丹参素对电离辐射损伤的防护作用及机制研究
摘要
研究背景：
近年来，核能和核技术广泛应用于能源、医疗、军事、食品加工、育种等国防和国民生活的方方面面，与人们日常生活息息相关。目前，可能受到放射线损伤的人群主要有接受放疗的癌症病人；从事与放射相关的工作人员，如放射科室的医务人员、核电站与辐射加工企业的工作人员、从事与核科学有关研究的科研工作者、各种核事故的波及人员以及宇航人员等，辐射在给人类造福的同时，对人们的健康也带来不同程度的威胁。
电离辐射对生物体造成损伤主要通过两种方式，一是直接作用，指由射线造成生物大分子的损伤，电离辐射的能量直接沉积于生物大分子，造成DNA链的断裂、蛋白酶失活或者破坏细胞内的膜的结构或通透性，从而影响细胞的正常功能。二是间接作用，指生物大分子的破坏和失活是由于电离辐射作用于水分子，继而水的辐射分解产物再作用于生物大分子，引起后者的物理和化学变化，从而诱导细胞凋亡。细胞经受辐射的直接作用和间接作用后，细胞膜和细胞内DNA发生改变以及大量ROS的产生，诱导细胞内多种信号分子的活化，或信号通路的激活，最终导致辐射后细胞凋亡、癌变，甚至机体死亡。
电离辐射对机体的损伤极大。迄今为止，虽然发现了一些有效预防损伤的化合物，如五十年前就已经发现了氨巯基化合物，三十年前合成的WR系列化合物等，这些已开发的抗辐射药物尽管拥有很好的抗辐射作用，但尚存在毒性大、不良反应多等多方面的不足，人们迫切希望能开发出毒性小，可用于临床防治电离辐射损伤的药物。丹参素(SalvianicacidA)是从唇形科植物丹参水溶性成分中提取的多酚
类物质，大量实验报道，丹参素具有抗炎、增强机体免疫力、抗肿瘤、保护肝脏、抑制肝纤维化、保护心肌细胞及中枢神经系统、防治心脑血管疾病的作用。我们前期实验发现，丹参素能广泛地清除各种自由基，降低小鼠经电离辐照后的死亡率，并明显减轻在体和离体实验中辐射引起的各种损伤。本实验拟进一步观察丹参素的辐射防护作用，并研究丹参素的辐射防护作用是否与其保护细胞DNA不受损伤、抑制细胞凋亡有关，进一步在细胞实验中探索丹参素发挥辐射防护作用时参与的信号通路及其可能的作用机制。
实验目的：
1.观察丹参素对辐射损伤小鼠的保护作用。
2.观察丹参素对辐射引起人正常细胞生长抑制的作用；并进一步研究丹参素对辐射损伤细胞形态、DNA、线粒体等的保护作用；
3.探索丹参素辐射防护作用的可能机制以及信号通路；
实验方法：
动物实验
雄性BALB/c小鼠(22±2g)适应环境后，随机分为五组:1)生理盐水对照组，2)单独照射组，3)丹参素10mg/Kg组，4)丹参素20mg/Kg组，5)阳性对照雌三醇（E3，2mg/Kg）组。不同浓度丹参素灌胃7天，阳性对照E3组在辐照前48h,24h,0h分别腹腔给药3次，然后给予不同剂量的γ射线照射。并于辐照后不同时间取材，观察丹参素对辐射损伤小鼠的生存率的影响，外周血中白细胞、红细胞、血红蛋白和血小板含量的变化，脾脏的病理学以及脾脏造血干细胞克隆形成率的改变，骨髓有核细胞数以及小鼠肝脏抗氧化能力的变化。
细胞实验
借助MTT法检测丹参素对四种人正常细胞的毒性和辐射防护作用，进一步采用克隆形成法观察丹参素对辐射损伤的L-02和HIEC细胞增殖能力的影响，然后用微核法，彗星尾实验，细胞免疫荧光观察丹参素对辐照细胞DNA的保护作用，最后分别使用荧光探针和生化方法检测丹参素对辐照损伤细胞的线粒体和氧化还原系统的保护作用。
为了研究丹参素对辐照损伤细胞中凋亡相关的信号通路的影响，取对数期生长的L-02细胞与丹参素共孵育1h后给予γ射线照射，继续分别培养不同时间，用WesternBlot方法检测细胞中凋亡通路、DNA修复、JNK通路和Ca~(2+)通路相关蛋白的表达，用比色法检测细胞凋亡通路中蛋白激酶的变化。
实验结果：
(1)丹参素对辐射损伤小鼠的保护作用
丹参素可以提高8Gy照射后30天小鼠的存活率，可以明显抑制4Gyγ射线照射后28天内外周血红细胞、白细胞、血红蛋白和血小板减少。
丹参素可减轻由辐照所致的小鼠脾脏重量的过度减轻，而且经丹参素预处理后，受照小鼠内源性脾结节较照射组均明显增多。形态学观察表明，4Gyγ射线照射后即可见脾脏体积明显缩小，脾切面上脾小体缩小或完全消失，大量淋巴细胞凋亡，而照前给予丹参素则能有效的抑制辐射引起的生发中心的萎缩，减少淋巴细胞凋亡。同时经丹参素处理过的小鼠骨髓细胞中的有核细胞计数明显多于照射组，表明丹参素能增强骨髓细胞对辐射的耐受性。丹参素预处理能增强小鼠肝脏抗氧化酶类的活性，减少肝脏蛋白的氧化水平并抑制肝脏的脂质过氧化水平。
(2)丹参素预处理对四种人正常细胞的毒性和辐射防护作用
MTT法实验结果显示，不同浓度的丹参素对L-02、HIEC、GES、HaCaT四种人正常细胞都没有产生明显毒性，而且在孵育1h时，丹参素对四种细胞均表现出促进增殖的作用，因此我们选择丹参素提前孵育1h做后续的辐射防护实验。在之后的辐射防护实验中，四种细胞的活力在经过γ射线的照射后都有不同程度的下降，而丹参素1h的预处理则能明显提高四种细胞的生存数量，其中10μg/ml丹参素的作用效果最明显。根据细胞对γ射线和丹参素的敏感性，我们选用了L-02和HIEC两种细胞做后续实验。
(3)丹参素对L-02和HIEC细胞辐射损伤的防护作用
首先细胞克隆形成和凋亡ER实验的结果表明细胞辐照前1h给予丹参素孵育能显著提高细胞的增殖能力，增加细胞的克隆形成数(P<0.01)，并能显著减少细胞凋亡(P<0.01)。而且丹参素预处理也能明显减少辐射损伤细胞形态的改变和细胞核的损伤。
与单纯照射组相比，丹参素预处理组γ-H2AX活化的阳性细胞数以及活化强度有明显减轻，说明丹参素降低了γ射线辐照后细胞DNA受损程度。另外微核实验中丹参素的预处理能够明显减少微核的形成率，减轻辐射诱导的细胞核的损伤。彗星尾实验中，丹参素预处理组较单纯照射组中细胞的尾长缩短、尾部DNA的百分比下降、尾矩减小，显示丹参素对辐射损伤的DNA有保护作用。
对细胞线粒体膜电位的测定结果显示丹参素预处理过的细胞表现出膜电位恢复的现象(P<0.01)。另外，我们的研究发现丹参素预处理能明显减少辐射在L-02和HIEC细胞内的ROS(P<0.05)，还能在一定程度上恢复细胞内抗氧化酶的活性，增加抗氧化物质的含量，减轻细胞脂质过氧化的程度，维持细胞氧化还原系统的正常功能。
(4)丹参素对辐射诱导细胞中凋亡相关通路活化的抑制作用
凋亡相关蛋白的检测结果显示照射后48h细胞内，与单纯照射组相比，Bax/Bcl-2的比值随着丹参素浓度的升高而有显著降低，而p53的表达随着预处理丹参素浓度的升高也是有显著降低，。这些说明丹参素能够促进抗凋亡蛋白的表达，抑制促凋亡蛋白的表达。细胞经过4Gyγ射线照射后，从线粒体释放到胞浆中的Cyt-C和AIF的蛋白含量显著上升，而经丹参素预处理之后再给予4Gyγ射线照射能够降低Cyt-C和AIF的释放，从而抑制由线粒体损伤引起的细胞凋亡。另外，细胞内由辐射引起的Caspase3/8/9蛋白激酶的活化在丹参素的干预下都有所减少，上述结果表明，丹参素能够通过抑制蛋白激酶的活化从而降低辐射诱导的细胞凋亡。
(5)丹参素对辐射损伤细胞DNA的保护作用
细胞内γ-H2AX在4Gy射线照射后，即刻开始出现，3h达到最高峰；而经过丹参素干预的实验组γ-H2AX在照后1h就达到最高峰；说明丹参素预处理能够加快H2AX的磷酸化，促进损伤DNA的修复。
辐照组γ-H2AX的上游分子ATM的表达水平从照后即刻开始到辐照后3h，逐渐增加，之后反而下降。而丹参素处理组中ATM从照后即刻开始直至照后3h一直处于活化状态。RT-PCR的结果显示经丹参素预处理的辐射损伤细胞中的ATM上调最多，但是4Gy单纯照射组和丹参素+辐照组与对照相比均无统计学差异。这说明丹参素对辐射损伤的细胞中ATM表达的影响主要集中在蛋白的合成和降解上。
(6)丹参素对辐射诱导细胞中JNK信号通路活化的抑制作用
丹参素预处理L-02细胞1h后经4Gyγ射线照射，继续培养24小时后，p38和JNK的表达均无明显变化，磷酸化的p38蛋白也没有明显变化，而磷酸化的JNK蛋白，p-JNK在受照后显著增加，而丹参素的预处理则能抑制辐射诱导的JNK蛋白的磷酸化，说明丹参素抑制辐射诱导的JNK激酶的磷酸化。经过丹参素预处理4Gyγ射线照射，继续培养12小时后，JNK激酶的上游分子p-MKK4的表达水平在辐照后显著升高，而丹参素预处理能降低p-MKK4的表达水平，MKK4的表达没有变化，NFκB的表达水平在细胞辐照后有所上升，但是丹参素对于辐照后NFκB的表达没有影响。
使用JNK的特异性抑制剂SP600125，辐照后36hJNK通路下游分子c-Jun和ATF-2的表达在丹参素作用下被抑制，而在正常情况下对它们的表达没有影响。此结果表明，丹参素能够抑制MKK4/JNK通路的活化，从而减少辐射诱导的细胞凋亡。
(7)丹参素对辐射诱导细胞中Ca~(2+)介导的凋亡通路活化的抑制
丹参素预处理L-02细胞1h后经4Gyγ射线照射，照后3h细胞内Ca~(2+)由内质网释放，与对照组相比，丹参素预处理组的Ca~(2+)释放强度和阳性细胞数则都有所减弱。Ca~(2+)通路的钙调蛋白calpain-2在4Gyγ射线照后表达量显著升高，而在丹参素预处理组中钙调蛋白calpain-2的表达量升高幅度明显降低。
结论：
1.在体实验结果说明丹参素能明显延长和提高辐照小鼠的生存时间和存活率，丹参素能明显降低受照小鼠外周血、脾脏、骨髓细胞的损伤程度，并能增强机体的抗氧化能力。
2.丹参素对L-02、HIEC、GES、HaCaT细胞没有明显的细胞毒性，并且其辐射防护效果显著。
3.丹参素能抑制γ射线导致的细胞凋亡，促进细胞增殖，其作用与保护细胞DNA，清除ROS，增强细胞的抗氧化力有关。
4．丹参素是通过抑制线粒体介导和钙离子诱导的细胞凋亡途径、促进细胞DNA损伤后修复分子的活化、抑制辐射诱导的JNK途径的激活发挥辐射保护作用的。
Background:
As the wide application of nuclear energy, the opportunity of people exposed to radiation is increasing, from the food irradiation, accidents in nuclear power plants, medical treatment to ionizing radiation in space, radiation is everywhere. High-dose radiation can cause a variety of the living beings injuries or damages. The risk of radiation induced malignancies as a result of genotoxicity to normal cells is one of the most serious problems during radiotherapy in patients with disease free survival. Tumor control without producing damage to the surrounding normal tissues through the use of chemical agents has a clinical relevance in radiotherapy. Therefore, protection of surrounding normal tissues has a practical relevance in improving the therapeutic outcome. Radioprotectors are needed to protect cancer patient with radiotherapy and other potentially exposed populations, such as workers in the nuchear-power industry, space travelers or operator on radioactive devices.
Ionizing radiation is considered as ubiquitous environmental carcinogen damaging DNA directly by energy deposition or indirectly by the generation of reactive oxygen species and free radicals. Reactive oxygen species and free radicals react with cellular macromolecules (i.e., proteins, carbohydrates, lipids and nucleic acids) leading to cell death or mutations and chromosome instability when they are not repaired or misrepaired. Several endogenous antioxidant enzymes (such as superoxide dismutase, catalase and glutathione peroxidase) are capable of scavenging reactive oxygen species and has shown pivotal role in the repair of DNA damage induced by reactive oxygen species.
Although many natural and synthetic/semi-synthetic chemicals have been investigated in the recent past for their efficacy to reduce adverse effects of ionizing radiation, the inherent toxicity of some of the synthetic agents at their radioprotective concentrations necessitated further search for secured and efficient compounds. In view of these limitations, an approach to evaluate the radiation protective ability of non-toxic and physiologically acceptable compounds seems to be hopeful and deserves investigation.
Salvianic acid A (SAA) is the major water-soluble components of Salvia miltiorrhiza Bunge and with the molecular structure of which is d(+)-(3,4-dihydroxyphenyl) lactic acid. SAA has been reported to possess several pharmacological activities, which includes protection of cardiac muscle, dilatation of blood vessel, inhibition of arteriosclerosis and thrombus, improvement of microcirculation, regulation of restoration and regeneration of tissue, antisepsis and anti-inflammation and so on. And our previous studies have shown that SAA could scavenge lipid free radicals and inhibit mice death and damage induced by radiation. Hence the aim of our present study was to investigate the radioprotective effect of SAA on γ-radiation induced damage and its possible mechanism.
Aims:
1. To explore the radioprotective activity of SAA in vivo;
2. To study on the radioprotection of SAA on multiple human normal cell types and to investigate on the protection of SAA on the cell morphology, DNA and mitochondria damaged by radiation;
3. To identify the mechanisms and involved signal pathway of the radioprotection of SAA.
Methods:
Animal experiment
Male BALB/c mice (22±2g), were randomly divided into five groups,1)saline control group,2) γ-radiation group,3)10mg/Kg SAA group,4)20mg/Kg SAA group,5) E3group. After treatment, the mice were fed for different times for the survival studies, hematological study, spleen histopathological and colony count study, nucleated cells analysis of mouse bone marrow and estimation of lipid peroxidation and antioxidant enzymes。
Cell experiment
In our present study, we investigate the radioprotective efficacy and cytotoxicity of SAA in vitro by using four types of human normal cells. Radiation-induced DNA damage, apoptosis and changes of cell morphology in human intestinal epithelial cells (HIEC) and human normal liver cells (L-02) in the presence and absence of SAA were examined. Finally, we observe the effect of SAA on ROS formation, the protection on mitochondria and the balance of the pro-oxidant and antioxidant in the cells.
Cells were allowed to attach for24h; and then treated with SAA1h before γ-radiation and after a further culture for different times. And then cells were investigated for the expressions of proteins in the apoptosis signal pathway, DNA repaired signal pathway, INK pathway and Ca2+pathway and the activities of caspases.
Results:
(1) The radioprotective activity of SAA in vivo.
SAA increased survival rate, the spleen index and clone of the irradiated mouse. The results also showed that the whole blood cell and the nucleated cells of bone marrow were decreased after the irradiation, and SAA can reverse these damages caused by radiation.
Splenic corpuscles had a uniform distribution and clear boundaries, and appeared uniformly blue in normal mice, in mice from the radiation-alone group splenic corpuscles were deflated and their number decreased. Compared with radiation-alone group, the splenic corpuscle in mice treated with10and20mg/kg SAA before irradiation, increased and distributed uniformly. In present of SAA, the activities of SOD and CAT and the content of GSH were increased, protein and lipid peroxidation were inhibited in liver.
(2) The cytotoxicity and radioprotection of SAA on multiple normal cell types
The results showed that different concentrations of SAA exhibited no significant cytotoxicities on L-02、HIEC、GES、HaCaT cells, and SAA could improve the viability of these cells after1h incubation. Pretreatment (1h) of cells with0.1,1,10μg/ml SAA provided protection compared to cells treated with radiation alone. Moreover, the maximum and marked protection achieved at10μg/ml SAA with radiation at4Gy.
(3) Pretreatment with SAA reduced radiation-induced cell damages
Compared with4Gy group, SAA could significantly increase the clone count and decrease apoptosis and promote proliferation on radiated cells (P<0.01). It can also maintain the morphology of cells and nuclear.
The foci positive cells and fluorescence intensity of y-H2AX were significantly reduced which means the DNA damage was alleviated. Micronuclei assay and the comet assay also showed the same results.
It is possible that SAA can protect mitochondrial from radiation induced damages by maintaining mitochondrial membrane potential and scavenging ROS. The results indicated that SAA can antagonize the reduction in the levels of both enzymatic and non-enzymatic antioxidants in irradiated cells. An increase in the level of TBARS induced by γ-radiation (4Gy) was effectively reduced by pretreatment with10μg/ml SAA (P<0.05).
(4) Inhibition of SAA on radiation-induced activated cell apoptosis related signal pathway
Western blotting results showed that the SAA-mediated radioprotection also involved down-regulating Bax and P53expressions and up-regulating Bcl-2expression, and suggested that SAA treatment shifted the balance between pro-and anti-apoptotic proteins towards cell survival.
Pretreatment of cells with SAA for1h before γ-radiation was found to significantly block the release of cytochrome c and AIF from mitochondria, and these changes were statistically significant (P<0.05). SAA treatment also significantly reversed the increase of caspase-3/8/9activity in irradiated cells (P<0.01). This result implied that inhibition of the mitochondria-mediated apoptotic pathway was involved in the radioprotective effect of SAA.
(5) Protection of SAA against radiation-induced DNA damage
When exposed to4Gy γ-radiation, cells showed a prompt and temporary increase in the expression of y-H2AX within3h. When the cells were pretreated with SAA before4Gy γ-radiation, the peak expression of y-H2AX occurred at1h. Western blotting results showed that SAA significantly promoted the expression of DNA repair-related factor, ATM. And the results of RT-PCR indicated that change of ATM expression was related to protein degradation.
(6) Inhibition of SAA on the radiation-induced activated JNK pathway
SAA was added to cultured cells maintaining the final concentration (1μg/ml) for1h before y-radiation (4Gy) and after a further culture for24h, the expressions of p38, JNK, p-p38had no significant changes. But JNK phosphorylation was greatly increased by radiation and SAA could inhibit the phosphorylation of JNK (p-JNK). And radiation-induced activity of the upstream molecular MKK4of the JNK pathway could also be inhibited by SAA. The NFκB pathway can not be influenced by SAA. The inhibitor of JNK had almost the same effect with SAA in radiated-cells. It is possible that SAA achieved its radioprotective action, at least in part, by suppressing the MKK4/JNK pathway and reduced the radiation-induced apoptosis.
(7) Inhibition of SAA on the radiation-induced activated Ca2+pathway
SAA was added to cultured cells maintaining the final concentration (lμg/ml) for1h before y-radiation (4Gy) and after a further culture for3h, the concentrations of Ca2+in cytoplasm were significantly increased in the irradiation group. The positive cells and fluorescence intensity of Ca2+in cells treated with SAA were decreased. Western blotting results showed that the level of calmodulin protein, calpain-2, was up-regulated by the radiation and down-regulated in the SAA pretreated cells.
Conclusions:
1. SAA increased survival rate, the spleen index and clone of the irradiated mouse and could reverse the decrease of the whole blood cell and the nucleated cells of bone marrow induced by radiation. It is clear that SAA could increase antioxidative ability and reduce lipid peroxidation induced by radiation in mice.
2. SAA exhibited no significant cytotoxicities on L-02、HIEC、GES、HaCaT cells, and preincubation1h of SAA could improve the viability of these cells after radiation.
3. SAA exerted its protective effect on the proliferative activity of L-02and HIEC cells as evidenced by decreased cytotoxicity after exposure to γ-radiation. It was possible that SAA achieved its radioprotective action, at least in part, by reducing DNA damage, enhancing the activity of antioxidant enzymes and scavenging ROS.
4. SAA exhibited its radioprotective activity via enhancing DNA repair and inhibiting the activities of JNK pathway and mitochondria and/or Ca2+-dependent apoptotic pathway in L-02cell line.
近年来，核能和核技术广泛应用于能源、医疗、军事、食品加工、育种等国防和国民生活的方方面面，与人们日常生活息息相关。目前，可能受到放射线损伤的人群主要有接受放疗的癌症病人；从事与放射相关的工作人员，如放射科室的医务人员、核电站与辐射加工企业的工作人员、从事与核科学有关研究的科研工作者、各种核事故的波及人员以及宇航人员等，辐射在给人类造福的同时，对人们的健康也带来不同程度的威胁。
电离辐射对生物体造成损伤主要通过两种方式，一是直接作用，指由射线造成生物大分子的损伤，电离辐射的能量直接沉积于生物大分子，造成DNA链的断裂、蛋白酶失活或者破坏细胞内的膜的结构或通透性，从而影响细胞的正常功能。二是间接作用，指生物大分子的破坏和失活是由于电离辐射作用于水分子，继而水的辐射分解产物再作用于生物大分子，引起后者的物理和化学变化，从而诱导细胞凋亡。细胞经受辐射的直接作用和间接作用后，细胞膜和细胞内DNA发生改变以及大量ROS的产生，诱导细胞内多种信号分子的活化，或信号通路的激活，最终导致辐射后细胞凋亡、癌变，甚至机体死亡。
电离辐射对机体的损伤极大。迄今为止，虽然发现了一些有效预防损伤的化合物，如五十年前就已经发现了氨巯基化合物，三十年前合成的WR系列化合物等，这些已开发的抗辐射药物尽管拥有很好的抗辐射作用，但尚存在毒性大、不良反应多等多方面的不足，人们迫切希望能开发出毒性小，可用于临床防治电离辐射损伤的药物。丹参素(SalvianicacidA)是从唇形科植物丹参水溶性成分中提取的多酚
类物质，大量实验报道，丹参素具有抗炎、增强机体免疫力、抗肿瘤、保护肝脏、抑制肝纤维化、保护心肌细胞及中枢神经系统、防治心脑血管疾病的作用。我们前期实验发现，丹参素能广泛地清除各种自由基，降低小鼠经电离辐照后的死亡率，并明显减轻在体和离体实验中辐射引起的各种损伤。本实验拟进一步观察丹参素的辐射防护作用，并研究丹参素的辐射防护作用是否与其保护细胞DNA不受损伤、抑制细胞凋亡有关，进一步在细胞实验中探索丹参素发挥辐射防护作用时参与的信号通路及其可能的作用机制。
实验目的：
1.观察丹参素对辐射损伤小鼠的保护作用。
2.观察丹参素对辐射引起人正常细胞生长抑制的作用；并进一步研究丹参素对辐射损伤细胞形态、DNA、线粒体等的保护作用；
3.探索丹参素辐射防护作用的可能机制以及信号通路；
实验方法：
动物实验
雄性BALB/c小鼠(22±2g)适应环境后，随机分为五组:1)生理盐水对照组，2)单独照射组，3)丹参素10mg/Kg组，4)丹参素20mg/Kg组，5)阳性对照雌三醇（E3，2mg/Kg）组。不同浓度丹参素灌胃7天，阳性对照E3组在辐照前48h,24h,0h分别腹腔给药3次，然后给予不同剂量的γ射线照射。并于辐照后不同时间取材，观察丹参素对辐射损伤小鼠的生存率的影响，外周血中白细胞、红细胞、血红蛋白和血小板含量的变化，脾脏的病理学以及脾脏造血干细胞克隆形成率的改变，骨髓有核细胞数以及小鼠肝脏抗氧化能力的变化。
细胞实验
借助MTT法检测丹参素对四种人正常细胞的毒性和辐射防护作用，进一步采用克隆形成法观察丹参素对辐射损伤的L-02和HIEC细胞增殖能力的影响，然后用微核法，彗星尾实验，细胞免疫荧光观察丹参素对辐照细胞DNA的保护作用，最后分别使用荧光探针和生化方法检测丹参素对辐照损伤细胞的线粒体和氧化还原系统的保护作用。
为了研究丹参素对辐照损伤细胞中凋亡相关的信号通路的影响，取对数期生长的L-02细胞与丹参素共孵育1h后给予γ射线照射，继续分别培养不同时间，用WesternBlot方法检测细胞中凋亡通路、DNA修复、JNK通路和Ca~(2+)通路相关蛋白的表达，用比色法检测细胞凋亡通路中蛋白激酶的变化。
实验结果：
(1)丹参素对辐射损伤小鼠的保护作用
丹参素可以提高8Gy照射后30天小鼠的存活率，可以明显抑制4Gyγ射线照射后28天内外周血红细胞、白细胞、血红蛋白和血小板减少。
丹参素可减轻由辐照所致的小鼠脾脏重量的过度减轻，而且经丹参素预处理后，受照小鼠内源性脾结节较照射组均明显增多。形态学观察表明，4Gyγ射线照射后即可见脾脏体积明显缩小，脾切面上脾小体缩小或完全消失，大量淋巴细胞凋亡，而照前给予丹参素则能有效的抑制辐射引起的生发中心的萎缩，减少淋巴细胞凋亡。同时经丹参素处理过的小鼠骨髓细胞中的有核细胞计数明显多于照射组，表明丹参素能增强骨髓细胞对辐射的耐受性。丹参素预处理能增强小鼠肝脏抗氧化酶类的活性，减少肝脏蛋白的氧化水平并抑制肝脏的脂质过氧化水平。
(2)丹参素预处理对四种人正常细胞的毒性和辐射防护作用
MTT法实验结果显示，不同浓度的丹参素对L-02、HIEC、GES、HaCaT四种人正常细胞都没有产生明显毒性，而且在孵育1h时，丹参素对四种细胞均表现出促进增殖的作用，因此我们选择丹参素提前孵育1h做后续的辐射防护实验。在之后的辐射防护实验中，四种细胞的活力在经过γ射线的照射后都有不同程度的下降，而丹参素1h的预处理则能明显提高四种细胞的生存数量，其中10μg/ml丹参素的作用效果最明显。根据细胞对γ射线和丹参素的敏感性，我们选用了L-02和HIEC两种细胞做后续实验。
(3)丹参素对L-02和HIEC细胞辐射损伤的防护作用
首先细胞克隆形成和凋亡ER实验的结果表明细胞辐照前1h给予丹参素孵育能显著提高细胞的增殖能力，增加细胞的克隆形成数(P<0.01)，并能显著减少细胞凋亡(P<0.01)。而且丹参素预处理也能明显减少辐射损伤细胞形态的改变和细胞核的损伤。
与单纯照射组相比，丹参素预处理组γ-H2AX活化的阳性细胞数以及活化强度有明显减轻，说明丹参素降低了γ射线辐照后细胞DNA受损程度。另外微核实验中丹参素的预处理能够明显减少微核的形成率，减轻辐射诱导的细胞核的损伤。彗星尾实验中，丹参素预处理组较单纯照射组中细胞的尾长缩短、尾部DNA的百分比下降、尾矩减小，显示丹参素对辐射损伤的DNA有保护作用。
对细胞线粒体膜电位的测定结果显示丹参素预处理过的细胞表现出膜电位恢复的现象(P<0.01)。另外，我们的研究发现丹参素预处理能明显减少辐射在L-02和HIEC细胞内的ROS(P<0.05)，还能在一定程度上恢复细胞内抗氧化酶的活性，增加抗氧化物质的含量，减轻细胞脂质过氧化的程度，维持细胞氧化还原系统的正常功能。
(4)丹参素对辐射诱导细胞中凋亡相关通路活化的抑制作用
凋亡相关蛋白的检测结果显示照射后48h细胞内，与单纯照射组相比，Bax/Bcl-2的比值随着丹参素浓度的升高而有显著降低，而p53的表达随着预处理丹参素浓度的升高也是有显著降低，。这些说明丹参素能够促进抗凋亡蛋白的表达，抑制促凋亡蛋白的表达。细胞经过4Gyγ射线照射后，从线粒体释放到胞浆中的Cyt-C和AIF的蛋白含量显著上升，而经丹参素预处理之后再给予4Gyγ射线照射能够降低Cyt-C和AIF的释放，从而抑制由线粒体损伤引起的细胞凋亡。另外，细胞内由辐射引起的Caspase3/8/9蛋白激酶的活化在丹参素的干预下都有所减少，上述结果表明，丹参素能够通过抑制蛋白激酶的活化从而降低辐射诱导的细胞凋亡。
(5)丹参素对辐射损伤细胞DNA的保护作用
细胞内γ-H2AX在4Gy射线照射后，即刻开始出现，3h达到最高峰；而经过丹参素干预的实验组γ-H2AX在照后1h就达到最高峰；说明丹参素预处理能够加快H2AX的磷酸化，促进损伤DNA的修复。
辐照组γ-H2AX的上游分子ATM的表达水平从照后即刻开始到辐照后3h，逐渐增加，之后反而下降。而丹参素处理组中ATM从照后即刻开始直至照后3h一直处于活化状态。RT-PCR的结果显示经丹参素预处理的辐射损伤细胞中的ATM上调最多，但是4Gy单纯照射组和丹参素+辐照组与对照相比均无统计学差异。这说明丹参素对辐射损伤的细胞中ATM表达的影响主要集中在蛋白的合成和降解上。
(6)丹参素对辐射诱导细胞中JNK信号通路活化的抑制作用
丹参素预处理L-02细胞1h后经4Gyγ射线照射，继续培养24小时后，p38和JNK的表达均无明显变化，磷酸化的p38蛋白也没有明显变化，而磷酸化的JNK蛋白，p-JNK在受照后显著增加，而丹参素的预处理则能抑制辐射诱导的JNK蛋白的磷酸化，说明丹参素抑制辐射诱导的JNK激酶的磷酸化。经过丹参素预处理4Gyγ射线照射，继续培养12小时后，JNK激酶的上游分子p-MKK4的表达水平在辐照后显著升高，而丹参素预处理能降低p-MKK4的表达水平，MKK4的表达没有变化，NFκB的表达水平在细胞辐照后有所上升，但是丹参素对于辐照后NFκB的表达没有影响。
使用JNK的特异性抑制剂SP600125，辐照后36hJNK通路下游分子c-Jun和ATF-2的表达在丹参素作用下被抑制，而在正常情况下对它们的表达没有影响。此结果表明，丹参素能够抑制MKK4/JNK通路的活化，从而减少辐射诱导的细胞凋亡。
(7)丹参素对辐射诱导细胞中Ca~(2+)介导的凋亡通路活化的抑制
丹参素预处理L-02细胞1h后经4Gyγ射线照射，照后3h细胞内Ca~(2+)由内质网释放，与对照组相比，丹参素预处理组的Ca~(2+)释放强度和阳性细胞数则都有所减弱。Ca~(2+)通路的钙调蛋白calpain-2在4Gyγ射线照后表达量显著升高，而在丹参素预处理组中钙调蛋白calpain-2的表达量升高幅度明显降低。
结论：
1.在体实验结果说明丹参素能明显延长和提高辐照小鼠的生存时间和存活率，丹参素能明显降低受照小鼠外周血、脾脏、骨髓细胞的损伤程度，并能增强机体的抗氧化能力。
2.丹参素对L-02、HIEC、GES、HaCaT细胞没有明显的细胞毒性，并且其辐射防护效果显著。
3.丹参素能抑制γ射线导致的细胞凋亡，促进细胞增殖，其作用与保护细胞DNA，清除ROS，增强细胞的抗氧化力有关。
4．丹参素是通过抑制线粒体介导和钙离子诱导的细胞凋亡途径、促进细胞DNA损伤后修复分子的活化、抑制辐射诱导的JNK途径的激活发挥辐射保护作用的。
Background:
As the wide application of nuclear energy, the opportunity of people exposed to radiation is increasing, from the food irradiation, accidents in nuclear power plants, medical treatment to ionizing radiation in space, radiation is everywhere. High-dose radiation can cause a variety of the living beings injuries or damages. The risk of radiation induced malignancies as a result of genotoxicity to normal cells is one of the most serious problems during radiotherapy in patients with disease free survival. Tumor control without producing damage to the surrounding normal tissues through the use of chemical agents has a clinical relevance in radiotherapy. Therefore, protection of surrounding normal tissues has a practical relevance in improving the therapeutic outcome. Radioprotectors are needed to protect cancer patient with radiotherapy and other potentially exposed populations, such as workers in the nuchear-power industry, space travelers or operator on radioactive devices.
Ionizing radiation is considered as ubiquitous environmental carcinogen damaging DNA directly by energy deposition or indirectly by the generation of reactive oxygen species and free radicals. Reactive oxygen species and free radicals react with cellular macromolecules (i.e., proteins, carbohydrates, lipids and nucleic acids) leading to cell death or mutations and chromosome instability when they are not repaired or misrepaired. Several endogenous antioxidant enzymes (such as superoxide dismutase, catalase and glutathione peroxidase) are capable of scavenging reactive oxygen species and has shown pivotal role in the repair of DNA damage induced by reactive oxygen species.
Although many natural and synthetic/semi-synthetic chemicals have been investigated in the recent past for their efficacy to reduce adverse effects of ionizing radiation, the inherent toxicity of some of the synthetic agents at their radioprotective concentrations necessitated further search for secured and efficient compounds. In view of these limitations, an approach to evaluate the radiation protective ability of non-toxic and physiologically acceptable compounds seems to be hopeful and deserves investigation.
Salvianic acid A (SAA) is the major water-soluble components of Salvia miltiorrhiza Bunge and with the molecular structure of which is d(+)-(3,4-dihydroxyphenyl) lactic acid. SAA has been reported to possess several pharmacological activities, which includes protection of cardiac muscle, dilatation of blood vessel, inhibition of arteriosclerosis and thrombus, improvement of microcirculation, regulation of restoration and regeneration of tissue, antisepsis and anti-inflammation and so on. And our previous studies have shown that SAA could scavenge lipid free radicals and inhibit mice death and damage induced by radiation. Hence the aim of our present study was to investigate the radioprotective effect of SAA on γ-radiation induced damage and its possible mechanism.
Aims:
1. To explore the radioprotective activity of SAA in vivo;
2. To study on the radioprotection of SAA on multiple human normal cell types and to investigate on the protection of SAA on the cell morphology, DNA and mitochondria damaged by radiation;
3. To identify the mechanisms and involved signal pathway of the radioprotection of SAA.
Methods:
Animal experiment
Male BALB/c mice (22±2g), were randomly divided into five groups,1)saline control group,2) γ-radiation group,3)10mg/Kg SAA group,4)20mg/Kg SAA group,5) E3group. After treatment, the mice were fed for different times for the survival studies, hematological study, spleen histopathological and colony count study, nucleated cells analysis of mouse bone marrow and estimation of lipid peroxidation and antioxidant enzymes。
Cell experiment
In our present study, we investigate the radioprotective efficacy and cytotoxicity of SAA in vitro by using four types of human normal cells. Radiation-induced DNA damage, apoptosis and changes of cell morphology in human intestinal epithelial cells (HIEC) and human normal liver cells (L-02) in the presence and absence of SAA were examined. Finally, we observe the effect of SAA on ROS formation, the protection on mitochondria and the balance of the pro-oxidant and antioxidant in the cells.
Cells were allowed to attach for24h; and then treated with SAA1h before γ-radiation and after a further culture for different times. And then cells were investigated for the expressions of proteins in the apoptosis signal pathway, DNA repaired signal pathway, INK pathway and Ca2+pathway and the activities of caspases.
Results:
(1) The radioprotective activity of SAA in vivo.
SAA increased survival rate, the spleen index and clone of the irradiated mouse. The results also showed that the whole blood cell and the nucleated cells of bone marrow were decreased after the irradiation, and SAA can reverse these damages caused by radiation.
Splenic corpuscles had a uniform distribution and clear boundaries, and appeared uniformly blue in normal mice, in mice from the radiation-alone group splenic corpuscles were deflated and their number decreased. Compared with radiation-alone group, the splenic corpuscle in mice treated with10and20mg/kg SAA before irradiation, increased and distributed uniformly. In present of SAA, the activities of SOD and CAT and the content of GSH were increased, protein and lipid peroxidation were inhibited in liver.
(2) The cytotoxicity and radioprotection of SAA on multiple normal cell types
The results showed that different concentrations of SAA exhibited no significant cytotoxicities on L-02、HIEC、GES、HaCaT cells, and SAA could improve the viability of these cells after1h incubation. Pretreatment (1h) of cells with0.1,1,10μg/ml SAA provided protection compared to cells treated with radiation alone. Moreover, the maximum and marked protection achieved at10μg/ml SAA with radiation at4Gy.
(3) Pretreatment with SAA reduced radiation-induced cell damages
Compared with4Gy group, SAA could significantly increase the clone count and decrease apoptosis and promote proliferation on radiated cells (P<0.01). It can also maintain the morphology of cells and nuclear.
The foci positive cells and fluorescence intensity of y-H2AX were significantly reduced which means the DNA damage was alleviated. Micronuclei assay and the comet assay also showed the same results.
It is possible that SAA can protect mitochondrial from radiation induced damages by maintaining mitochondrial membrane potential and scavenging ROS. The results indicated that SAA can antagonize the reduction in the levels of both enzymatic and non-enzymatic antioxidants in irradiated cells. An increase in the level of TBARS induced by γ-radiation (4Gy) was effectively reduced by pretreatment with10μg/ml SAA (P<0.05).
(4) Inhibition of SAA on radiation-induced activated cell apoptosis related signal pathway
Western blotting results showed that the SAA-mediated radioprotection also involved down-regulating Bax and P53expressions and up-regulating Bcl-2expression, and suggested that SAA treatment shifted the balance between pro-and anti-apoptotic proteins towards cell survival.
Pretreatment of cells with SAA for1h before γ-radiation was found to significantly block the release of cytochrome c and AIF from mitochondria, and these changes were statistically significant (P<0.05). SAA treatment also significantly reversed the increase of caspase-3/8/9activity in irradiated cells (P<0.01). This result implied that inhibition of the mitochondria-mediated apoptotic pathway was involved in the radioprotective effect of SAA.
(5) Protection of SAA against radiation-induced DNA damage
When exposed to4Gy γ-radiation, cells showed a prompt and temporary increase in the expression of y-H2AX within3h. When the cells were pretreated with SAA before4Gy γ-radiation, the peak expression of y-H2AX occurred at1h. Western blotting results showed that SAA significantly promoted the expression of DNA repair-related factor, ATM. And the results of RT-PCR indicated that change of ATM expression was related to protein degradation.
(6) Inhibition of SAA on the radiation-induced activated JNK pathway
SAA was added to cultured cells maintaining the final concentration (1μg/ml) for1h before y-radiation (4Gy) and after a further culture for24h, the expressions of p38, JNK, p-p38had no significant changes. But JNK phosphorylation was greatly increased by radiation and SAA could inhibit the phosphorylation of JNK (p-JNK). And radiation-induced activity of the upstream molecular MKK4of the JNK pathway could also be inhibited by SAA. The NFκB pathway can not be influenced by SAA. The inhibitor of JNK had almost the same effect with SAA in radiated-cells. It is possible that SAA achieved its radioprotective action, at least in part, by suppressing the MKK4/JNK pathway and reduced the radiation-induced apoptosis.
(7) Inhibition of SAA on the radiation-induced activated Ca2+pathway
SAA was added to cultured cells maintaining the final concentration (lμg/ml) for1h before y-radiation (4Gy) and after a further culture for3h, the concentrations of Ca2+in cytoplasm were significantly increased in the irradiation group. The positive cells and fluorescence intensity of Ca2+in cells treated with SAA were decreased. Western blotting results showed that the level of calmodulin protein, calpain-2, was up-regulated by the radiation and down-regulated in the SAA pretreated cells.
Conclusions:
1. SAA increased survival rate, the spleen index and clone of the irradiated mouse and could reverse the decrease of the whole blood cell and the nucleated cells of bone marrow induced by radiation. It is clear that SAA could increase antioxidative ability and reduce lipid peroxidation induced by radiation in mice.
2. SAA exhibited no significant cytotoxicities on L-02、HIEC、GES、HaCaT cells, and preincubation1h of SAA could improve the viability of these cells after radiation.
3. SAA exerted its protective effect on the proliferative activity of L-02and HIEC cells as evidenced by decreased cytotoxicity after exposure to γ-radiation. It was possible that SAA achieved its radioprotective action, at least in part, by reducing DNA damage, enhancing the activity of antioxidant enzymes and scavenging ROS.
4. SAA exhibited its radioprotective activity via enhancing DNA repair and inhibiting the activities of JNK pathway and mitochondria and/or Ca2+-dependent apoptotic pathway in L-02cell line.
引文
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