兰州大学机构库 >基础医学院
多肽DR3penA 改善二氧化硅和百草枯所致肺纤维化的作用及机制研究
Alternative TitleThe effect and mechanism study of peptide DR3penA on alleviating silica- and paraquat-induced pulmonary fibrosis
李洁茹
Subtype硕士
Thesis Advisor王锐
2023-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学硕士
Degree Discipline生物化学与分子生物学
Keyword多肽DR3penA Peptide DR3penA 肺纤维化 Pulmonary fibrosis 二氧化硅 Silica 百草枯 Paraquat
Abstract

间质性肺病(ILD)是一组以肺间质炎症和纤维化为特征的限制性肺疾病,患者最终因呼吸衰竭而死亡。其中特发性肺纤维化(IPF)和多种危害因素暴露相关的ILD的发病率呈逐年升高态势。但现有的小分子治疗药物仅能缓解疾病进展,无法有效逆转肺纤维化,且副作用明显,成本高昂,导致该类疾病患者5年生存率很低,预后极差,因此研发新型安全有效的抗肺纤维化药物具有重要意义。

本课题组前期对抗氧化肽DR8进行构效关系研究后设计改造出了血清稳定性明显提升的新型低毒多肽DR3penA,并证实该多肽在模拟IPF的经典模型——博来霉素小鼠肺纤维化模型中具有良好的抗肺纤维化活性,具备进一步研发的潜力。由于单一的动物模型预测人体疗效往往具有局限性,运用多种模型进行研究可从多角度评价药效,进而推动先导化合物的成药。因此,基于研发针对矽肺和百草枯肺有效药物的紧迫性以及多模型评价DR3penA临床前药效的必要性,本课题在SiO2和PQ诱导的肺纤维化模型中进一步研究了多肽DR3penA的抗肺纤维化效果。

实验结果显示,在SiO2诱导的A549细胞模型和TGF-β1诱导的BEAS-2B细胞模型中,DR3penA可明显抑制纤维化标志物α-SMA、Fibronectin和Collagen I的异常高表达。在SiO2诱导的矽肺小鼠模型和PQ诱导的大鼠肺纤维化模型中,运用H&E和Masson染色、免疫组化、Western blot以及RT-qPCR等多种手段评价了DR3penA的体内抗肺纤维化活性。结果表明DR3penA(0.5、1.6和5.0 mg/kg)可降低矽肺小鼠肺部纤维化标志物Fibronectin和Collagen I的异常高表达,减轻肺纤维化程度,在小鼠体内的抗肺纤维化效果与阳性对照药物吡非尼酮(PFD)(100 mg/kg)相比无明显差异;在大鼠肺纤维化模型中,DR3penA(0.200、0.632和2.000 mg/kg)可减少PQ引起的细胞外基质(ECM)的过度沉积,降低肺间质实变程度,且对Fibronectin和Collagen I的抑制效果与阳性对照药物PFD(100 mg/kg)相比无显著差异。

由于SiO2和PQ均可诱导机体产生氧化应激并引发上皮间充质转化(EMT)过程从而导致肺纤维化进展,因此本课题在体内外实验中均检测了氧化应激标志物烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)和EMT标志物(E-Cadherin与N-Cadherin)的表达情况,同时检测了细胞中活性氧(ROS)的生成以及动物血清中过氧化氢酶(CAT)活力、抗氧化物谷胱甘肽(GSH)和脂质过氧化产物丙二醛(MDA)的含量。结果提示DR3penA可显著抑制SiO2和PQ诱导的氧化应激和EMT过程。

初步机制研究发现,在矽肺纤维化中,DR3penA能够通过抑制氧化应激,减少ROS的产生进而负调控PI3K/AKT信号通路的激活,从而阻碍EMT过程,减少ECM的过度积累,最终实现抗肺纤维化;在PQ所致的肺纤维化中,DR3penA能够通过抑制氧化应激和TGF-β的异常高表达来降低ERK和p38的磷酸化水平,从而阻碍EMT过程,发挥抗肺纤维化作用。

综上,本研究的结果表明DR3penA可有效抑制SiO2和PQ诱导的肺纤维化进展,根据不同给药途径下药物生物利用度的转换,DR3penA的体内抗肺纤维化活性与对照药物PFD相当,并且具有低剂量优势。相关机制研究表明DR3penA可通过抑制PI3K/AKT和MAPK通路的激活来发挥抗肺纤维化作用。因此,多肽DR3penA可作为治疗纤维化性ILDs的候选化合物进行深入的成药性评价,本研究为开发具有自主知识产权的用于治疗纤维化相关疾病的多肽药物提供了研究基础。

Other Abstract

Interstitial lung disease (ILD) is a group of restrictive lung diseases characterized by interstitial inflammation and fibrosis, with patients eventually dying of respiratory failure. Among them, the incidence of idiopathic pulmonary fibrosis (IPF) and ILDs associated with multi-hazard exposure is increasing yearly. However, available small molecule therapeutic agents only mitigate disease progression and cannot effectively reverse pulmonary fibrosis with the disadvantages of significant side effects and high cost. Therefore, there is a low five-year survival rate and poor prognosis for patients with these diseases. It is of great importance to develop new safe and effective against pulmonary fibrosis drugs.

Our group developed a novel and low toxicity peptide DR3penA with significantly improved serum stability by designing and modifying the antioxidant peptide DR8, and confirmed that the DR3penA has good antifibrotic activity in bleomycin murine model, with potential for further development. Since a single animal model is often limited in predicting efficacy in human, studies using multiple models can evaluate efficacy from various perspectives and thus promote the development of lead compounds into drugs. Therefore, in view of the urgency of developing effective drugs against silicosis and paraquat lung and the need for preclinical efficacy evaluation of DR3penA in multiple models, this study evaluated the efficacy of DR3penA in other two pulmonary fibrosis models induced by silica (SiO2) and paraquat (PQ) respectively.

The experimental results showed that the peptide DR3penA significantly inhibited the abnormally high expression of fibrosis markers α-SMA, Fibronectin and Collagen I in the SiO2-induced A549 cell model and the TGF-β1-induced BEAS-2B cell model. The antifibrotic activity of DR3penA was tested by multiple methods including H&E and Masson staining, immunohistochemistry, western blot and RT-qPCR in SiO2- and PQ-induced lung fibrosis model. The results suggested that DR3penA (0.5, 1.6 and 5.0 mg/kg) reduced the abnormally high expression of the fibrosis markers Fibronectin and Collagen I in silicosis mice and alleviate the extent of pulmonary fibrosis, and its anti-pulmonary fibrosis effect in mice was not significantly different from that of the positive control drug pirfenidone (PFD) (100 mg/kg). In the PQ-induced rat lung fibrosis model, DR3penA (0.200, 0.632 and 2.000 mg/kg) decreased the excessive deposition of extracellular matrix (ECM) and mitigated the extent of interstitial lung solidification, and its inhibitory effect on Fibronectin and Collagen I was not significantly different compared to the positive control drug PFD (100 mg/kg).

As both SiO2 and PQ can contribute to the progression of lung fibrosis by inducing oxidative stress and triggering the Epithelial-mesenchymal transition (EMT) process in organisms. Therefore, we examined the expression of oxidative stress markers nicotinamide adenine dinucleotide phosphate oxidases 4 (NOX4) and EMT markers (E-Cadherin and N-Cadherin) both in vitro and in vivo, as well as the production of reactive oxygen species (ROS) in cells and the levels of antioxidant glutathione (GSH) and lipid peroxidation product malondialdehyde (MDA) and the catalase (CAT) activity in the serum of the animals. The results suggested that DR3penA depressed SiO2- and PQ-induced oxidative stress and EMT processes obviously. The preliminary mechanistic study revealed that in silicosis, DR3penA negatively regulated PI3K/AKT pathway by inhibiting oxidative stress with reducing ROS production and thus hindered the EMT process to ultimately fight pulmonary fibrosis. In PQ-induced pulmonary fibrosis, DR3penA was able to reduce the phosphorylation levels of ERK and p38 by inhibiting oxidative stress and abnormally high expression of TGF-β, thereby impeding the EMT process and thus exerting an anti-pulmonary fibrosis effect.

In summary, this study found that DR3penA could effectively inhibit the progression of SiO2- and PQ-induced pulmonary fibrosis. And through the conversion of drug bioavailability under different routes of administration, DR3penA exerts antifibrotic activity equivalent to that of the positive control drug pirfenidone (PFD) at lower doses. The mechanistic studies suggested that DR3penA can exert anti-pulmonary fibrosis effects by inhibiting the activation of PI3K/AKT and MAPK signaling pathways. Therefore, DR3penA can be used as a candidate compound for the treatment of fibrotic ILDs to evaluate in greater depth on medicinal properties. This study provides a research basis for the development of peptide drugs with independent intellectual property rights for the treatment of fibrotic diseases.

MOST Discipline Catalogue理学 - 生物学 - 生物化学与分子生物学
URL查看原文
Language中文
Other Code262010_220200924590
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/536081
Collection基础医学院
Affiliation
兰州大学基础医学院
Recommended Citation
GB/T 7714
李洁茹. 多肽DR3penA 改善二氧化硅和百草枯所致肺纤维化的作用及机制研究[D]. 兰州. 兰州大学,2023.
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