| 地龙素对缺血性脑卒中神经保护作用及机制的研究 | |
| Alternative Title | Neuroprotective effect and underlying mechanism of lumbritin on ischemic stroke |
李娟 | |
| Subtype | 博士 |
| Thesis Advisor | 李应东 |
| 2018-12-12 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 博士 |
| Degree Discipline | 中西医结合临床 |
| Keyword | 地龙素 缺血性脑卒中 血管内皮生长因子 血管新生 PI3K/Akt/mTOR信号通路 |
| Abstract | 目的：对中药地龙治疗缺血性脑卒中进行系统评价/Mata分析，为地龙素治疗缺血性脑血管病提供理论及实验依据。研究地龙素制备工艺，确定地龙素质量控制标准，探讨地龙素对缺血性脑卒中小鼠及体外培养的神经细胞（PC12细胞）的神经保护作用及机制。 方法：1、由2位培训合格的研究员严格按照纳入/排除标准，独立筛选文献和提取资料，通过系统评价和Meta分析全面评价现有的中药地龙治疗缺血性脑卒中的实验证据，探讨中药地龙对缺血性脑卒中动物的神经功能缺失评分、神经系统症状体征疗效、血液流变学、血凝的影响和作用。2、研究地龙素制备工艺；通过酸度计检测酸碱度确定地龙素的纯净度；通过紫外风光光度计检测紫外可见吸收光谱；通过高效液相色谱法分析地龙素不同组分；相同方法下制备不同批次的地龙素，并进行药效稳定性试验及小鼠药物安全性试验。3、光化学栓塞法制备小鼠局部缺血性脑卒中模型，随机分为假手术组、模型组、地龙素低剂量组（40mg/mL）、地龙素中剂量组（60mg/mL）、地龙素高剂量组（80mg/mL）。依照Bederson评分标准进行神经功能缺失评分；行为学测试中用疲劳转移棒来评价动物运动的协调平衡能力，前肢抓力测试用来测试和评价前肢的抓力；采集心脏血检测各组小鼠的凝血功能；术后7天用TTC染色检测梗死体积。4、光化学栓塞法制备小鼠局部缺血性脑卒中模型，随机分为假手术组、模型组、地龙素组。采用HE染色观察实验各组小鼠脑组织病理形态学结果；通过免疫组织化学染色法观察地龙素对缺血性脑卒中脑组织VEGF、CD34的表达水平及微血管密度的影响；通过Western-blot技术检测地龙素对PI3K/Akt/mTOR信号通路的调节作用。5、制备地龙素含药血清，利用 CoCl2建立 PC12 细胞缺氧模型，用地龙素含药血清进行干预，实验分为对照组、模型组、地龙素含药血清低剂量组、地龙素含药血清中剂量组、地龙素含药血清高剂量组。采用MTT法检测PC12 细胞缺氧损伤后的OD值计算细胞存活率；EdU法检测细胞增殖率；通过乳酸脱氢酶试验检测PC12细胞的损伤程度；通过Western-blot技术检测地龙素对HIF-1α、 VEGF蛋白表达的影响，以探讨对PI3K/AKT/mTOR信号通路的调节作用。结果：1、通过系统评价和Meta分析全面评价现有的实验证据，结果表明：相比于生理盐水组或蒸馏水组，地龙和（或）含地龙药物组大鼠的神经功能缺损有显著改善（P<0.05）；大鼠运动的协调和整合功能、躯体感觉功能、前肢肌力测试等均得到改善；血小板聚集性降低，凝血功能降低（P<0.05），血液流变学得到改善（P<0.01）。2、我们用鲜地龙成功提取了地龙素，确定了制备工艺流程。制备的地龙素进行酸碱度检测，pH值固定在5.5～7.5，显示药物较纯净。经检测地龙素的最大吸收波长集中在200～280nm之间，多次实验发现最稳定的特征吸收峰为220nm、254nm、280nm。高效液相色谱法波长220nm的显示峰值最高，最能反映产品的特征，因此将波长220nm的吸收峰设定为地龙素的特征吸收峰。按照最佳制备工艺流程，得出制备过程中质量控制的关键参数。药物长期稳定性试验及小鼠药物安全性试验结果显示本提取物稳定、安全。3、各组小鼠于术后4小时观察其神经系统症状，绝大多数动物评分都达到3分以上，挑选3分以上的动物用于后续实验。疲劳转移棒实验显示三种剂量的地龙素在小鼠脑卒中后7天增加了在疲劳转棒上的时间，与模型组比较具有统计学意义（P < 0.05），在缺血后3天高剂量组在疲劳转移棒上增加的时间最多，在缺血后7天，高剂量组在疲劳转移棒上的时间比低剂量组及中剂量组明显增加（P< 0.05），但低剂量组和中剂量组之间没有显著差异。卒中后7天内前肢抓力测试的结果显示三种剂量的地龙素均可使前肢抓力提高，高剂量组明显好于低剂量组和中剂量组（P < 0.05）。4、凝血功能检测结果显示不同剂量地龙素给药1周后PT和TT均有所延长。低剂量组与模型组的PT比较差异无统计学意义（P> 0.05），中剂量组、高剂量组比模型组的PT明显延长（P< 0.05）。TT 检测显示，高剂量组比模型组明显延长（P< 0.05），而低剂量组、中剂量组与模型组差异无统计学意义（P> 0.05）。国际标准化比率（INR）显示高剂量组增高，与模型组相比有统计学意义（P< 0.05），低剂量组、中剂量组与模型组差异无统计学意义（P> 0.05）。相比模型组，不同剂量地龙素组的纤维蛋白原（Fbg）含量显示，高剂量组差异有统计学意义（P< 0.05），而低剂量组和中剂量组与模型组差异无统计意义（P> 0.05）。5、TTC染色结果显示，三种剂量的地龙素均显著降低了光化学法诱导小鼠脑缺血后的梗死体积，与模型组比较，均有统计学意义(P< 0.01)；低剂量组使梗死体积降低了36.29%，中剂量组使梗死体积降低了56.91%，高剂量组使梗死体积降低了68.32%，高剂量组在梗死体积的降低上明显优于低剂量组与中剂量组（P< 0.01)。综合行为学和组织学结果，在后期动物实验中地龙素干预选择高剂量组的剂量（80mg/ mL）。6、HE染色观察病理形态学结果显示，假手术组：小鼠脑组织细胞形态基本正常，神经元细胞胞膜光滑，胞浆丰富，胞核清晰可见。神经细胞排列整齐，极向规则, 横纹清晰，颗粒层细胞与锥体细胞界限分明，细胞间隙无明显水肿。模型组：缺血损伤部位细胞数量较少，细胞肿胀，可见核固皱、核溶解，间质有明显水肿，缺血半影区可见少量毛细血管。地龙素组：与假手术组比较，梗死区细胞分布稀少，且出现空泡变性；与模型组比较，细胞间质水肿不明显，可见神经纤维走行，缺血半影区可见较多毛细血管。7、免疫组化结果显示：与假手术组相比，模型组脑组织缺血半影区VEGF表达增多（P<0.05），地龙素组脑组织缺血半影区VEGF表达明显增多（P<0.01），与模型组相比，地龙素组脑组织缺血半影区中VEGF表达水平增高，具有统计学意义（P<0.05）。与假手术组相比，模型组脑组织缺血半影区CD34表达增多（P<0.05），地龙素组脑组织缺血半影区CD34表达明显增多（P<0.01）；与模型组相比，地龙素组（80 mg/mL）脑组织缺血半影区中CD34表达水平增高，差异具有统计学意义（P<0.01）。微血管密度值：与假手术组相比，模型组与地龙素组微血管密度增多（P<0.05）；与模型组相比，地龙素组脑组织缺血半影区中微血管密度增高，差异具有统计学意义（P< 0.05）。8、Western印迹法定量分析结果如下：VEGF：与假手术组比较，模型组蛋白表达差异无统计学意义（p >0.05），地龙素治疗组蛋白水平明显增高（P<0.01）。与模型组比较，地龙素治疗组蛋白表达水平明显增高（P<0.01）。PI3K：与假手术组比较，模型组蛋白表达差异无统计学意义（P>0.05），地龙素治疗组蛋白水平明显增高（P<0.05）；与模型组相比，地龙素治疗组蛋白表达水平明显增高（P<0.05）。AKT：与假手术组相比，模型组蛋白表达差异无统计学意义（P>0.05），地龙素治疗组蛋白水平明显增高（P<0.05）；与模型组比较，地龙素治疗组蛋白表达水平明显增高（P<0.05）。P-AKT（Ser473）：与假手术组比较，模型组蛋白表达差异无统计学意义（P>0.05），地龙素治疗组蛋白水平明显增高（P<0.05）；与模型组比较，地龙素治疗组蛋白表达水平明显增高（P<0.05）。m-TOR：与假手术组相比，模型组、地龙素治疗组蛋白水平明显增高（P<0.05）；与模型组相比，地龙素治疗组蛋白表达水平明显增高（P<0.05）。9、MTT法检测结果显示，缺氧24小时后，与正常对照组比较, 模型组的 OD值显著降低，细胞存活率为57%；地龙素低剂量组（含药血清5%），地龙素中剂量组（含药血清10%）、地龙素高剂量组（含药血清15%）细胞存活率分别为73%， 80%及81%。 结果表明模型组的存活率与正常对照组相比明显下降，差异显著(P <0.01 )；与模型组相比，地龙素低剂量组、地龙素中剂量组、地龙素高剂量组细胞存活率均升高，差异具有统计学意义（P <0.01）；地龙素低剂量组与中剂量组比较，差异显著（P <0.01），地龙素中剂量组与高剂量组相比，差异无统计学意义（P >0.05）。10、EdU 法检测实验各组PC12细胞的增殖能力结果如下：模型组细胞增殖率为35.7%，地龙素低剂量组增殖率为42%，地龙素中剂量组增殖率为51%，地龙素高剂量组增殖率为52%。与正常组相比，模型组的增殖率明显下降 , 差异有统计学意义(P <0.01 )；与模型组相比，地龙素低剂量组增殖率增高（P <0.05）、地龙素中剂量组、地龙素高剂量组细胞增殖率均升高，差异具有统计学意义（P <0.01）。11、采用酶联免疫吸附法检测地龙素对缺氧PC12细胞LDH漏出的影响来判断细胞的损伤程度。结果显示，与正常对照组比较，模型组LDH漏出水平升高，差异具有统计学意义（P <0.01 )；与模型组比较，地龙素高、中、低剂量组LDH的漏出水平明显降低，差异具有统计学意义（P <0.01 )，地龙素高、中、低剂量组之间LDH的漏出水平差异没有统计学意义（P >0.05 )。12、用Western印迹法定量分析结果如下：HIF-1α：与正常对照组比较，缺氧模型组和地龙素组HIF-1α蛋白表达量明显增高，具有统计学意义（P<0.01）；与模型组比较，地龙素治疗组蛋白表达水平明显增高，差异具有统计学意义（P<0.05）。VEGF：与正常对照组比较，缺氧模型组和地龙素组VEGF蛋白表达量明显增高（P<0.01）；与模型组比较，地龙素治疗组蛋白表达水平明显增高，差异具有统计学意义（P<0.01）。结论：系统评价及Mata分析表明中药地龙治疗能有效改善缺血性脑卒中动物的神经系统症状及体征，同时地龙能改善血流环境和血凝状态，有利于血栓的溶解。但由于纳入研究质量的限制，有必要进行更多高质量的实验研究进一步证实，为开展临床治疗提供实验依据和新的思路。以祛痰化瘀、活血通络为治则的地龙素对缺血性脑卒中小鼠具有改善行为学功能、降低凝血功能、减少梗死体积，从而保护神经功能的作用；地龙素能够改善缺血性脑卒中后脑组织缺血损伤的病理反应，促进缺血半影区VEGF、CD34的表达，有助于新生血管的生成，产生这种效应的机制与地龙素上调PI3K/Akt/mTOR信号通路有关，进而对缺血性脑卒中具有神经保护作用；地龙素可以促进缺氧损伤的PC12细胞存活率及增殖能力，降低缺氧对细胞的损伤程度，通过激活PI3K/AKT/mTOR信号通路上调HIF-1α、VEGF表达，从而对缺氧损伤的神经细胞起保护作用。 |
| Other Abstract | Purpose:This paper systematically evaluates the effect of Earthworm on ischemic stroke/Mata analysis, so as to provide theoretical and experimental basis for Lumbritin in treating ischemic cerebrovascular disease.The purpose of the research is to study the preparation technology and quality control standard of Lumbritin, and explore the neuroprotective effect and mechanism of Lumbritin on ischemic stroke mice and nerve cells cultured in vitro. Method:1. Two trained and qualified researchers independently screened the literature and extracted data according to inclusion/exclusion criteria. Through systematic evaluation and Meta analysis,the purpose is to explore the influence that earthworm exerts on neurological deficit score and nervous system in ischemic stroke animals, and to record the effects and functions of symptoms and signs, blood rheology and blood coagulation.2. In this paper, the preparation technology of Lumbritin was established; the purity of Lumbritin was determined by acidity meter; the ultraviolet-visible absorption spectrum was detected by ultraviolet spectrophotometer; the different components of Lumbritin were analyzed by high performance liquid chromatography; different batches of Lumbritin were prepared by the same method, and pharmacodynamic stability test and mouse safety test were carried out.3. The model of local ischemic stroke in mice was established by photochemical embolization. The research objects were divided into sham operation group, model group, low dose group, middle dose group and high dose group. Neurological deficits were scored according to Bederson's score criteria; fatigue transfer rods were used in behavioral tests to evaluate the motor coordination and the balance of the animals; forelimb grip tests were used to test and evaluate forelimb grip; heart blood was collected to test the coagulation function of the mice in each group; and infarct volume was measured by TTC staining 7 days after operation. 4. The model of local ischemic stroke in mice was prepared by photochemical embolization , The research objects were randomlydivided into sham operation group, model group and Lumbritin group. HE staining and light microscopy were used to observe the pathological morphology of the brain tissues of mice in each group; immunohistochemical staining was used to observe the effect of Lumbritin on the expression of VEGF、CD34 and microvessel density in ischemic stroke brain tissues; Western blot was used to detect the regulation of Dilongsu on PI3K/Akt/mTOR signaling pathway.Result: 1. The present experimental evidence was comprehensively evaluated by systematic evaluation and Meta analysis. The results showed that compared with normal saline group or distilled water group, the neurological impairment was significantly improved in Earthworm and/or Earthworm-containing drug group (P < 0.05); the motor coordination and integration function, somatosensory function and forelimb muscle strength were measured in rats. The platelet aggregation rate and coagulation function were decreased (P < 0.05), and the hemorheology was improved (P < 0.01). 2.We successfully extracted the Lumbritin from fresh earthworm, and determined the preparation process. The pH value of Lumbritin was fixed at 5.5-7.5, indicating purity of Lumbritin. The maximum absorption wavelength of Lumbritin was 200-280 nm, and the most stable characteristic absorption peaks were 220 nm, 254 nm and 280 nm. The peak value of 220 nm in high performance liquid chromatography was the highest, which could best reflect the characteristics of the product. Therefore, the absorption peak of 220 nm was set as the characteristic absorption peak of Lumbritin. According to the best preparation process, the key parameters of quality control during the preparation process were obtained. The results of long-term stability test and mouse safety test showed that the extract was stable and safe. 3.The neurological symptoms of the mice in each group were observed 4 hours after operation. Most of the animals scored 3 points. The animals with 3 points were selected for follow-up experiments. Rotarod test showed that the mice of three different doses Lumbritin increased the retention time on Rod at day 3 and day 7 after stroke, which was statistically significant compared with the model group (P < 0.05). The effect of high dosage group was the bestonday 3 after ischemia.Onday 7 after ischemia, high dosage group was significantly better than low dosage group and middle dose group (P < 0.05), but there was no significant difference between low dose group and middle dose group. The results of forelimb grip strength test within one week after stroke showed that three different doses of Lumbritin significantly increased forelimb grip strength, whether at day3 or day 7 after ischemia, the high dose group was significantly better than the low dose group and the middle dose group (P < 0.05). 4.Coagulation function test showed that PT and TT were prolonged after one week of administration of different doses of Lumbritin. There was no significant difference in PT between the low dose group and the model group (P > 0.05). The PT of the middle dose group and the high dose group was significantly longer than that of the model group (P < 0.05). TT test showed that the high dose group was significantly longer than the model group (P < 0.05), but there was no significant difference between the low dose group, the middle dose group and the model group (P > 0.05). The international standardized ratio (INR) showed that the high dose group increased significantly compared with the model group (P < 0.05). There was no significant difference between the low dose group and the middle dose group and the model group (P > 0.05). Fibrinogen ( Fbg ) levels in different doses of Lumbritin group were significantly higher than those in high doses group (P < 0.05), but there was no significant difference between low and middle doses group and model group (P > 0.05).5. The results of TTC staining after 7 days of ischemia showed that all three doses of Lumbritin significantly reduced the infarct volume induced by ischemia (P < 0.01), the infarct volume decreased by 36.29% in the low dose group, 56.91% in the middle dose group and 68.32% in the high dose group. The high dose group was significantly better than the low dose group and the middle dose group in the reduction of infarct volume (P < 0.01). The high dose group (80mg/ml) was selected for the intervention of Lumbritin in the later study.6. The results of HE staining showedthe morphology of the brain cells.Sham-operated group: The morphology of brain cells was basically normal, and the capsule of neurons was smooth, rich in plasma and clear nucleus. The nerve cells arranged neatly, the boundary between granular layer cells and pyramidal cells was clear, the staining was uniform, the striation was regular and clear, and there was no obvious edema in the space around the cells. Model group: the structure of ischemic brain tissue was disordered, arranged irregularly, the number of cells was reduced, the cells were swollen, the nucleus was shrinked or dissolved, chromatin condensation, interstitial edema and loosening. Some new capillaries were seen in the ischemic penumbra. Lumbritin group: Compared with sham operation group, the cells in infarct area were sparsely distributed and vacuolar degeneration was observed; compared with model group, the changes in ischemic area were not as obvious as that in cerebral ischemia model group, there was no obvious edema in the interstitial cells, the nerve fibers were visible, some fibers were wavy, the number of brain cells increased, and there are more neovascularization in the ischemic penumbra. 7. The results of immunohistochemistry showed that the expression of VEGF in ischemic penumbra increased in model group compared with sham operation group (P < 0.05), and the expression of VEGF in ischemic penumbra increased significantly in Lumbritin group (P < 0.01). 0.05). Compared with sham operation group, the expression of CD34 in ischemic penumbra increased in model group (P < 0.05), and that in Lumbritin group (P < 0.01). Compared with the model group, the expression of CD34 in the ischemic penumbra in the Lumbritin group (80 mg/ml) was significantly higher (P < 0.01). Microvessel density: Compared with sham operation group, microvessel density increased in model group and Lumbritin group (P < 0.05). Compared with the model group, the microvessel density in the ischemic penumbra of brain tissue in the Lumbritin group increased, and the difference was statistically significant (P < 0.05). 8. The results of Western-blot analysis were as follows: VEGF: Compared with sham operation group, the expression of protein in model group had no significant difference (p > 0.05), and the protein level in Lumbritin treatment group increased significantly (p < 0.01). Compared with model group, the expression level of Lumbritin treated group was significantly higher (P < 0.01). PI3K: Compared with sham operation group, there was no significant difference in protein expression in model group (p > 0.05), and the protein level in Lumbritin treatment group was significantly higher (p < 0.05). Compared with model group, the expression level of Lumbritin treated group was significantly higher (P < 0.05). AKT: Compared with sham operation group, the expression of protein in model group had no significant difference (p > 0.05), and the protein level in Lumbritin treatment group was significantly higher (p < 0.05). Compared with model group, the expression level of Lumbritin treated group was significantly higher (P < 0.05). P-AKT (Ser473): Compared with sham operation group, there was no significant difference in protein expression in model group (p > 0.05), and the protein level in Lumbritin treatment group was significantly higher (p < 0.05). Compared with model group, the expression level of Lumbritin treated group was significantly higher (P < 0.05). M-TOR: Compared with sham operation group, the protein levels in model group and Lumbritin treatment group were significantly higher (p < 0.05). Compared with model group, the expression level of Lumbritin treated group was significantly higher (P < 0.05). 9. MTT assay showed that after 24 hours of hypoxia, compared with the normal control group, the OD value of the model group decreased significantly, and the cell survival rate was 57%; the cell survival rates of the low dose group, the middle dose group and the high dose group were 73%, 80% and 81%. The results showed that the survival rate of the model group was significantly lower than that of the normal group (P < 0.01); compared with the model group, the cell survival rate of the low-dose group, the middle-dose group and the high-dose group were significantly higher (P < 0.01). The difference was significant (P < 0.01). There was no significant difference between the middle dose group and the high dose group (P > 0.05).10.The proliferation rate of PC12 cells was 35.7% in the model group, 42% in the low dose group, 51% in the medium dose group and 52% in the high dose group. Compared with the normal group, the proliferation rate of the model group decreased significantly (P < 0.01); compared with the model group, the proliferation rate of the low dose group increased (P < 0.05), the medium dose group and the high dose group increased significantly (P < 0.01). 11. Enzyme-linked immunosorbent assay (ELISA) was used to detect the effect of earthworm extract on LDH leakage in hypoxic PC12 cells. The results showed that the OD value of the model group was higher than that of the normal control group (P < 0.01). Compared with the model group, the leakage level of LDH in the high, middle and low dosage groups was significantly lower (P < 0.01). There was no significant difference in the leakage level of LDH among the high, middle and low dosage groups (P > 0.05). 12.The results of Western blot analysis were as follows: HIF-1a: Compared with the normal control group, the expression of HIF-1a protein in hypoxia model group and Lumbritin group was significantly higher (p < 0.01). Compared with the model group, the protein expression level of the treatment group was significantly higher ,the difference was statistically significant (P < 0.05). VEGF: Compared with normal control group, the expression of VEGF protein in hypoxia model group and Lumbritin group was significantly higher (p < 0.01). Compared with the model group, the protein expression level of the treatment group was significantly higher, the difference was statistically significant (P < 0.01).Conclusion：Systematic evaluation and MaTa analysis showed that earthworm could effectively improve neurological symptoms and signs of ischemic stroke animals. At the same time, earthworms improve the blood flow and coagulation state, and are conducive to the dissolution of thrombus. However, due to the limitation of research quality, it is necessary to carry out more high-quality experimental studies to further confirm, and provide experimental basis and new ideas for clinical treatment.Lumbritin improved the behavioral function of cerebral ischemic mice, reduced the coagulation function, reduced the infarct volume, and thus protected the nerve function. Lumbritin improved the pathological reaction of ischemic brain injury after ischemic stroke and promoted the expression of VEGF and CD34 in ischemic penumbra. The mechanism is related to the up-regulation of PI3K/Akt/mTOR signaling pathway by lumbritin, which has neuroprotective effect on ischemic stroke.Lumbritin can promote the survival rate and proliferation ability of PC12 cells damaged by hypoxia, and enhance the hypoxia tolerance of PC12 cells damaged by hypoxia. It can protect the nerve cells damaged by up-regulating the expression of HIF-1a and VEGF and activating PI3K/AKT/mTOR/HIF-1 signaling pathway. |
| Pages | 149 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/340866 |
| Collection | 基础医学院 |
| Affiliation | 基础医学院 |
| First Author Affilication | School of Basic Medical Sciences |
| Recommended Citation GB/T 7714 | 李娟. 地龙素对缺血性脑卒中神经保护作用及机制的研究[D]. 兰州. 兰州大学,2018. |
| Files in This Item: | There are no files associated with this item. | ||||
