+

WO2018171279A1 - 化合物ss-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用 - Google Patents

化合物ss-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用 Download PDF

Info

Publication number
WO2018171279A1
WO2018171279A1 PCT/CN2017/118835 CN2017118835W WO2018171279A1 WO 2018171279 A1 WO2018171279 A1 WO 2018171279A1 CN 2017118835 W CN2017118835 W CN 2017118835W WO 2018171279 A1 WO2018171279 A1 WO 2018171279A1
Authority
WO
WIPO (PCT)
Prior art keywords
preparation
compound
aortic
development
medicament
Prior art date
Application number
PCT/CN2017/118835
Other languages
English (en)
French (fr)
Inventor
李宽钰
乔彤
Original Assignee
南京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京大学 filed Critical 南京大学
Publication of WO2018171279A1 publication Critical patent/WO2018171279A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides

Definitions

  • the invention belongs to the field of chemical medicine and relates to the application of a compound Szeto-Schiller-31 (SS-31) for preparing a medicament for treating atherosclerotic diseases.
  • Atherosclerosis is the pathological basis of cardiovascular disease, including carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease.
  • the clinical manifestations and pathological features of these diseases are not the same, but they have the common characteristics: the slow development of the lesions, the narrowing of the lumen of the affected arteries, and the lack of blood supply to the distal tissues. So far, there is no ideal preventive drug, which has become a worldwide cause. The most important cause of human death. At present, the pathogenesis of these diseases is still controversial. Genetic, inflammatory, high-fat diet, aging and other complex factors are the common pathogenic factors of these diseases.
  • the pathological mechanisms include vascular endothelial injury and monocyte adhesion migration.
  • Atherosclerotic diseases are a serious hazard to human health, which brings great psychological and economic burden to patients, their families and society.
  • the development of drugs for the prevention and treatment of atherosclerosis has received much attention.
  • AS Arterial endothelial injury is the initial step of AS, and damaged endothelial cells secrete adhesion molecules and cytokines to recruit white blood cells.
  • the persistence of inflammation upregulates the expression of macrophage scavenger receptors, increases ox-LDL uptake, and promotes foam cell formation.
  • Foam cells are the most prominent inflammatory cells in AS plaques.
  • adhesion molecules such as intracellular adhesion molecules, vascular cell adhesion molecules and monocyte chemotactic factors are highly expressed in AS plaques. After adhering to endothelial cells via adhesion molecules, leukocytes are mediated by chemokines and penetrate into the intima of the blood vessels.
  • CD36 and LOX-1 are oxidative low-density lipoprotein (ox-LDL) receptors, which are lipoproteins on the surface of macrophages; ABCA1 and ABCG1 are lipoproteins on the surface of macrophages, and cholesterol is excreted and receptor HDL Or apolipoprotein A-I binding.
  • Szeto-Schiller-31 (SS-31) is a novel mitochondrial-targeting compound synthesized by Szeto in 2005. It can act on mitochondrial inner membrane cardiolipin to protect mitochondrial integrity in an animal model of renal ischemia. Reduce or inhibit the production of mitochondria-derived reactive oxygen species (ROS), increase the level of adenosine triphosphate (ATP), and reduce oxidative stress. Obviously these findings are not directly related to anti-atherosclerosis. At present, SS-31 has been applied to many human disease models and has shown certain effects.
  • ROS mitochondria-derived reactive oxygen species
  • ATP adenosine triphosphate
  • SS-31 has a protective effect on heart failure and hypertensive cardiomyopathy, although heart failure and hypertensive myocardium
  • the disease and the atherosclerotic disease involved in this application are completely different from the pathogenesis of the disease, the pathophysiological basis, the corresponding therapeutic drugs, therapeutic targets and treatment strategies, and it is impossible to give guidance and enlightenment, but we still want to Boldly try to see if SS-31 has a protective effect on atherosclerotic disease.
  • the object of the present invention is to discover a new medical use of SS-31 in the preparation of atherosclerosis and related diseases through a disease model associated with atherosclerotic diseases.
  • SS-31 is used in an animal model of atherosclerotic disease, and the results show that subcutaneous injection of SS-31 delays the development of mouse AS, blocks the formation of plaque, and stabilizes vulnerable plaque on the other hand.
  • SS-31 can reduce the level of aortic ROS in mice, reduce oxidative damage, increase ATP levels, improve systemic inflammation in mice, and decrease the expression of lipid uptake proteins (CD36 and LOX-1) in mouse aortic plaques. These results indicate that SS-31 can be used clinically as a therapeutic drug for AS or related diseases.
  • SS-31 itself or its main component is used in the treatment of atherosclerotic diseases including carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease.
  • SS-31 itself or its use as a main component in the preparation of the following drugs:
  • the present invention is the first to apply the novel mitochondrial-targeting compound SS-31 for the treatment of atherosclerosis and related diseases, and can be used as a new application for preparing drugs for treating atherosclerosis and related diseases, and has a huge market. Value and social benefits.
  • the present invention provides the use of SS-31 as a main component of a drug in atherosclerotic diseases.
  • SS-31 delays the development of atherosclerotic disease in mice, reduces the formation of plaques, and stabilizes vulnerable plaques.
  • FIG. 6 Effect of SS-31 on aortic reactive oxygen species (ROS) in ApoE -/- mice.
  • ROS reactive oxygen species
  • ApoE -/- mice Male 8-week-old ApoE -/- mice (genetic background: C57BL/6) were purchased from the Institute of Model Animals of Nanjing University. ApoE -/- mice were housed in SPF-class animal rooms and fed a high-fat diet. High-fat diet formula: 0.2% cholesterol and 20% fat mixed with conventional feed. ApoE -/- mice were randomly divided into control group (P), low-dose drug group (M1, 1 mg/kg/d) and high-dose drug group (M3, 3 mg/kg/d), with 30 rats in each group.
  • P control group
  • M1, 1 mg/kg/d low-dose drug group
  • M3, 3 mg/kg/d high-dose drug group
  • Group P was injected subcutaneously with 5 mL ⁇ kg -1 ⁇ d -1 saline, and M1 group was injected subcutaneously with SS-31 5 mL ⁇ kg -1 ⁇ d -1 (SS-31 powder was dissolved in physiological saline at a concentration of 0.2 mg ⁇ mL -1 , Shanghai Qiang Yao Biotechnology Co., Ltd. synthesis, dose reference), M3 group subcutaneous injection of SS-31 5mL ⁇ kg -1 ⁇ d -1 (concentration 0.6mg ⁇ mL -1 ).
  • mice were anesthetized by intraperitoneal injection of pentobarbital (40 mg ⁇ kg -1 ), blood was taken through the inferior vena cava, the anesthetic dose (80 mg ⁇ kg -1 ) was added, and the neck dislocation was sacrificed, and the heart and aorta were collected.
  • Cardiac specimens were fixed in 4% paraformaldehyde for 24 hours, embedded in OCT or embedded in paraffin, and 10 slices of 6 ⁇ m thick paraffin or frozen sections were continuously cut in the microtome. The aortic sinus paraffin sections were stored at room temperature for use.
  • TG serum triglyceride
  • TC total cholesterol
  • Enzyme-linked immunosorbent assay (ELISA) kit detects intracellular adhesion molecule-1 (ICAM-1) and monocyte chemotaxis Factor-1 (Monocyte chemoattractant protein, MCP-1), interleukin (IL-6) and C-reactive protein (CRP) levels.
  • ICM-1 intracellular adhesion molecule-1
  • MCP-1 monocyte chemotaxis Factor-1
  • IL-6 interleukin
  • CRP C-reactive protein
  • the aorta was embedded in OCT immediately after excision. After cryopreservation, a 6 ⁇ m thick slice was sliced on a glass slide. The fluorescent probe (Dihydroethidium, DHE, 10 ⁇ M, Sigma-Aldrich, USA) was incubated at 37 ° C for 30 min in the dark. Focusing microscope (ZEISS HB050, ZEISS, Germany) observed fluorescence, and fluorescence strongly reacted with ROS levels. Immediately after the aorta was isolated, the ATP level and total SOD activity were measured using the ATP test kit (Shanghai Biyuntian Biotechnology Co., Ltd.) and the SOD test kit (Nanjing Institute of Bioengineering).
  • aorta was lysed by lysate (Thermo Fisher Scientific, UK), the protein concentration determined by BCA method, 35 ⁇ g of total protein extract was electrophoresed on a 12% SDS-PAGE gel, and transferred to a nitrocellulose transfer membrane (Nitrocellulose).
  • tissue sections of the mice were dewaxed, hematoxylin staining for 4 min, Masson's modified trichrome dyeing for 8 min, bright green staining for 8 min, 0.2% ammonium acetate wash to no dye shedding, dehydration, transparency, sealing, observation under light microscope And take pictures.
  • Image J software was used to analyze the amount of collagen (blue) in the plaque.
  • mice Tissue sections of mice were dewaxed, antigen-repaired, 3% H 2 O 2 inhibited endogenous peroxidase, primary antibody (1:200 dilution) was incubated for 1.5 h at room temperature, and secondary antibody (1:200 dilution) was incubated for 30 min at room temperature. DAB coloration, hematoxylin counterstaining. After the staining was completed, it was observed under an optical microscope and photographed.
  • CD68 macrophage molecular marker
  • ⁇ -SMA smooth muscle cell molecular marker
  • CD36 antibody was purchased from Proteintech Group, USA
  • LOX-1 antibody was purchased from Santa Cruz Biotech, USA
  • ABCA1 antibody was purchased from US Signalway Antibody LLC
  • secondary antibody goat anti-rabbit or oxygen anti-mouse
  • the P group ApoE -/- mice were similar in weight to the administration group (M1 and M3) at 8 and 20 weeks, and the serum TC and TG in the administration group were similar to the P group at 20 weeks, as shown in Table 1.
  • Gross aortic red staining of the aorta revealed a significant reduction in plaque area in the ApoE -/- mice of the M1 and M3 groups, as shown in Figure 1, and quantified on the right. Oil red staining of the frozen section of the aortic sinus area showed that the plaque size of the ApoE -/- mice in the M1 and M3 groups was significantly reduced, as shown in Fig. 2, and the quantified map below.
  • the plaque composition changes were studied by CD68, ⁇ -SMA immunohistochemistry and Masson special staining. As shown in Fig. 3, the immunohistochemical positive areas of CD68 in M1 and M3 groups were significantly reduced, and the quantitative map was below; Fig. 4 is ⁇ -SMA. Immunohistochemistry showed a significant increase in smooth muscle cells at the plaques of ApoE -/- mice in the M1 and M3 groups, with quantified maps below; Figure 5 is a special staining of Masson, blue area is quantified below, and ApoE -/- is small in M1 and M3 groups. Collagen increased significantly at the plaque of the mouse. The above results indicate that the plaque area of the SS-31 group is significantly reduced and the plaque is more early and more stable. Therefore, SS-31 can be used to block the formation of atherosclerotic plaque and delay the development of AS.
  • 2.2SS-31 reduces aortic oxidative stress levels in ApoE -/- mice and increases aortic ATP synthesis
  • FIG. 6 is a DHE staining of aortic frozen sections.
  • the red positive areas of ApoE -/- mice in the M1 and M3 groups were significantly reduced, indicating a decrease in ROS levels and a decrease in oxidative stress in ApoE -/- mice.
  • Aortic ATP results showed a significant increase in aortic ATP levels in the M1 and M3 groups, as shown in Figure 7.
  • SOD is the main protein for ROS clearance in cells, including SOD1 in cells and SOD2 in mitochondria.
  • SOD2 plays a major role.
  • Aortic Western blotting showed no change in SOD2 protein levels, as shown in Figure 8, below which is a quantitative map; but the total aortic SOD activity was significantly increased, as shown in Figure 9.
  • Increased ROS levels impair DNA, and 8-OHDG immunohistochemistry was used to detect arterial DNA damage.
  • the results showed that the 8-OHDG-positive area of mice in M1 and M3 groups was significantly reduced, as shown in Figure 10, and the upper part was 8-OHDG immunized.
  • Histochemical image below is a quantitative map of positive areas. The above results indicate that SS-31 reduces the level of arterial oxidative stress and can therefore be used to reduce aortic oxidative damage and further expansion of atheromatous plaques.
  • ICAM-1 and MCP-1 are the major inflammatory factors in AS, promoting the adhesion of monocytes/macrophages to endothelial cells and migration to the inner membrane.
  • the serum levels of ICAM-1 and MCP-1 in the M1 and M3 mice were significantly reduced (see Table 1).
  • Macrophages that migrate to the endothelium secrete pro-inflammatory factors, including IL-6, IL-1 ⁇ and Tumor necrosis factor alfa (TNF- ⁇ ). These inflammatory factors mediate systemic inflammatory responses, such as activation of acute phase proteins encoded by the liver gene, including CRP and serum amyloid A (SAA).
  • Serum IL-6 was significantly decreased in the ApoE -/- mice of the M1 and M3 groups, and CRP was slightly decreased, as shown in Table 1.
  • the above results indicate that SS-31 can improve the systemic inflammation level of ApoE -/- mice, which is beneficial to delay the further development of atheroma.
  • 2.4SS-31 reduces the expression of aortic lipid uptake protein in ApoE -/- mice
  • Foam cell formation is the key to the emergence of early plaques in AS. Excessive intake of Oxidized low-density lipoprotein (ox-LDL), excessive cholesterol esterification, and impaired cholesterol release lead to accumulation of cholesterol esters, formation of lipid droplets, and the cells gradually transform into foam cells.
  • CD36 Cluster of differentiation 36
  • LOX-1 Lectin-like ox-LDL receptor-1
  • ABCA1 ATP-binding cassette A1 is a lipid.
  • Excrete protein is a lipid.
  • Table 1 mouse body weight and serum lipid index.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

化合物Szeto-Schiller-31(SS-31)在制备治疗动脉粥样硬化及相关疾病药物上的应用，如颈动脉狭窄、下肢动脉硬化闭塞症及冠心病。通过实验发现SS-31减轻动脉粥样硬化程度：显著降低小鼠动脉ROS水平，减少氧化损伤，提高ATP水平；显著减少动脉粥样硬化斑块处巨噬细胞含量，显著增加斑块处平滑肌细胞含量和胶原含量，稳定动脉粥样硬化斑块；显著改善炎症水平；显著降低主动脉斑块处脂质摄取蛋白的表达。结果表明SS-31具有治疗动脉粥样硬化疾病及其相关疾病的作用。

Description

化合物SS-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用 技术领域
本发明属于化学药物领域，涉及一种化合物Szeto-Schiller-31(SS-31)在制备治疗动脉粥样硬化疾病药物上的应用。
背景技术
动脉粥样硬化(Atherosclerosis,AS)是心血管疾病的病理基础，主要包括颈动脉狭窄、下肢动脉硬化闭塞症及冠心病。这类疾病的临床表现、病变特征虽不尽相同，但它们有共同的特征：病变缓慢发展，受累动脉管腔狭窄，远端组织血供不足，至今无理想预防药物，已成为世界范围内导致人类死亡的最主要原因。目前这类疾病的发病机制尚存争议，遗传、炎症、高脂饮食、老化等多种复合因素是该类疾病的共同致病因素，病理机制均包括血管内皮损伤、单核细胞粘附迁移形成巨噬细胞、巨噬细胞吞噬脂质形成泡沫细胞和代谢通路受损等。动脉粥样硬化疾病严重危害人类健康，给患者、家属和社会带来了极大的心理和经济负担，发展防治动脉粥样硬化的药物备受重视。
慢性炎症状态和高胆固醇血症是AS的重要特征。动脉内皮损伤是AS的起始步骤，受损的内皮细胞分泌粘附分子和细胞因子，募集白细胞。炎症持续存在会上调巨噬细胞清除剂受体的表达，增加ox-LDL摄取，促进泡沫细胞形成。泡沫细胞是AS斑块中最主要的炎症细胞。研究发现AS斑块处细胞内粘附分子、血管细胞粘附分子和单核细胞趋化因子等粘附分子高表达。白细胞经粘附分子粘附于内皮细胞后，经趋化因子介导，渗透进入血管内膜。
巨噬细胞脂质摄取和排出不平衡，细胞内脂质累积，泡沫细胞形成，是AS斑块形成的关键。CD36和LOX-1是氧化性低密度脂蛋白(ox-LDL)受体，是巨噬细胞表面的吸脂蛋白；ABCA1和ABCG1是巨噬细胞表面的排脂蛋白，胆固醇排出并与受体HDL或载脂蛋白A-Ⅰ结合。
Szeto-Schiller-31(SS-31)是一种新型靶向线粒体的化合物，由Szeto于2005年合成，在肾脏缺血动物模型中，可以作用于线粒体内膜心磷脂，保护线粒体的完整性，减少或抑制线粒体来源的活性氧(ROS)的产生，提高三磷酸腺苷(ATP)水平，达到减少氧化应激的目的。显然这些发现与抗动脉粥样硬化没有直接联系。 目前SS-31已应用于多个人类疾病模型中并显示出一定的效果，比如研究显示SS-31对心衰和高血压性心肌病均有一定的保护作用，虽然心衰和高血压性心肌病与本申请所涉及的动脉粥样硬化疾病无论从疾病的发病机制，病理生理基础，还是对应的治疗药物、治疗靶点和治疗策略都完全不同，无法给出教导和启示，但是我们还是想大胆的尝试一下SS-31对动脉粥样硬化疾病是否具有保护作用。
但到目前为止，关于SS-31抗动脉粥样硬化及其相关疾病方面尚无报道。本申请首次通过模型评价SS-31在抗动脉粥样硬化方面的活性，并发现了其在控制动脉粥样硬化方面多方面的调控活性和功能。
Figure PCTCN2017118835-appb-000001
发明内容
发明目的
本发明的目的是通过动脉粥样硬化疾病相关的疾病模型，发现SS-31在制备治疗动脉粥样硬化及相关疾病方面新的医药用途。
技术方案
本发明将SS-31用于动脉粥样硬化疾病动物模型，结果显示皮下注射SS-31一方面延缓小鼠AS的发展，阻滞斑块的形成，另一方面可以稳定易损斑块。SS-31可以降低小鼠主动脉ROS水平、减少氧化损伤、提高ATP水平，改善小鼠全身炎症水平，降低小鼠主动脉斑块处脂质摄取蛋白(CD36和LOX-1)的表达。这些结果表明SS-31可以作为AS或者相关疾病的治疗药物用于临床。
SS-31本身或者其作为主要成分在治疗动脉粥样硬化疾病药物上的应用，所 述动脉粥样硬化疾病包括颈动脉狭窄、下肢动脉硬化闭塞症及冠心病。
进一步的，SS-31本身或者其作为主要成分在制备下列药物上的应用：
(1)阻滞动脉粥样硬化斑块形成、延缓AS的发展；
(2)稳定易损动脉粥样硬化斑块、防止不稳定型心绞痛或/和心肌梗死的发生；
(3)降低主动脉ROS水平、减少主动脉氧化损伤、提高主动脉ATP水平，提高主动脉的生理机能与功能；
(4)降低主动脉或者全身炎症水平，阻滞AS的发生与发展；
(5)降低主动脉斑块处脂质摄取蛋白CD36的表达，阻滞AS的发生与发展；
(6)降低主动脉斑块处脂质摄取蛋白LOX-1的表达，阻滞AS的发生与发展。
本发明的优点：
1、本发明首次将新型靶向线粒体的化合物SS-31用于动脉粥样硬化及其相关疾病的治疗，可作为制备治疗动脉粥样硬化及其相关疾病药物的新用途申请，具有巨大的市场价值和社会效益。
2、本发明提供了SS-31作为药物主要成分在动脉粥样硬化疾病中的应用。SS-31延缓小鼠动脉粥样硬化疾病的发展，减少斑块的形成，另一方面可以稳定易损斑块。
3、降低主动脉ROS水平、减少主动脉氧化损伤、提高主动脉ATP水平，提高主动脉的生理机能与功能；
4、降低主动脉或者全身炎症水平，阻滞AS的发生与发展；
5、降低主动脉斑块处脂质摄取蛋白CD36的表达，阻滞AS的发生与发展；
6、降低主动脉斑块处脂质摄取蛋白LOX-1的表达，阻滞AS的发生与发展。
附图说明：
图1SS-31对ApoE -/-小鼠主动脉斑块形成的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000002
表示。
图2SS-31对ApoE -/-小鼠主动脉窦区斑块形成的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000003
表示。
图3SS-31对ApoE -/-小鼠主动脉窦区斑块成分(巨噬细胞)的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000004
表示。
图4SS-31对ApoE -/-小鼠主动脉窦区斑块成分(平滑肌细胞)的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000005
表示。
图5SS-31对ApoE -/-小鼠主动脉窦区斑块成分(胶原)的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000006
表示。
图6SS-31对ApoE -/-小鼠主动脉活性氧(ROS)的影响。
图7SS-31对ApoE -/-小鼠主动脉ATP的影响(n＝13)，数据均以
Figure PCTCN2017118835-appb-000007
表示。
图8SS-31对ApoE -/-小鼠主动脉SOD2蛋白水平的影响(n＝14)，数据均以
Figure PCTCN2017118835-appb-000008
表示。
图9SS-31对ApoE -/-小鼠主动脉总SOD2酶活性的影响(n＝13)，数据均以
Figure PCTCN2017118835-appb-000009
表示。
图10SS-31对ApoE -/-小鼠主动脉窦区DNA损伤的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000010
表示。
图11SS-31对ApoE -/-小鼠主动脉窦区CD36的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000011
表示。
图12SS-31对ApoE -/-小鼠主动脉窦区LOX-1的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000012
表示。
图13SS-31对ApoE -/-小鼠主动脉窦区ABCA1的影响(n＝15)，数据均以
Figure PCTCN2017118835-appb-000013
表示。
具体实施方式：
SS-31对ApoE-/-小鼠的动脉粥样硬化的影响
1材料和方法
1.1实验分组与标本收集
雄性8周大ApoE -/-小鼠(遗传背景：C57BL/6)90只，从南京大学模式动物研究所购买。ApoE -/-小鼠饲养于SPF级动物房，并予高脂饮食。高脂饮食配方：0.2％胆固醇和20％脂肪混合常规饲料。ApoE -/-小鼠随机分为对照组(P)、低剂量药物组(M1，1mg/kg/d)和高剂量药物组(M3,3mg/kg/d)，每组30只。P组皮下注射生理盐水5mL·kg -1·d -1，M1组皮下注射SS-31 5mL·kg -1·d -1(SS-31 粉末溶于生理盐水，浓度0.2mg·mL -1，上海强耀生物科技有限公司合成，剂量参考文献)，M3组皮下注射SS-31 5mL·kg -1·d -1(浓度0.6mg·mL -1)。12周后，小鼠腹腔注射戊巴比妥(40mg·kg -1)麻醉，经下腔静脉取血，增加麻醉剂量(80mg·kg -1)后颈脱位处死，收集心脏及主动脉。心脏标本4％多聚甲醛固定24h，OCT包埋或石蜡包埋，在切片机连续切10张6μm厚石蜡或冰冻切片，主动脉窦石蜡切片常温保存备用。主动脉窦冰冻切片置于丙二醇中静置2min，油红O染色缸中染色16h，85％丙二醇分化1min，苏木素染色3min，封片，尽快在光学显微镜(德国ZEISS公司)下观察并拍照。每组小鼠主动脉进行大体油红染色(具体步骤见参考文献)，主动脉检测超氧化物歧化酶2(Superoxide dismutase 2,SOD2)蛋白水平，主动脉检测总SOD活性，主动脉检测ATP水平。
1.2血液标本处理
血液标本4℃过夜，2500g离心20min，取上清，获得血清标本。自动生化分析仪(Beckman Coulter AU542)检测血清中甘油三酯(Triglyceride,TG)和胆固醇(total cholesterol，TC)的含量。酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)试剂盒(武汉博士德生物工程有限公司)检测血清中细胞内粘附分子-1(Intracellular adhesion molecule，ICAM-1)、单核细胞趋化因子-1(Monocyte chemoattractant protein，MCP-1)、白介素6(Interleukin,IL-6)和C-反应蛋白(C-reactive protein，CRP)水平。
1.3DHE染色、ATP检测及SOD活性检测
主动脉离体后立即OCT包埋，急冻后冰冻切片机切6μm厚切片于载玻片上，荧光探针(Dihydroethidium,DHE,10μM,美国Sigma-Aldrich公司)37℃避光孵育30min，激光共聚焦显微镜(ZEISS HB050，德国ZEISS公司)观察荧光，荧光强弱反应ROS水平。主动脉离体后立即利用ATP检测试剂盒(上海碧云天生物技术有限公司)及SOD检测试剂盒(南京建成生物工程研究所)检测ATP水平及总SOD活性。
1.4Western blotting
主动脉通过裂解液(英国Thermo Fisher Scientific公司)研磨裂解后，BCA法测定的蛋白浓度，35μg总蛋白提取物在12％SDS-PAGE凝胶中电泳分离，转至硝酸纤维素转印膜(Nitrocellulose membranes，NC)后一抗(SOD2，1：1000 稀释，英国Abcam公司；Tubulin，1:2000稀释，美国Sigma-Aldrich公司)4℃过夜，二抗(羊抗兔或羊抗鼠，1:10000稀释)孵育1h。NC膜与ECL化学发光底物(上海碧云天生物技术有限公司)孵育后，立即用Western blotting化学发光检测系统(英国Thermo Fisher Scientific公司)进行曝光拍摄。采用Image J软件检测SOD2含量。
1.5Masson特殊染色
小鼠的组织切片脱蜡，苏木素精染色4min，Masson氏改良三色染料染色8min，亮绿染色液染色8min，0.2％醋酸铵洗至无染料脱落，脱水，透明，封片，光学显微镜下观察并拍照。采用Image J软件分析斑块中胶原(蓝色)的含量。
1.6免疫组织化学(Immunohistochemistry，IHC)
小鼠的组织切片脱蜡，抗原修复，3％H 2O 2抑制内源性过氧化酶，一抗(1:200稀释)室温孵育1.5h，二抗(1:200稀释)室温孵育30min，DAB显色，苏木素复染。染色完成后，在光学显微镜镜下观察并拍照。CD68(巨噬细胞分子标记)、α-SMA(平滑肌细胞分子标记)抗体购于英国Abcam公司；CD36抗体购于美国Proteintech Group公司；LOX-1抗体购于美国Santa Cruz Biotech公司；ABCA1抗体购于美国Signalway Antibody LLC公司；二抗(羊抗兔或氧抗鼠)购于美国Santa Cruz Biotech公司。采用Image J软件分析IHC阳性区域的强度。
1.7数据处理
采用SPSS 22.0软件进行统计分析。计量资料以均数±标准差
Figure PCTCN2017118835-appb-000014
表示，组间两两比较采用Student t检验。p<0.05表示差异有统计学意义。
2结果
2.1SS-31可以延缓ApoE -/-小鼠AS发展
P组ApoE -/-小鼠8周和20周时体重与给药组(M1和M3)相似，给药组20周血清TC和TG与P组相似，如表1所示。主动脉大体油红染色发现M1和M3组ApoE -/-小鼠斑块面积显著减小，如图1所示，右方为其量化图。主动脉窦区冰冻切片油红染色发现M1和M3组ApoE -/-小鼠斑块大小显著减小，如图2所示，下方为其量化图。利用CD68、α-SMA免疫组织化学和Masson特殊染色研究斑块成分变化，如图3所示，M1和M3组CD68免疫组化阳性区域显著减 少，下方为其量化图；图4为α-SMA免疫组化，M1和M3组ApoE -/-小鼠斑块处平滑肌细胞显著增多，下方为量化图；图5为Masson特殊染色，下方为蓝色面积量化，M1和M3组ApoE -/-小鼠斑块处胶原显著增多。以上结果表明SS-31组小鼠斑块面积明显减少减小，斑块表现更早期并且更加稳定，因此SS-31能够用于阻滞动脉粥样硬化斑块形成、延缓AS的发展。
2.2SS-31减少ApoE -/-小鼠主动脉氧化应激水平并提高主动脉ATP合成
AS斑块处氧化应激水平升高，而SS-31作为一种抗氧化剂可以减少氧化应激水平，于是我们检测了各小鼠主动脉的ROS水平和ATP水平。图6是主动脉冰冻切片的DHE染色，M1和M3组ApoE -/-小鼠红色阳性区域明显减少，表明ApoE -/-小鼠主动脉细胞内ROS水平下降，氧化应激降低。主动脉ATP检测结果显示M1和M3组主动脉ATP含量显著升高，如图7所示。SOD是细胞清除ROS的主要蛋白，包括细胞中的SOD1和线粒体中的SOD2，SOD2发挥主要作用。主动脉Western blotting检测发现SOD2蛋白水平无变化，如图8所示，下方为其量化图；但是主动脉总SOD酶活性显著升高，如图9所示。ROS水平升高会损伤DNA，利用8-OHDG免疫组织化学检测动脉的DNA损伤，结果显示M1和M3组小鼠8-OHDG阳性区域面积显著降低，如图10所示，上方为8-OHDG免疫组织化学图像，下方为阳性区域量化图。以上结果表明SS-31降低了动脉氧化应激水平,因此可用于减少主动脉氧化损伤和粥样斑块的进一步扩张。
2.3SS-31改善ApoE -/-小鼠全身炎症水平
AS是一种慢性炎症性疾病，ICAM-1和MCP-1是AS中主要的炎症因子，促进单核细胞/巨噬细胞向内皮细胞的粘附和向内膜的迁移。M1和M3组小鼠血清中ICAM-1和MCP-1显著降低(见表1)。迁移至血管内膜的巨噬细胞会分泌促炎因子，包括IL-6，IL-1β和肿瘤坏死因子(Tumor necrosis factor alfa，TNF-α)。这些炎症因子会介导全身的炎症反应，比如激活肝基因编码的急性期蛋白，包括CRP和血清淀粉样蛋白A(serum amyloid A，SAA)。M1和M3组ApoE -/-小鼠血清IL-6显著降低，CRP轻微降低，见表1。以上结果表明SS-31可以改善ApoE -/-小鼠全身炎症水平，有利于延缓粥样斑块的进一步发展。
2.4SS-31降低ApoE -/-小鼠主动脉脂质摄取蛋白的表达
泡沫细胞形成是AS早期斑块出现的关键。氧化性低密度脂蛋白(Oxidized  low-density lipoprotein，ox-LDL)摄取过多、胆固醇酯化过度和胆固醇释放受损均会导致胆固醇酯累积，形成脂滴，细胞逐渐向泡沫细胞转化。CD36(Cluster of differentiation 36)和LOX-1(Lectin-like ox-LDL receptor-1)为ox-LDL受体，是重要的脂质摄取蛋白，ABCA1(ATP-binding cassette A1)是一种脂质排出蛋白。图11-13所示，M1和M3组ApoE -/-小鼠主动脉斑块处CD36和LOX-1表达显著降低，而ABCA1表达无改变。以上结果表明SS-31降低主动脉CD36和LOX-1的表达，抑制ox-LDL的摄取，预防脂质累积，减少泡沫细胞形成，因此SS-31可用于阻滞AS的发生和发展。
表1 小鼠体重和血清脂质指标.
Figure PCTCN2017118835-appb-000015
注：数据均用
Figure PCTCN2017118835-appb-000016
表示，*p<0.05，n＝15。

Claims (10)

  1. 化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用。
  2. 根据权利要求1所述的化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用，其特征在于所述动脉粥样硬化疾病包括颈动脉狭窄、下肢动脉硬化闭塞症及冠心病。
  3. 根据权利要求1或2所述的化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用，其特征在于化合物SS-31在制备阻滞动脉粥样硬化斑块形成、延缓AS的发展药物或者制剂上的应用。
  4. 根据权利要求1或2所述的化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用，其特征在于化合物SS-31在制备稳定易损动脉粥样硬化斑块、防止不稳定型心绞痛和心肌梗死发生药物或者制剂上的应用。
  5. 根据权利要求1或2所述的化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用，其特征在于化合物SS-31在制备缓解机体具有的全身性的氧化应激和慢性炎症过程的药物或者制剂上的应用。
  6. 根据权利要求1或2所述的化合物SS-31在制备治疗动脉粥样硬化疾病药物或者制剂上的应用，其特征在于化合物SS-31在制备血管老化的病理特征的药物或者制剂上的应用。
  7. 化合物SS-31在制备降低主动脉ROS水平、减少主动脉氧化损伤、提高主动脉ATP水平、提高主动脉的生理机能与功能药物或者制剂上的应用。
  8. 化合物SS-31在制备降低主动脉或者全身炎症水平、阻滞AS的发生与发展药物或者制剂上的应用。
  9. 化合物SS-31在制备降低主动脉斑块处脂质摄取蛋白CD36的表达、阻滞AS的发生与发展药物或者制剂上的应用。
  10. 化合物SS-31在制备降低主动脉斑块处脂质摄取蛋白LOX-1的表达、阻滞AS的发生与发展药物或者制剂上的应用。
PCT/CN2017/118835 2017-03-24 2017-12-27 化合物ss-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用 WO2018171279A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710186245.1 2017-03-24
CN201710186245.1A CN107041946A (zh) 2017-03-24 2017-03-24 化合物ss‑31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用

Publications (1)

Publication Number Publication Date
WO2018171279A1 true WO2018171279A1 (zh) 2018-09-27

Family

ID=59544864

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/118835 WO2018171279A1 (zh) 2017-03-24 2017-12-27 化合物ss-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用

Country Status (2)

Country Link
CN (1) CN107041946A (zh)
WO (1) WO2018171279A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107041946A (zh) * 2017-03-24 2017-08-15 南京大学 化合物ss‑31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用
EP3771467A1 (en) * 2019-07-30 2021-02-03 Fundacio Institut de Recerca de l'Hospital de la Santa Creu i sant Pau Ss-31 for the prevention and/or treatment of aneurysm
CN113143922B (zh) * 2021-05-07 2022-12-20 广州医科大学 Dhc在制备动脉粥样硬化治疗制剂中的应用
CN115400201A (zh) * 2021-05-26 2022-11-29 四川大学华西医院 Ss-31在制备预防和/或治疗香烟诱导的气道炎症及慢性阻塞性肺疾病的药物中的用途
CN113476587B (zh) * 2021-07-19 2022-07-12 河南大学 一种脑靶向的神经保护剂ss31-ha-rt及其制备方法和应用
CN113975402B (zh) * 2021-07-19 2023-05-16 河南大学 靶向脑缺血区线粒体的神经保护药物ss31-ha-qt及其荧光探针和应用
CN116602952A (zh) * 2023-03-28 2023-08-18 浙江大学 脂肪代谢因子lipoxinB4在制备动脉粥样硬化治疗药物中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1938042A (zh) * 2004-01-23 2007-03-28 科内尔研究基金会 用于减轻氧化性损伤的方法
CN102791279A (zh) * 2009-12-31 2012-11-21 康肽德生物医药技术有限公司 用于进行冠状动脉旁路移植手术的方法
CN104203262A (zh) * 2012-02-23 2014-12-10 康奈尔大学 芳香族阳离子肽及其用途
CN104324360A (zh) * 2005-09-16 2015-02-04 科内尔研究基金会 用于减少cd36表达的方法
WO2016007921A1 (en) * 2014-07-10 2016-01-14 Rhodel Island Hospital Treating arrhythmia with mitochondrial-targeted antioxidants
CN107041946A (zh) * 2017-03-24 2017-08-15 南京大学 化合物ss‑31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3485897A1 (en) * 2012-08-02 2019-05-22 Stealth Peptides International, Inc. D-arg-2'6'-dmt-lys-phe-nh2 for decreasing statin side effects

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1938042A (zh) * 2004-01-23 2007-03-28 科内尔研究基金会 用于减轻氧化性损伤的方法
CN104324360A (zh) * 2005-09-16 2015-02-04 科内尔研究基金会 用于减少cd36表达的方法
CN102791279A (zh) * 2009-12-31 2012-11-21 康肽德生物医药技术有限公司 用于进行冠状动脉旁路移植手术的方法
CN104203262A (zh) * 2012-02-23 2014-12-10 康奈尔大学 芳香族阳离子肽及其用途
WO2016007921A1 (en) * 2014-07-10 2016-01-14 Rhodel Island Hospital Treating arrhythmia with mitochondrial-targeted antioxidants
CN107041946A (zh) * 2017-03-24 2017-08-15 南京大学 化合物ss‑31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAO, SHUANGYING: "Mitochondrion-Targeted Peptide SS-31 Inhibited Oxidi- zed Low-Density Lipoproteins-Induced Foam Cell Formation through both ROS Scavenging and Inhibition of Cholesterol Influx in RAW264.7 Cells", MOLECULES, vol. 20, no. 12, 1 December 2015 (2015-12-01), XP055610656 *
ZHANG, MENG: "Chronic administration of mitochondrion-targeted peptide SS-31 prevents atherosclerotic development in ApoE knockout mice fed Western diet", PLOS ONE, vol. 12A, no. 9, e0185688, 29 September 2017 (2017-09-29), pages 1 - 15, XP055610654 *

Also Published As

Publication number Publication date
CN107041946A (zh) 2017-08-15

Similar Documents

Publication Publication Date Title
WO2018171279A1 (zh) 化合物ss-31在制备治疗动脉粥样硬化及相关疾病药物或制剂上的应用
Bi et al. Irisin alleviates liver ischemia-reperfusion injury by inhibiting excessive mitochondrial fission, promoting mitochondrial biogenesis and decreasing oxidative stress
Meng et al. Estrogen prevent atherosclerosis by attenuating endothelial cell pyroptosis via activation of estrogen receptor α-mediated autophagy
Kim et al. Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4–MD2 complex
Rayner et al. Extracellular release of the atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor-A
Kim et al. TFEB–GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response
De Gaetano et al. M1-and M2-type macrophage responses are predictive of adverse outcomes in human atherosclerosis
Zhang et al. Fasting exacerbates hepatic growth differentiation factor 15 to promote fatty acid β-oxidation and ketogenesis via activating XBP1 signaling in liver
Xing et al. Salidroside decreases atherosclerosis plaque formation via inhibiting endothelial cell pyroptosis
Wang et al. Protective effect of shikonin in experimental ischemic stroke: attenuated TLR4, p-p38MAPK, NF-κB, TNF-α and MMP-9 expression, up-regulated claudin-5 expression, ameliorated BBB permeability
Salazar et al. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells
Liu et al. Sulconazole induces PANoptosis by triggering oxidative stress and inhibiting glycolysis to increase radiosensitivity in esophageal cancer
Petrović et al. Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver
Yaman et al. Effects of resveratrol on incisional wound healing in rats
Wu et al. C1QTNF1 attenuates angiotensin II-induced cardiac hypertrophy via activation of the AMPKa pathway
Song et al. Essential role for UVRAG in autophagy and maintenance of cardiac function
Ren et al. Identification of irisin as a therapeutic agent that inhibits oxidative stress and fibrosis in a murine model of chronic pancreatitis
Wang et al. κ-opioid receptor activation promotes mitochondrial fusion and enhances myocardial resistance to ischemia and reperfusion injury via STAT3-OPA1 pathway
Zheng et al. SAP deficiency mitigated atherosclerotic lesions in ApoE−/− mice
Liu et al. Irisin Attenuates Apoptosis Following Ischemia–Reperfusion Injury Through Improved Mitochondria Dynamics and ROS Suppression Mediated Through the PI3K/Akt/mTOR Axis
Wang et al. Nuclear receptor subfamily 4 group A member 1 promotes myocardial ischemia/reperfusion injury through inducing mitochondrial fission factor-mediated mitochondrial fragmentation and inhibiting FUN14 domain containing 1-depedent mitophagy
Wang et al. Blocking CXCR3 with AMG487 ameliorates the blood-retinal barrier disruption in diabetic mice through anti-oxidative
Li et al. Role of mechanistic target of rapamycin and autophagy in alcohol-induced adipose atrophy and liver injury
Zhang et al. SAP130 released by ferroptosis tubular epithelial cells promotes macrophage polarization via Mincle signaling in sepsis acute kidney injury
Nakladal et al. Perivascular adipose tissue-derived nitric oxide compensates endothelial dysfunction in aged pre-atherosclerotic apolipoprotein E-deficient rats

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17902255

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17902255

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载