义。丹参是属于唇形科鼠尾草植物丹参（Salvia miltiorrhiza，Labiataefamily）的干燥根，是中国传统的中草药之一，具有活血化瘀、凉血、去痈、平燥等功效，现代研究证实丹参还具有降低氧自由基生成、抑制钙离子通道等作用。丹参注射液（丹参的水提物）在临床上极为常用，广泛用于治疗心血管疾病和肝、肾功能障碍等疾病，是一种简便价廉的中药制剂。近年来对丹参注射液的基础和临床研究已逐渐成为热点。我们的前期研究表明，丹参能减少斑马鱼肝脏、心脏和肾脏铁沉积，抑制脂质过氧化；此外，前期研究还证实丹参注射液对其它一些二价金属离子如铅中毒小鼠肾脏损伤也有保护作用。因此，丹参是潜在的治疗铁超载和其他金属离子所致脏器损伤的药物，其作用及机制值得进一步深入研究证实。综上所述，近年来有关铁超载引起的健康问题尤其是对心脏、肝脏等重要脏器的损伤逐渐引起人们关注。除DFO等铁螯合剂外，其它治疗铁超载的药物报道亦逐年增加，但丹参注射液对铁超载致心脏及肝脏损伤的保护作用及其机制的研究尚未见报道。本研究论文主要从分子生物学、形态学、细胞学、离子通道水平深入探讨丹参注射液对铁超载致脏器损伤的保护作用及其机制，主要包括以下内容：（1）丹参注射液对铁超载致小鼠急性心脏损伤的保护作用及机制；（2）丹参注射液对铁超载致急性肝脏损伤的保护作用及机制；（3）丹参注射液对大鼠心肌细胞钙电流及钙瞬变的影响。论文具体内容如下：第一部分丹参注射液对铁超载所致心脏损伤的保护作用及机制目的：测定丹参注射液的活性成分及其含量；深入探讨丹参注射液对铁超载所致小鼠心脏损伤的保护作用及机制。方法：通过高效液相色谱-紫外检测（HPLC-UV)定量测定丹参注射液的主要活性成分及其含量。将60只实验小鼠随机分为5组：对照组、铁超载组、丹参高剂量组、丹参低剂量组和DFO组。除对照组腹腔注射生理盐水外，其它各组均腹腔注射右旋糖酐铁注射液（50mg/kg/d）。给予右旋糖酐铁4h后，丹参高、低剂量组小鼠再分别注射丹参注射液（4ml/kg/d、2ml/kg/d）及DFO（100mg/kg/d）。连续给药2周。结果：（1）丹参注射液的主要活性成分及其含量分别是丹参素（2.15mg/mL）、原儿茶醛（0.44mg/mL）和丹酚酸B（1.01mg/mL）。（2）H&E染色结果表明，丹参注射液可明显改善铁超载所致的心脏损伤；（3）普鲁士蓝染色及组织铁含量测定的结果显示，丹参注射液可以显著降低铁在心脏的沉积；（4）血清生化指标检测结果显示，铁超载模型组的CK、CK-MB及LDH水平明显升高，丹参高剂量组和低剂量组使其活性下调，并且具有剂量依赖性。（5）心肌组织氧化指标测定结果显示：丹参可明显提高小鼠抗氧化能力，丹参高剂量组和低剂量组的MDA浓度显著降低，同时SOD和GSH-Px水平显著升高，并且具有剂量依赖性。结论：上述结果表明丹参注射液对铁超载所致小鼠心脏损伤具有明显的改善作用，机制与丹参可减少铁在心脏过量沉积，抑制脂质过氧化作用有关。第二部分丹参注射液对铁超载所致肝脏损伤的保护作用及机制目的：研究丹参注射液对铁超载所致小鼠的肝脏损伤保护作用及机制方法：分组及给药方法同“第一部分”。结果：（1）体重和肝系数统计结果显示，铁模型组的体重和肝系数明显升高，丹参注射液可有效缓解体重和肝系数的上升，对肝脏具有一定的保护作用；（2）H&E染色结果表明，丹参注射液可明显改善铁超载所致的肝脏损伤；（3）普鲁士蓝染色结果显示，丹参注射液可以显著降低铁在肝脏的沉积；（4）血清生化指标检测结果显示，铁模型组的AST和ALT水平明显降低，丹参高剂量组和低剂量组使其活性均有所上调，并且具有剂量依赖性；（5）肝脏组织氧化指标测定结果显示：丹参可明显提高小鼠抗氧化能力，丹参高剂量组和低剂量组的MDA浓度显著降低，同时SOD和GSH-Px水平显著升高，并且具有剂量依赖性；（6）TUNEL染色结果显示，丹参注射液可显著降低肝细胞的凋亡。结论：上述结果表明丹参对铁超载所致小鼠肝脏损伤具有明显保护作用，其机制与丹参注射液可以减少铁在肝脏的沉积，抑制脂质过氧化和降低细胞凋亡有关。第三部分丹参注射液对心肌细胞钙电流及钙瞬变的作用目的：研究丹参注射液对心肌细胞膜L-型钙电流（ICa-L）及细胞内钙离子浓度的影响。方法：采用全细胞膜片钳技术研究丹参对大鼠心肌细胞膜ICa-L的作用；采用细胞收缩与离子浓度同步测量系统研究丹参注射液对心肌细胞内钙离子浓度变化及对心肌细胞收缩力的影响。结果：（1）100μM维拉帕米可完全阻断所记录的电流，说明此电流即为ICa-L。（2）100mg/L丹参注射液可显著抑制心肌ICa-L并且冲洗后可恢复至给药前水平，说明该抑制作用确由丹参注射液引起而非其自身的衰减。（3）丹参注射液可浓度依赖性降低ICa-L。量效曲线结果显示，不同浓度的丹参注射液（0.1、0.3、1、3及10g/L），对ICa-L的抑制率分别为11.6%、19.1%、26.7%、32.1%及35.2%。（4）丹参注射液对I-V关系及反转ICa,L电位无明显作用。（5）丹参注射液对心肌钙离子激活和失活无明显作用。（6）丹参注射液（3g/L）可以显著抑制心肌细胞收缩（抑制率为21.0%）和降低细胞内钙离子浓度（钙瞬变的峰值降低23.3%）。（7）丹参注射液可明显的缩短从心肌细胞舒张开始至达到90%基线所需的时间（the timeto90%of the baseline，Tr），但对从收缩开始至到达收缩峰值10%的时间（the time to10%of the peak，Tp）无影响。结论：结果表明丹参注射液可明显地抑制心肌细胞ICa-L，降低心肌细胞内钙离子浓度，减弱心肌收缩力，发挥其心肌保护作用。由于铁离子可通过细胞膜钙离子通道进入心肌细胞内，提示由于丹参抑制ICa-L作用使铁离子进入细胞减少，从而降低铁在心肌细胞内的沉积。更多还原

【Abstract】 Iron is one of indispensable microelement of our body, and it hasimportant physiological functions as an important component of hemoglobin,myoglobin and enzymes necessary for normal cell proliferation. Although ironcatalyzes critical redox reactions, detoxification and other processes neededfor energy production, it has highly cytotoxic in excess quantities includingtriggering to product large amount of free radicals, even damaging multipleimportant organs especially heart and liver. Swanson reported that progressvieiron depoistion in body.is one of the risk factors of coronary heart disease. Inaddition, liver is the significant organ to keep iron storage and its metabolism,in vitro experiment hepatocytes cultured mixs non-transferrin-bound ironcould change its shape. Studies show that with the extreme increasement offree iron, OH··is also rise in body, which is one of most damaging the freeradical, and then the latter would attack the unsaturated fattyacid inmitochondria and produce lipid peroxide, thereby result in the damage ofmembrance in mitochondria and eventually cause the death of the cells andtissues, which maybe one of the mechanisms of excessive iron-inducedorganic injuried.Deferoxamine (DFO) and1,2-dimethyl-3-hydroxypyrid-4-one(deferiprone, L1) is currently prescribed for the treatment of iron overloaddiseases. As iron-chelating agents, DFO and deferiprone/L1have been shownto inhibit free radical formation and the consequent tissue damage in someexperimental systems. However, they have several side effects anddisadvantages. In addition, the need for lifelong treatment of daily ironchelation therapy has a notable negative impact on patients’ perceived andactual quality of life. In rencent years, the reported activities lend some support to the use of calcium channel blockers such as verapamil to restrainthe lipid peroxidation of hepatocyte membranes and to protect against liverinjury, but the protective mechanisms did not illustrate clearly. Besides, it hasrecently been reported that supplementation with quercetin or baicalinextracted from Scutellaria baicalensis Georgi, which possess both antioxidantand iron chelation activities, can increase iron excretion in iron overloadedmice and hence protect the liver from oxidative stress and fibrosis. However,the content of baicalin in the root of S. baicalensis Georgi is lower than10%and is difficult to extract. Therefore, a less toxic and more widely availabledrug is needed to improve therapy outcomes for patients with iron overloaddiseases.The dried root of SM belonging to the family Labiatae is one of the mostversatile Chinese herbal drugs. Its traditional curative functions are toinvigorate blood circulation, remove stagnation, cool blood, reduce carbunclesand soothe irritability. Modern researches have proved that SM could decreasethe produce of oxygen radical and inhibit the L-type calcium channels. As theSM decoction is a commonly used preparation in traditional Chinese medicine,recent studies have focused on its aqueous fraction and constituents. SMinjection, the aqueous extracts of SM, is widely used in experimental andclinical treatment of cardiovascular diseases, liver diseases and renal disease.Meanwhile, it is very cheap and convenient. Our preliminary studies show thatSM could reduce the iron deposition in liver, heart and kidney and inhibit theoccurrence of lipid peroxidation. Moreover, we also proved that SM injectioncould protect the kidney injured induced by divalent metal ion such as lead.Therefore, SM is the potential drug that could treat iron overload and metalion, and it deserves to further studies.In recent years the health issue results from concerns about the importantorgans damage especial heart and liver induced by iron overload in patients. Inaddition to DFO, the reports of other therapies about iron overload diseasesincreased by years. However, the protective effects involves its mechanism ofSM injection against organs injury induced by iron overload remain unknown. Based upon the above mentioned SM injection, in my thesis, we usetechniques such as molecular biology, pathomorphology, patch clamp andothers to systematically study the following subjects:(1) Protective effect andpotential mechanisms of SM injection on iron overload-induced heart injury;(2) Protective effects and potential mechanismsof SM injection on ironoverload-induced liver injury;(3) Effects of SM injection on calcium currentand calcium transient of ventricular myocytes.Part1Protective effect and potential mechanisms of SM injection on ironoverload-induced heart injuryObjective: Determination the active ingredient and its content of SMinjection. Research the protective effect and potential mechanism of SM oniron overload-induced cardiac damage in mice.Methods: Determination the active ingredient and its content of SMinjection using high performance liquid chromatography-ultraviolet detection(HPLC-UV). All mice were randomized divided into five groups: control, ironoverload, low-dose Danshen, high-dose Danshen and DFO groups. Mice ofthe latter four groups were intraperitoneally (i.p.) injected with a single doseof iron dextran at50mg·kg1per day. Mice of the control group received an i.p.injection of isovolumic saline. Mice of the L-Danshen, H-Danshen groups andDFO group were given i.p. SM injection at3,6g·kg1and DFO100mg·kg1per day respectively. The entire experimental period lasted for2weeks.Results:(1) The three main constituents and the actual concentrations ofSM injection were danshensu (2.15mg·mL1), protocatechuic (0.44mg·mL1)and aldehyde salvianolic acid B (1.01mg·mL1).(2) H&E staining showedthat the cardiac damage induced by iron overload was attenuated by SMinjection significantly.(3) The results of Prussian blue staining anddetermination of total iron in heart tissues showed that the accumulation ofexcess iron in the heart diminished following treatment with SM injection.(4)The testing results of serum biochemical indicators showed that serum levelsof CK, CK-MB and LDH in the iron-overloaded mice were significantlyelevated, whereas their activities were down-regulated by SM Injection in a dose-dependent manner.(5) The testing results of three oxidative stressmarkers in myocardial tissues showed that SM can obviously improve theantioxidant ability of mice. SM injection reduced the myocardial MDAcontent and improved cardiac SOD and GSH-Px levels in a dose-dependentmanner.Conclusions: Altogether, these results illustrated that SM injection couldattenuate the cardiac damage induced by iron overload significantly. Themechanisms may be attributed to its ability to decrease iron deposition andsuppress lipid peroxidation.Part2Protective effect and potential mechanisms of SM injection on ironoverload-induced liver injuryObjective: Investigation the protective effects and the potentialmechanism of SM injection on liver injury induced by iron overload in mice.Methods: Methods of subgrouping and administration are same with “thefirst part”.Results:(1) Statistical results showed that the mean body weight andliver coefficients of iron overloaded mice was significantly lower than that ofthe control group, but they were improved by SM injection.(2) H&stainingshowed that the liver injury induced by iron overload was attenuated by SMinjection.(3) The results of Prussian blue staining showed that theaccumulation of excess iron in the liver diminished following treatment withSM injection.(4) The testing results of serum biochemical indicators showedthat serum levels of AST and ALT in the iron-overloaded mice weresignificantly down-regulated, whereas their activities were elevated by SMInjection in a dose-dependent manner.(5) The testing results of three oxidativestress markers in myocardial tissues showed that SM can obviously improvethe antioxidant ability of mice. SM injection reduced the hepatic MDA contentand improved cardiac SOD and GSH-Px levels in a dose-dependent manner.(6) The results of TUNEL staining showed that SM injection suppressed thehepatocyte apoptosis significantly.Conclusions: SM injection demonstrated significant protective effects towards the liver of iron overloaded mice, which may due to the decrease ofiron deposition and inhibition of lipid peroxidation and hepatocyte apoptosis.Part3Effects of SM injection on calcium current and calcium transientof ventricular myocytesObjective: To investigate the effects of SM on L-type calcium current(ICa-L) and intracellular calcium ([Ca2+]i).Methods: we used the whole-cell patch-clamp techniques to study ICa-L,and used myocyte calcium and contractility systems to measured calciumtransient and contractility in isolated adult rat ventricular myocytes of SMinjection.Results:(1) The recored currents were completely blocked by100μMVerapamil, which indicated that the currents were calcium currents.(2) Thecalcium currents were blocked by100μM SM injection and it could recoverafter washed, which indicated that the inhibition effects were induced by SMinjection not “run down”.(3) SM reduced the ICa-Lin aconcentration-dependent manner.0.1,0.3,1,3,10g/L SM reduced the ICa-Lby11.6%、19.1%、26.7%、32.1%and35.2%respectively.(4) The SM injectionhave no significant effect on the I-V relationship or reversal potential of ICa,L.(5) The SM injection have no significant effect on the activation andinactivation.(6) SM (3g/L) decreased the amplitudes of myocyte shortening21.02%and lessened the peak value of Ca2+transient23.33%respectively.(7)From the time parametes of cell shortening, SM injection was found tosignificantly shorten the time to90%of the baseline (Tr), however, the time to10%of the peak (Tp) was not dramatically prolonged.Conclusions: SM injection not only significantly inhibited ICa-Lin aconcentration-dependent manner but also suppressed calcium transient andcontraction in rat ventricular myocytes under physiological conditions. Sinceiron can enter into cardiomyocytes via myocardial calcium currents, theinhibition of calcium currents may be one of the intracellular mechenisms ofSM injection to prevent Fe2+into cardiomyocytes.更多还原