丹参素对电离辐射损伤的防护作用及机制研究

发布时间：2018-05-12 10:49

本文选题：丹参素 + ROS　；参考：《第四军医大学》2012年博士论文

【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(P0.01)，并能显著减少细胞凋亡(P0.01)。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(P0.01)。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(P0.05)，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。
[Abstract]:Research background:
In recent years, nuclear and nuclear technology has been widely used in all aspects of national defense and national life, such as energy, medical treatment, military, food processing, breeding, and people's daily life. At present, people who are likely to be damaged by radiation are mainly cancer patients receiving radiotherapy; workers engaged in radiological related work, such as the medical department's medicine. Personnel, workers in nuclear power plants and radiation processing enterprises, scientific researchers involved in nuclear science research, all kinds of nuclear accidents, and Astronautics, and so on. Radiation brings benefits to human beings and poses a different degree of threat to people's health.
The damage of the ionizing radiation to the organism is mainly through two ways, one is the direct action, which refers to the damage of the biological macromolecules by the rays. The energy of the ionizing radiation is directly deposited in the biological macromolecules, causing the breakage of the DNA chain, the protease inactivating or destroying the structure or permeability of the membrane in the cells, thus affecting the normal function of the cells. Two It is an indirect effect, which means that the destruction and inactivation of biological macromolecules is due to the effect of ionizing radiation on water molecules, and then the radiation decomposition products of water are reacting on the biological macromolecules, causing the physical and chemical changes of the latter to induce apoptosis. After the cells undergo the direct and indirect effects of radiation, the changes in the cell membrane and intracellular DNA are changed. Change and the production of a large number of ROS, induce the activation of a variety of signal molecules in the cell, or the activation of the signal pathway, which eventually leads to cell apoptosis, canceration and even the body death after radiation.
Ionizing radiation has been a great damage to the body. So far, although some effective compounds have been discovered, such as the ammonia sulfhydryl compound and the WR series synthesized thirty years ago, the developed anti radiation drugs have good radiation resistance, but there are still toxic and adverse reactions. In many aspects, people are eager to develop drugs with small toxicity and can be used to prevent the damage of ionizing radiation in clinical. SalvianicacidA is a polyphenol extracted from the water-soluble constituents of Salvia miltiorrhiza in the lip family.
In a large number of experiments, a large number of experiments have reported that Danshensu has the effect of anti-inflammatory, enhancing the body's immunity, anti tumor, protecting the liver, inhibiting the liver fibrosis, protecting the myocardial cells and the central nervous system, preventing the cardiovascular and cerebrovascular diseases. This experiment is to further observe the radiation protection of Danshensu and to investigate whether the radiation protection of Danshensu is related to the protection of cell DNA from the cell apoptosis and the inhibition of cell apoptosis, and to further explore the radiation protection effect of Danshensu in the cell experiment. The signaling pathways involved and their possible mechanisms of action.
Objective:
1. to observe the protective effect of Danshensu on radiation injured mice.
2. observe the effect of Danshensu on the growth inhibition of human normal cells induced by radiation, and further study the protective effect of Danshensu on radiation damaged cell morphology, DNA, mitochondria and so on.
3. explore the possible mechanisms and signaling pathways of Danshensu radiation protection.
Experimental methods:
Animal experiment
After the male BALB/c mice (22 + 2G) adapted to the environment, they were randomly divided into five groups: 1) normal saline control group, 2) single irradiation group, 3) Danshensu 10mg/Kg group, 4) Salvia miltiorrhizin 20mg/Kg group, 5) positive control female three alcohol (E3,2mg/Kg) group. Different concentration of Salvia miltiorrhiza was fed on the stomach for 7 days, and the positive control group E3 was given 3 times before irradiation, 48h, 24h, and 0h, respectively, and then given the difference in the abdominal cavity, and then given the difference, and then given the difference, and then the difference of the E3 group. The effects of Danshensu on the survival rate of irradiated mice, the changes of white blood cells, red blood cells, hemoglobin and platelets in peripheral blood, the pathological changes of spleen and the change of the clone formation rate of splenic hematopoietic stem cells, the number of nucleated cells in bone marrow and the liver of mice, were observed at different time after irradiation. Changes in antioxidant capacity.
Cell experiment
The toxicity and radiation protection of Danshensu on four human normal cells were detected by MTT method, and the effect of Danshensu on the proliferation of L-02 and HIEC cells damaged by radiation was observed by clonogenic method. Then, micronuclear method, comet tail experiment, and cell immunofluorescence were used to observe the protective effect of Danshensu on irradiated cells DNA. The protective effects of Danshensu on mitochondria and redox system of irradiated cells were detected by fluorescence probe and biochemical method.
In order to study the effect of Danshensu on the signal pathway related to apoptosis in irradiated cells, the logarithmic phase of L-02 cells and Salvia miltiorrhiza were incubated for 1H and irradiated by gamma ray, and the apoptosis pathway in cells was detected by WesternBlot method, DNA repair, JNK pathway and Ca~ (2+) pathway related protein expression were used. The changes of protein kinase in apoptotic pathway were detected by colorimetry.
Experimental results:
(1) protective effect of Danshensu on Radiation Injured Mice
Danshensu could increase the survival rate of mice at 30 days after 8Gy irradiation, and obviously inhibit the decrease of erythrocyte, white blood cell, hemoglobin and platelets at 28 days after 4Gy gamma ray irradiation.
Danshensu alleviated the excessive lightened weight of spleen in mice induced by irradiation, and the endogenous spleen nodules in the irradiated mice increased significantly after the pretreatment with Danshensu. The morphological observation showed that the spleen volume was obviously reduced after 4Gy gamma ray irradiation, and the splenic corpuscle was narrowed or completely disappeared, and a large number of lymphocytes withered. In addition, Danshensu could effectively inhibit the atrophy of the germinal center caused by radiation and reduce the apoptosis of lymphocytes. At the same time, the number of nucleated cells in the bone marrow cells treated by Danshensu was obviously more than that in the irradiated group, indicating that Danshensu could enhance the tolerance of the bone marrow cells to the radiation. The salvia miltiorrhizin pretreatment could enhance the liver of mice. The activities of visceral antioxidant enzymes reduce the oxidation level of liver protein and inhibit the level of lipid peroxidation in liver.
(2) toxicity and radioprotective effect of Danshensu pretreatment on four kinds of human normal cells.
The results of MTT assay show that Danshensu has no obvious toxicity to four normal cells of L-02, HIEC, GES and HaCaT, and danshensu promotes proliferation when incubating 1H, so we choose Danshensu to incubate 1h in advance to do follow-up radiation protection experiment. In the test, the vitality of the four cells decreased in varying degrees after gamma ray irradiation, and the pretreatment of Danshensu 1H could significantly increase the number of four cells, of which 10 mu g/ml Danshensu was most effective. According to the sensitivity of the cells to gamma ray and danshensu, we chose two cells of L-02 and HIEC after being used. Continue the experiment.
(3) the protective effect of Danshensu on radiation injury of L-02 and HIEC cells
First, the results of cell clone formation and apoptosis ER show that Salvia miltiorrhiza incubation before irradiation can significantly improve cell proliferation, increase the number of cell clones (P0.01), and significantly reduce cell apoptosis (P0.01), and danshensu preconditioning can also significantly reduce the morphological changes of radiation damaged cells and nucleus. Damage.
Compared with the simple irradiation group, the number of positive cells activated by gamma -H2AX in the salvia miltiorrhizin preconditioning group and the activation intensity were significantly reduced, indicating that Danshensu reduced the damage degree of DNA after gamma ray irradiation. In addition, the pretreatment of Danshensu could reduce the formation rate of micronucleus obviously and reduce the damage of radiation induced nuclei. Comet comet. In the tail experiment, the tail length of the cells in the salvia miltiorrhiza preconditioning group shortened, the percentage of the tail DNA decreased and the tail moment decreased, indicating the protective effect of Danshen on the radiation damage of DNA.
The results of cell mitochondrial membrane potential showed that the cells pretreated by Danshensu showed the phenomenon of membrane potential recovery (P0.01). In addition, our study found that Danshensu preconditioning could significantly reduce the ROS (P0.05) of radiation in L-02 and HIEC cells, and also restore the activity of intracellular antioxidant enzymes and increase antioxidant activity at a certain range. The content of substance can reduce the degree of lipid peroxidation and maintain the normal function of cell redox system.
(4) Danshensu inhibits activation of apoptosis related pathways in radiation-induced cells.
The detection of apoptosis related proteins showed that the ratio of Bax/Bcl-2 in 48h cells decreased significantly with the increase of Salvia miltiorrhizin, while the expression of p53 was significantly decreased with the increase of the concentration of Salvia miltiorrhiza, which indicated that the expression of anti apoptotic protein and inhibition of the expression of anti apoptotic protein could be promoted. The expression of apoptotic protein. After 4Gy gamma ray irradiation, the protein content of Cyt-C and AIF released from mitochondria to cytoplasm increased significantly, and 4Gy gamma ray irradiation after Danshensu pretreatment could reduce the release of Cyt-C and AIF, thus inhibiting apoptosis induced by mitochondrial damage. In addition, intracellular radiation caused by radiation. The activation of Caspase3/8/9 protein kinase is reduced in the intervention of Danshensu. The above results show that Danshensu can reduce the apoptosis of radiation induced cells by inhibiting the activation of protein kinase.
(5) Danshensu protects DNA cells from radiation injury.
The intracellular gamma -H2AX began to appear immediately after 4Gy ray, and 3H reached the peak, while the experimental group after Danshensu intervention reached the peak of 1h after irradiation, indicating that the pretreatment of Danshensu could accelerate the phosphorylation of H2AX and promote the repair of the injured DNA.
The expression level of the upstream molecule ATM of the irradiated group gamma -H2AX begins immediately after irradiation to the irradiated 3h, and gradually increases, and then decreases, while the ATM from the Danshen treatment group begins immediately after the illumination until 3H has been in the activated state.RT-PCR, indicating that the ATM in the irradiated cells pretreated by Danshensu is up to the most, but 4Gy There was no significant difference in the comparison between the single irradiated group and the irradiated group of Danshensu + and the irradiated group. This indicated that the effect of Danshensu on the expression of ATM in the irradiated cells was mainly concentrated on the synthesis and degradation of the protein.
(6) Danshensu inhibits activation of JNK signaling pathway in radiation-induced cells.
After irradiated by Danshensu L-02 cell 1h after 4Gy gamma ray irradiation for 24 hours, the expression of p38 and JNK had no obvious changes, and the phosphorylated p38 protein had no obvious change, but the phosphorylated JNK protein, p-JNK was significantly increased after exposure, and the pretreatment of Danshensu could inhibit the phosphorylation of the radiation induced JNK protein, indicating the salvia miltiorrhiza. The expression level of p-MKK4 in the upstream molecule of JNK kinase increased significantly after irradiated by Danshensu pretreated 4Gy gamma ray after irradiation for 12 hours after irradiation. The expression level of p-MKK4 could be reduced and the expression of MKK4 was not changed. The expression level of NF kappa B was irradiated by cells after irradiation for 12 hours. However, Danshensu has no effect on the expression of NF kappa B after irradiation.
Using the specific inhibitor of JNK, SP600125, the expression of c-Jun and ATF-2 of the downstream molecules of the 36hJNK pathway was inhibited under the action of Danshensu after irradiation, but it did not affect their expression in normal conditions. The results showed that Danshensu could inhibit the activation of MKK4/JNK pathway and reduce the apoptosis induced by less radiation.
(7) Danshensu inhibits activation of Ca~ (2+) - mediated apoptosis pathway in radiation-induced cells.
Danshensu pretreated L-02 cell 1H irradiated by 4Gy gamma ray, and Ca~ (2+) was released from endoplasmic reticulum in 3H cells after irradiation. Compared with the control group, the Ca~ (2+) release strength and the number of positive cells in the salvia miltiorrhiza preconditioning group decreased the.Ca~ (2+) pathway of the calmodulin significantly increased after the 4Gy gamma ray, and the salvia miltiorrhiza pretreatment The expression of calmodulin calpain-2 increased significantly in the group.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R142

【参考文献】