本文選題：丹參素 + ROS��； 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究背景： 近年來(lái)，核能和核技術(shù)廣泛應(yīng)用于能源、醫(yī)療、軍事、食品加工、育種等國(guó)防和國(guó)民生活的方方面面，與人們?nèi)粘Ｉ钕⑾⑾嚓P(guān)。目前，可能受到放射線(xiàn)損傷的人群主要有接受放療的癌癥病人；從事與放射相關(guān)的工作人員，如放射科室的醫(yī)務(wù)人員、核電站與輻射加工企業(yè)的工作人員、從事與核科學(xué)有關(guān)研究的科研工作者、各種核事故的波及人員以及宇航人員等，輻射在給人類(lèi)造福的同時(shí)，對(duì)人們的健康也帶來(lái)不同程度的威脅。 電離輻射對(duì)生物體造成損傷主要通過(guò)兩種方式，一是直接作用，指由射線(xiàn)造成生物大分子的損傷，電離輻射的能量直接沉積于生物大分子，造成DNA鏈的斷裂、蛋白酶失活或者破壞細(xì)胞內(nèi)的膜的結(jié)構(gòu)或通透性，從而影響細(xì)胞的正常功能。二是間接作用，指生物大分子的破壞和失活是由于電離輻射作用于水分子，繼而水的輻射分解產(chǎn)物再作用于生物大分子，引起后者的物理和化學(xué)變化，從而誘導(dǎo)細(xì)胞凋亡。細(xì)胞經(jīng)受輻射的直接作用和間接作用后，細(xì)胞膜和細(xì)胞內(nèi)DNA發(fā)生改變以及大量ROS的產(chǎn)生，誘導(dǎo)細(xì)胞內(nèi)多種信號(hào)分子的活化，或信號(hào)通路的激活，最終導(dǎo)致輻射后細(xì)胞凋亡、癌變，甚至機(jī)體死亡。 電離輻射對(duì)機(jī)體的損傷極大。迄今為止，雖然發(fā)現(xiàn)了一些有效預(yù)防損傷的化合物，如五十年前就已經(jīng)發(fā)現(xiàn)了氨巰基化合物，三十年前合成的WR系列化合物等，這些已開(kāi)發(fā)的抗輻射藥物盡管擁有很好的抗輻射作用，但尚存在毒性大、不良反應(yīng)多等多方面的不足，人們迫切希望能開(kāi)發(fā)出毒性小，可用于臨床防治電離輻射損傷的藥物。丹參素(SalvianicacidA)是從唇形科植物丹參水溶性成分中提取的多酚 類(lèi)物質(zhì)，大量實(shí)驗(yàn)報(bào)道，丹參素具有抗炎、增強(qiáng)機(jī)體免疫力、抗腫瘤、保護(hù)肝臟、抑制肝纖維化、保護(hù)心肌細(xì)胞及中樞神經(jīng)系統(tǒng)、防治心腦血管疾病的作用。我們前期實(shí)驗(yàn)發(fā)現(xiàn)，丹參素能廣泛地清除各種自由基，降低小鼠經(jīng)電離輻照后的死亡率，并明顯減輕在體和離體實(shí)驗(yàn)中輻射引起的各種損傷。本實(shí)驗(yàn)擬進(jìn)一步觀(guān)察丹參素的輻射防護(hù)作用，并研究丹參素的輻射防護(hù)作用是否與其保護(hù)細(xì)胞DNA不受損傷、抑制細(xì)胞凋亡有關(guān)，，進(jìn)一步在細(xì)胞實(shí)驗(yàn)中探索丹參素發(fā)揮輻射防護(hù)作用時(shí)參與的信號(hào)通路及其可能的作用機(jī)制。 實(shí)驗(yàn)?zāi)康模?1.觀(guān)察丹參素對(duì)輻射損傷小鼠的保護(hù)作用。 2.觀(guān)察丹參素對(duì)輻射引起人正常細(xì)胞生長(zhǎng)抑制的作用；并進(jìn)一步研究丹參素對(duì)輻射損傷細(xì)胞形態(tài)、DNA、線(xiàn)粒體等的保護(hù)作用； 3.探索丹參素輻射防護(hù)作用的可能機(jī)制以及信號(hào)通路； 實(shí)驗(yàn)方法： 動(dòng)物實(shí)驗(yàn) 雄性BALB/c小鼠(22±2g)適應(yīng)環(huán)境后，隨機(jī)分為五組:1)生理鹽水對(duì)照組，2)單獨(dú)照射組，3)丹參素10mg/Kg組，4)丹參素20mg/Kg組，5)陽(yáng)性對(duì)照雌三醇（E3，2mg/Kg）組。不同濃度丹參素灌胃7天，陽(yáng)性對(duì)照E3組在輻照前48h,24h,0h分別腹腔給藥3次，然后給予不同劑量的γ射線(xiàn)照射。并于輻照后不同時(shí)間取材，觀(guān)察丹參素對(duì)輻射損傷小鼠的生存率的影響，外周血中白細(xì)胞、紅細(xì)胞、血紅蛋白和血小板含量的變化，脾臟的病理學(xué)以及脾臟造血干細(xì)胞克隆形成率的改變，骨髓有核細(xì)胞數(shù)以及小鼠肝臟抗氧化能力的變化。 細(xì)胞實(shí)驗(yàn) 借助MTT法檢測(cè)丹參素對(duì)四種人正常細(xì)胞的毒性和輻射防護(hù)作用，進(jìn)一步采用克隆形成法觀(guān)察丹參素對(duì)輻射損傷的L-02和HIEC細(xì)胞增殖能力的影響，然后用微核法，彗星尾實(shí)驗(yàn)，細(xì)胞免疫熒光觀(guān)察丹參素對(duì)輻照細(xì)胞DNA的保護(hù)作用，最后分別使用熒光探針和生化方法檢測(cè)丹參素對(duì)輻照損傷細(xì)胞的線(xiàn)粒體和氧化還原系統(tǒng)的保護(hù)作用。 為了研究丹參素對(duì)輻照損傷細(xì)胞中凋亡相關(guān)的信號(hào)通路的影響，取對(duì)數(shù)期生長(zhǎng)的L-02細(xì)胞與丹參素共孵育1h后給予γ射線(xiàn)照射，繼續(xù)分別培養(yǎng)不同時(shí)間，用WesternBlot方法檢測(cè)細(xì)胞中凋亡通路、DNA修復(fù)、JNK通路和Ca~(2+)通路相關(guān)蛋白的表達(dá)，用比色法檢測(cè)細(xì)胞凋亡通路中蛋白激酶的變化。 實(shí)驗(yàn)結(jié)果： (1)丹參素對(duì)輻射損傷小鼠的保護(hù)作用 丹參素可以提高8Gy照射后30天小鼠的存活率，可以明顯抑制4Gyγ射線(xiàn)照射后28天內(nèi)外周血紅細(xì)胞、白細(xì)胞、血紅蛋白和血小板減少。 丹參素可減輕由輻照所致的小鼠脾臟重量的過(guò)度減輕，而且經(jīng)丹參素預(yù)處理后，受照小鼠內(nèi)源性脾結(jié)節(jié)較照射組均明顯增多。形態(tài)學(xué)觀(guān)察表明，4Gyγ射線(xiàn)照射后即可見(jiàn)脾臟體積明顯縮小，脾切面上脾小體縮小或完全消失，大量淋巴細(xì)胞凋亡，而照前給予丹參素則能有效的抑制輻射引起的生發(fā)中心的萎縮，減少淋巴細(xì)胞凋亡。同時(shí)經(jīng)丹參素處理過(guò)的小鼠骨髓細(xì)胞中的有核細(xì)胞計(jì)數(shù)明顯多于照射組，表明丹參素能增強(qiáng)骨髓細(xì)胞對(duì)輻射的耐受性。丹參素預(yù)處理能增強(qiáng)小鼠肝臟抗氧化酶類(lèi)的活性，減少肝臟蛋白的氧化水平并抑制肝臟的脂質(zhì)過(guò)氧化水平。 (2)丹參素預(yù)處理對(duì)四種人正常細(xì)胞的毒性和輻射防護(hù)作用 MTT法實(shí)驗(yàn)結(jié)果顯示，不同濃度的丹參素對(duì)L-02、HIEC、GES、HaCaT四種人正常細(xì)胞都沒(méi)有產(chǎn)生明顯毒性，而且在孵育1h時(shí)，丹參素對(duì)四種細(xì)胞均表現(xiàn)出促進(jìn)增殖的作用，因此我們選擇丹參素提前孵育1h做后續(xù)的輻射防護(hù)實(shí)驗(yàn)。在之后的輻射防護(hù)實(shí)驗(yàn)中，四種細(xì)胞的活力在經(jīng)過(guò)γ射線(xiàn)的照射后都有不同程度的下降，而丹參素1h的預(yù)處理則能明顯提高四種細(xì)胞的生存數(shù)量，其中10μg/ml丹參素的作用效果最明顯。根據(jù)細(xì)胞對(duì)γ射線(xiàn)和丹參素的敏感性，我們選用了L-02和HIEC兩種細(xì)胞做后續(xù)實(shí)驗(yàn)。 (3)丹參素對(duì)L-02和HIEC細(xì)胞輻射損傷的防護(hù)作用 首先細(xì)胞克隆形成和凋亡ER實(shí)驗(yàn)的結(jié)果表明細(xì)胞輻照前1h給予丹參素孵育能顯著提高細(xì)胞的增殖能力，增加細(xì)胞的克隆形成數(shù)(P0.01)，并能顯著減少細(xì)胞凋亡(P0.01)。而且丹參素預(yù)處理也能明顯減少輻射損傷細(xì)胞形態(tài)的改變和細(xì)胞核的損傷。 與單純照射組相比，丹參素預(yù)處理組γ-H2AX活化的陽(yáng)性細(xì)胞數(shù)以及活化強(qiáng)度有明顯減輕，說(shuō)明丹參素降低了γ射線(xiàn)輻照后細(xì)胞DNA受損程度。另外微核實(shí)驗(yàn)中丹參素的預(yù)處理能夠明顯減少微核的形成率，減輕輻射誘導(dǎo)的細(xì)胞核的損傷。彗星尾實(shí)驗(yàn)中，丹參素預(yù)處理組較單純照射組中細(xì)胞的尾長(zhǎng)縮短、尾部DNA的百分比下降、尾矩減小，顯示丹參素對(duì)輻射損傷的DNA有保護(hù)作用。 對(duì)細(xì)胞線(xiàn)粒體膜電位的測(cè)定結(jié)果顯示丹參素預(yù)處理過(guò)的細(xì)胞表現(xiàn)出膜電位恢復(fù)的現(xiàn)象(P0.01)。另外，我們的研究發(fā)現(xiàn)丹參素預(yù)處理能明顯減少輻射在L-02和HIEC細(xì)胞內(nèi)的ROS(P0.05)，還能在一定程度上恢復(fù)細(xì)胞內(nèi)抗氧化酶的活性，增加抗氧化物質(zhì)的含量，減輕細(xì)胞脂質(zhì)過(guò)氧化的程度，維持細(xì)胞氧化還原系統(tǒng)的正常功能。 (4)丹參素對(duì)輻射誘導(dǎo)細(xì)胞中凋亡相關(guān)通路活化的抑制作用 凋亡相關(guān)蛋白的檢測(cè)結(jié)果顯示照射后48h細(xì)胞內(nèi)，與單純照射組相比，Bax/Bcl-2的比值隨著丹參素濃度的升高而有顯著降低，而p53的表達(dá)隨著預(yù)處理丹參素濃度的升高也是有顯著降低，。這些說(shuō)明丹參素能夠促進(jìn)抗凋亡蛋白的表達(dá)，抑制促凋亡蛋白的表達(dá)。細(xì)胞經(jīng)過(guò)4Gyγ射線(xiàn)照射后，從線(xiàn)粒體釋放到胞漿中的Cyt-C和AIF的蛋白含量顯著上升，而經(jīng)丹參素預(yù)處理之后再給予4Gyγ射線(xiàn)照射能夠降低Cyt-C和AIF的釋放，從而抑制由線(xiàn)粒體損傷引起的細(xì)胞凋亡。另外，細(xì)胞內(nèi)由輻射引起的Caspase3/8/9蛋白激酶的活化在丹參素的干預(yù)下都有所減少，上述結(jié)果表明，丹參素能夠通過(guò)抑制蛋白激酶的活化從而降低輻射誘導(dǎo)的細(xì)胞凋亡。 (5)丹參素對(duì)輻射損傷細(xì)胞DNA的保護(hù)作用 細(xì)胞內(nèi)γ-H2AX在4Gy射線(xiàn)照射后，即刻開(kāi)始出現(xiàn)，3h達(dá)到最高峰；而經(jīng)過(guò)丹參素干預(yù)的實(shí)驗(yàn)組γ-H2AX在照后1h就達(dá)到最高峰；說(shuō)明丹參素預(yù)處理能夠加快H2AX的磷酸化，促進(jìn)損傷DNA的修復(fù)。 輻照組γ-H2AX的上游分子ATM的表達(dá)水平從照后即刻開(kāi)始到輻照后3h，逐漸增加，之后反而下降。而丹參素處理組中ATM從照后即刻開(kāi)始直至照后3h一直處于活化狀態(tài)。RT-PCR的結(jié)果顯示經(jīng)丹參素預(yù)處理的輻射損傷細(xì)胞中的ATM上調(diào)最多，但是4Gy單純照射組和丹參素+輻照組與對(duì)照相比均無(wú)統(tǒng)計(jì)學(xué)差異。這說(shuō)明丹參素對(duì)輻射損傷的細(xì)胞中ATM表達(dá)的影響主要集中在蛋白的合成和降解上。 (6)丹參素對(duì)輻射誘導(dǎo)細(xì)胞中JNK信號(hào)通路活化的抑制作用 丹參素預(yù)處理L-02細(xì)胞1h后經(jīng)4Gyγ射線(xiàn)照射，繼續(xù)培養(yǎng)24小時(shí)后，p38和JNK的表達(dá)均無(wú)明顯變化，磷酸化的p38蛋白也沒(méi)有明顯變化，而磷酸化的JNK蛋白，p-JNK在受照后顯著增加，而丹參素的預(yù)處理則能抑制輻射誘導(dǎo)的JNK蛋白的磷酸化，說(shuō)明丹參素抑制輻射誘導(dǎo)的JNK激酶的磷酸化。經(jīng)過(guò)丹參素預(yù)處理4Gyγ射線(xiàn)照射，繼續(xù)培養(yǎng)12小時(shí)后，JNK激酶的上游分子p-MKK4的表達(dá)水平在輻照后顯著升高，而丹參素預(yù)處理能降低p-MKK4的表達(dá)水平，MKK4的表達(dá)沒(méi)有變化，NFκB的表達(dá)水平在細(xì)胞輻照后有所上升，但是丹參素對(duì)于輻照后NFκB的表達(dá)沒(méi)有影響。 使用JNK的特異性抑制劑SP600125，輻照后36hJNK通路下游分子c-Jun和ATF-2的表達(dá)在丹參素作用下被抑制，而在正常情況下對(duì)它們的表達(dá)沒(méi)有影響。此結(jié)果表明，丹參素能夠抑制MKK4/JNK通路的活化，從而減少輻射誘導(dǎo)的細(xì)胞凋亡。 (7)丹參素對(duì)輻射誘導(dǎo)細(xì)胞中Ca~(2+)介導(dǎo)的凋亡通路活化的抑制 丹參素預(yù)處理L-02細(xì)胞1h后經(jīng)4Gyγ射線(xiàn)照射，照后3h細(xì)胞內(nèi)Ca~(2+)由內(nèi)質(zhì)網(wǎng)釋放，與對(duì)照組相比，丹參素預(yù)處理組的Ca~(2+)釋放強(qiáng)度和陽(yáng)性細(xì)胞數(shù)則都有所減弱。Ca~(2+)通路的鈣調(diào)蛋白calpain-2在4Gyγ射線(xiàn)照后表達(dá)量顯著升高，而在丹參素預(yù)處理組中鈣調(diào)蛋白calpain-2的表達(dá)量升高幅度明顯降低。 結(jié)論： 1.在體實(shí)驗(yàn)結(jié)果說(shuō)明丹參素能明顯延長(zhǎng)和提高輻照小鼠的生存時(shí)間和存活率，丹參素能明顯降低受照小鼠外周血、脾臟、骨髓細(xì)胞的損傷程度，并能增強(qiáng)機(jī)體的抗氧化能力。 2.丹參素對(duì)L-02、HIEC、GES、HaCaT細(xì)胞沒(méi)有明顯的細(xì)胞毒性，并且其輻射防護(hù)效果顯著。 3.丹參素能抑制γ射線(xiàn)導(dǎo)致的細(xì)胞凋亡，促進(jìn)細(xì)胞增殖，其作用與保護(hù)細(xì)胞DNA，清除ROS，增強(qiáng)細(xì)胞的抗氧化力有關(guān)。 4．丹參素是通過(guò)抑制線(xiàn)粒體介導(dǎo)和鈣離子誘導(dǎo)的細(xì)胞凋亡途徑、促進(jìn)細(xì)胞DNA損傷后修復(fù)分子的活化、抑制輻射誘導(dǎo)的JNK途徑的激活發(fā)揮輻射保護(hù)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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R142

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