兰州大学机构库 >药学院
Pgk1激动剂筛选及其治疗细胞死亡相关疾病动物模型分子机理研究
Alternative TitleScreening and mechanism action of Pgk1 agonists in animal models for the treatment of cell death-related diseases
刘菁菁
Subtype博士
Thesis Advisor陈新平
2023-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学博士
Degree Discipline药物化学生物学
Keyword特拉唑嗪 Terazosin 拉米夫定 Lamivudine Pgk1 Pgk1 胃肠道疾病 Gastrointestinal disease 心脑血管疾病 Cardiovascular diseases 糖代谢 Glucose metabolism 细胞死亡 Cell death
Abstract

     糖代谢主要分为无氧糖代谢、有氧糖代谢和磷酸戊糖途径,是机体产能的主要方式。其中,糖的无氧糖代谢又称为“糖酵解”,作为一种细胞质途径,主要通过磷酸甘油酸激酶1(Pgk1)和丙酮酸激酶(PK)催化,将葡萄糖分解为丙酮酸或乳酸。在疾病发生过程中,糖酵解能为身体所有细胞产生能量,其最终产物在有氧条件下是丙酮酸,无氧条件下是乳酸。同时,文献表明激活Pgk1之后,继而激活糖代谢通路以抑制细胞死亡,而多种疾病与能量代谢和细胞死亡相关。据此,本论文主要以Pgk1蛋白入手,根据生物学方法筛选Pgk1激动剂,并借助体外细胞模型和体内动物模型确定其激动剂的活性。然后结合基因敲除、基因过表达等技术,研究小分子激动剂与Pgk1的相互作用,揭示调控胃肠道疾病和心脑血管疾病的分子信号网络,深刻理解新型细胞死亡抑制剂治疗胃肠道疾病和心脑血管疾病的机制。

     本论文第一章主要探讨了特拉唑嗪对胃肠道疾病的保护作用及机制研究。胃肠道疾病聚焦于胃溃疡和溃疡性结肠炎,以乙醇诱导的 GES-1细胞和乙醇诱导的小鼠胃溃疡模型,同时建立H2O2诱导的Caco-2细胞和DSS诱导的小鼠溃疡性结肠炎模型,结合体外体内实验研究特拉唑嗪的保护机制。本实验结果表明,特拉唑嗪能够激活Pgk1,增加ATP的产生和LDH的活性,从而激活糖代谢;此外,通过细胞转染构建过表达Pgk1的稳定细胞系,并检测了在多重压力刺激下的细胞存活率,实验结果表明激活Pgk1能提高细胞存活率;同时,特拉唑嗪还表现出极强的抗氧化和抗炎活性,显著提高p-AKT的表达量,抑制NF-κB p65活性,同时降低p-IKBα、Caspase-1和GSDMD的表达量,表明特拉唑嗪通过激活Pgk1,增强糖代谢抑制NF-κB-GSDMD信号轴介导的细胞焦亡来治疗胃肠道疾病,并证明Pgk1即为可能成为治疗胃肠道疾病的新靶标。

     根据 “肠-脑轴” 理论及相关文献调研,我们猜测靶向Pgk1可能对治疗缺血性脑卒中有效,因此本论文第二章研究内容以Pgk1蛋白为靶标,筛选其新型激动剂拉米夫定,建立缺血性脑卒中大鼠MCAO模型,探讨拉米夫定对缺血性脑卒中的神经保护作用及机制研究。实验结果表明拉米夫定能够抑制由H2O2和OGD/R损伤引起的细胞死亡,同时改善MCAO大鼠的神经行为,减少脑梗死体积和脑组织病理学变化;此外,Western Blot实验结果表明拉米夫定能提高Pgk1表达量,激活糖代谢,从而抑制Bcl-2/Bax介导的细胞凋亡,阻断Caspase-1/GSDMD介导的细胞焦亡,提高细胞存活率;通过构建过表达和敲除Pgk1的稳定细胞系,证明了拉米夫定是通过作用于Pgk1起效的; 此外,构建的过表达Pgk1的腺相关病毒(AAVs)定位注射到脑内之后,能够改善大鼠脑缺血和再灌注损伤导致的神经元功能障碍,减少脑梗死体积和组织病理学变化的程度,证明了Pgk1为治疗缺血性脑卒中的靶标。综上所述,拉米夫定通过激活Pgk1,上调糖代谢,从而抑制细胞凋亡和细胞焦亡以防治缺血性脑卒中,证明拉米夫定为Pgk1的新型激动剂,且为治疗缺血性脑卒中的新靶标。

      由于心肌梗死和缺血性脑卒中有相似的发病机理,且学术界有“心脑同治”理念,据此本论文第三章聚焦于拉米夫定对心肌缺血再灌注损伤的潜在保护作用及机制研究。建立冠状动脉结扎致心肌缺血再灌注损伤模型,同时利用细胞转染和基因敲除构建过表达和敲除Pgk1的稳定细胞系。结果表明拉米夫定可显著改善缺血再灌注损伤情况下心肌酶变化,缩小心肌梗死体积,减少心肌组织病理学变化;此外,拉米夫定可上调Pgk1的表达量,增加丙酮酸的含量,减少乳酸的含量和LDH的活性,同时抑制心肌缺血再灌注损伤诱导的铁死亡,主要表现为亚铁离子的积累减少,MDA和COX2的水平下降,GSH和GPX4的水平增强,进一步表明拉米夫定通过激活Pgk1上调糖代谢而抑制细胞铁死亡,同时激活Keap-1/Nrf2-HO-1信号通路来阻断心肌梗死,进而证明Pgk1为治疗心肌梗死新靶标。

      综上所述,本研究发现Pgk1为治疗胃肠道疾病和心脑血管疾病的新靶标;其次,特拉唑嗪和拉米夫定可通过激活Pgk1,上调糖代谢抑制多种类型细胞死亡治疗胃肠道疾病和心脑血管疾病,为临床治疗胃肠道疾病和心脑血管疾病提供新思路。

Other Abstract

      Glucose metabolism acts as the most important way for the body to obtain energy, mainly including the anaerobic and aerobic glucose metabolism, as well as pentose phosphate pathway. Glucose oxidation, also known as “glycolysis”, is a cytoplasmic pathway that is catalyzed by phosphoglycerate kinase 1 (Pgk1) and pyruvate kinase (PK), which break down glucose into pyruvate or lactate. Under diseased condition, glycolysis produces energy to support cell metabolism, and its end product is pyruvate under aerobic condition while lactate works as the end product under anaerobic condition. At the same time, activating Pgk1 enhances glucose metabolism and blocks cell death, while many diseases are associated with energy metabolism and cell death. Therefore, this thesis focuses on screening Pgk1 agonists and explore their activities with the help of in vitro cellular and in vivo animal models, followed by the confirmation of its action target Pgk1 by gene editing, gene expression and other strategies. By uncovering how Pgk1 along with its agonists regulate gastrointestinal and cardiovascular diseases, we would identify novel cell death inhibitors to develop new therapeutics of gastrointestinal and cardiovascular diseases.

       The first chapter of this thesis focuses on the protective effect and mechanism study of terazosin on gastrointestinal diseases. Gastrointestinal diseases are typified by gastric ulcer and ulcerative colitis, and the protective effects and mechanism studies of terazosin were carried out by combining with ethanol-induced GES-1 cell death model and ethanol-induced mouse gastric ulcer model, as well as H2O2-induced Caco-2 cell death model and DSS-induced mouse ulcerative colitis model. The results showed that terazosin could activate Pgk1, increase ATP production and LDH activity, and thus enhance glucose metabolism. In addition, stable cells overexpressing Pgk1 constructed by cell transfection survived better than wild type and EGFP expressing cells under multiple stresses. Meanwhile, terazosin also exhibited strong antioxidant and anti-inflammatory activities, by significantly upregulating the expression of p-AKT and inhibiting the activation of NF-κB p65, and lowering the expression of p-IKBα, Caspase-1 and GSDMD, indicating that terazosin inhibits NF-κB-GSDMD-mediated cell pyroptosis to treat gastrointestinal diseases by activating Pgk1 to enhance glucose metabolism, further suggesting that Pgk1 is a new target for the treatment of gastrointestinal diseases.

      Based on the theory of “Gut-Brain axis” and literature reports, we speculate that targeting Pgk1 is effective in treating ischemic stroke. Hence, the second chapter of this dissertation is devoted to screening of the novel agonists of Pgk1, and to exploration of the neuroprotective effect of lamivudine on stroke using a rat model of ischemic stroke called MCAO. The experimental results demonstrate that lamivudine could inhibit cell death caused by H2O2- and OGD/R injuries, as well as improve neurobehavior, reduce brain infarct volume and brain histopathological changes in MCAO rats. In addition, Western Blot results show that lamivudine augmentes Pgk1 expression and upregulates glucose metabolism, thereby inhibiting Bcl-2/Bax-mediated apoptosis and blocking Caspase-1/GSDMD-mediated pyroptosis to improve cell survival. Finally, expression of Pgk1 by adeno-associated viruses (AAVs) reduced neuronal dysfunction, brain infarct volume and histopathological changes after cerebral ischemia and reperfusion injury in rats. In conclusion, lamivudine inhibits apoptosis and pyroptosis by activating Pgk1, upregulating glucose metabolism to protect ischemic stroke, and the findings demonstrate that lamivudine works as a novel agonist of Pgk1, as well as Pgk1 acts as a promising target for the development of new therapeutics of ischemic stroke.

      Since myocardial infarction and cerebral stroke share similar pathogenesis and the idea of "treating the cerebral and cardiac diseases together" comes to clinic, chapter 3 of this thesis focuses on the protective effect and mechanism of lamivudine on myocardial ischemia/reperfusion injury. The results show that lamivudine significantly improved myocardial enzymatic changes, myocardial infarct volume and myocardial histopathological changes. In addition, lamivudine can upregulate the expression of Pgk1, increase the content of pyruvate, decrease the content of lactate and LDH activity, and also inhibited iron death induced by myocardial ischemia-reperfusion injury, which mainly shows that the accumulation of ferrous ions, a decrease in the levels of MDA and COX2, and an enhancement in the levels of GSH and GPX4. In short, lamivudine inhibited ferroptosis through activating Pgk1 to upregulate glucose metabolism, followed by stimulating Keap-1/Nrf2-HO-1 signaling pathway to benefit myocardial infarction. In conclusion, Pgk1 should be a new target for the treatment of myocardial infarction.

      In summary, this study identifies Pgk1 as a new target for the treatment of gastrointestinal and cardiovascular diseases; secondly, terazosin and lamivudine can inhibit multiple types of cell death through activating Pgk1 and upregulating glucose metabolism to treat gastrointestinal and cardiovascular diseases, providing new direction for the clinical treatment of gastrointestinal and cardiovascular diseases.

MOST Discipline Catalogue理学 - 化学 - 分析化学
URL查看原文
Language中文
Other Code262010_120190907410
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/535794
Collection药学院
Affiliation
兰州大学药学院
Recommended Citation
GB/T 7714
刘菁菁. Pgk1激动剂筛选及其治疗细胞死亡相关疾病动物模型分子机理研究[D]. 兰州. 兰州大学,2023.
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