WO2023216280A1 - 一种铁死亡抑制剂在制备治疗胃炎的药物中的应用 - Google Patents

一种铁死亡抑制剂在制备治疗胃炎的药物中的应用 Download PDF

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WO2023216280A1
WO2023216280A1 PCT/CN2022/093212 CN2022093212W WO2023216280A1 WO 2023216280 A1 WO2023216280 A1 WO 2023216280A1 CN 2022093212 W CN2022093212 W CN 2022093212W WO 2023216280 A1 WO2023216280 A1 WO 2023216280A1
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ferroptosis
cells
ges
grim
ferrostatin
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French (fr)
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黄轶
杨梅华
冯金梅
曾欣
徐小惠
臧金宝
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重庆医科大学附属儿童医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of medical technology, and specifically relates to the application of a ferroptosis inhibitor in the preparation of medicines for treating gastritis.
  • CAG Chronic Atrophic Gastritis
  • Metaplasia ⁇ metaplasia
  • Intestinal GC intestinal gastric cancer
  • metaplasia as an important node in the pathological process of CAG, is the key to the early prevention and control of intestinal-type gastric cancer, but the formation mechanism is not completely clear. Therefore, research on the causes and mechanisms of metaplasia is of great significance for the early prevention and control of intestinal-type gastric cancer.
  • Chinese patent document CN113940309A discloses a highly simulated, stable and long-lasting, specific phenotype, and efficient method for simulating chronic atrophic gastritis lesions by the research group: editing the gene-edited mice GRIM-19flox/flox and ATP4b-Cre Mice were crossed to obtain a gastric mucosa-specific parietal cell GRIM-19 knockout mouse strain (GRIM-19-/-/ATP4b-Cre, SPF grade). This gene knockout mouse has chronic atrophic gastritis at the age of 8 months.
  • the early stage of intestinal metaplasia is spontaneous spasmolytic polypeptide expressing metaplasia (SPEM), which can be stably expressed without recurrence and can be used as a pathological model of chronic atrophic gastritis in mice.
  • SPEM spontaneous spasmolytic polypeptide expressing metaplasia
  • Ferroptosis also known as iron-dependent death, is a new form of regulated cell death that is different from apoptosis, necrosis and autophagy. It is closely related to a variety of diseases, such as neurodegeneration, tumors, and ischemia. Reperfusion injury, kidney damage, liver fibrosis, etc. Ferroptosis is typically characterized by the accumulation of intracellular lipid peroxides, in which excessive intracellular Fe 2+ leads to depletion of antioxidant glutathione (GSH) and loss of glutathione peroxidase 4 (GPX4) through Fenton reaction. activity, thereby inducing the accumulation of lipid peroxides and causing ferroptosis.
  • GSH antioxidant glutathione
  • GPX4 glutathione peroxidase 4
  • Ferrostain-1 is currently recognized as a ferroptosis inhibitor. It is a synthetic antioxidant that prevents membrane lipid damage and inhibits cell death through a reduction mechanism.
  • Chinese patent document CN111529518A discloses that the ferroptosis inhibitor Ferrostatin-1 significantly improves lung injury and cell damage in mice caused by drowning, and has a significant protective effect on lung injury caused by drowning;
  • Chinese patent document CN113440509A discloses that ferroptosis inhibition The agent Ferrostatin-1 significantly increased the intracellular GPX4 content, significantly inhibited the generation of ROS in osteoblasts stimulated by wear particles, and reduced the ferroptosis of osteoblasts.
  • the present invention provides a new use of a ferroptosis inhibitor in the preparation of drugs for the treatment of gastritis.
  • the present invention first discovered that GRIM-19 deletion induces a slowdown in the proliferation and vitality of human gastric mucosal epithelial cells.
  • the mechanism of reduction is ferroptosis, not other forms of death such as apoptosis, necrosis, autophagy, etc.
  • the purpose of the present invention is to provide a ferroptosis inhibitor Ferrostatin-1 that can be used as a therapeutic target for chronic atrophic gastritis disease characterized by GRIM-19 deficiency.
  • Ferrostatin-1 can specifically catalyze glutathione by increasing the level of GPX4 in gastric mucosal tissue.
  • Selenoproteins that convert lipid peroxides into lipid alcohols remove toxic products during ferroptosis, thereby further inhibiting ferroptosis.
  • Ferrostatin-1 can significantly reduce the infiltration of inflammatory cells and inflammatory factors in gastric mucosal tissue caused by deletion of GRIM-19 in mouse gastric mucosal parietal cells, reduce SPEM indicators, effectively inhibit the pathological process of chronic atrophic gastritis, and is a drug for chronic atrophic gastritis R&D and innovation provide new ideas.
  • ferroptosis inhibitors in the preparation of drugs for the treatment of gastritis.
  • ferroptosis inhibitor uses Ferrostatin-1 as the active ingredient, supplemented by pharmaceutically acceptable carriers, excipients, diluents, adjuvants, vehicles or combinations thereof.
  • the active ingredient in the ferroptosis inhibitor is a Ferrostatin-1 derivative, isomer or pharmaceutically acceptable salt.
  • gastritis is chronic atrophic gastritis.
  • chronic atrophic gastritis is chronic atrophic gastritis induced by GRIM-19 gene deficiency.
  • ferroptosis inhibitors can inhibit the expression of SPEM genes.
  • ferroptosis inhibitors can increase the level of ferroptosis marker protein glutathione peroxidase 4 (GPX4) in the chronic atrophic gastritis model.
  • GPX4 ferroptosis marker protein glutathione peroxidase 4
  • ferroptosis inhibitors can reduce the expression of inflammatory factors such as L-6, TNF- ⁇ , VEGF, GM-CSF, IL-1 ⁇ , IL-33 and other inflammatory factors in the gastric mucosal tissue of mice in chronic atrophic gastritis models.
  • ferroptosis inhibitors can protect gastric mucosal tissue.
  • the medicine is a tablet, suppository, injection or capsule.
  • the drug is an injection, which is administered by intraperitoneal injection, and the dosage is 1 to 1.5 mg/kg.
  • the present invention finds that in vivo injection of the ferroptosis inhibitor Ferrostatin-1 can significantly increase the level of GPX4 in the gastric mucosal tissue of mice and inhibit the pathological process of ferroptosis in gastric mucosal parietal cells, thereby effectively reducing the levels of L-6, TNF- ⁇ ,
  • the expression of inflammatory factors such as VEGF, GM-CSF, IL-1 ⁇ , IL-33, and SPEM indicators were significantly reduced, which effectively inhibited the pathological process of chronic atrophic gastritis and can be used for the treatment of chronic atrophic gastritis.
  • Ferrostatin-1 treatment of GES-1-134 cells in vitro can increase cellular GPX4 levels, inhibit the pathological process of ferroptosis, and thereby enhance cell viability.
  • Ferroptosis inhibitor Ferrostatin-1 can be used to treat chronic atrophic gastritis, and can provide new ideas for the development and innovation of chronic atrophic gastritis drugs.
  • Figure 1 shows the monoclonal construction diagram of GRIM-19 knockout human gastric mucosal epithelial GES-1 cells (GES-1-134);
  • A is the monoclonal flow cytometry GFP positive detection picture;
  • B is the Western blot detection of GES -1-134 cell GRIM-19 protein expression analysis;
  • Figure 2 shows the results of GES-1-134 cell viability detection
  • Figure 3 shows the detection of apoptosis, necrosis and autophagy of GES-1-134 cells; A is the detection of apoptosis and necrosis of GES-1-134 cells by flow cytometry; B is the detection of GES-1-134 cell autophagy by Western blotting. Analysis of protein expression of phagocytosis marker LC3I/LC3II;
  • Figure 4 shows the detection of ferroptosis marker proteins in GES-1-134 cells and GRIM-19 -/- mouse gastric mucosal tissue;
  • A is the Western blot detection of ferroptosis marker proteins in GES-1-134 cells (COX2, GPX4, FTH1, TFR) expression levels;
  • B is Western blotting to detect the expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR) in gastric mucosal tissue of GRIM-19 -/- mice;
  • Figure 5 is a transmission electron microscope image of the gastric mucosal tissue of GES-1-134 cells and GRIM-19 -/- mice;
  • A is a transmission electron microscope analysis of mitochondrial phenotype of GES-1-134 cells;
  • B is a GRIM-19 -/- Transmission electron microscopy analysis of mitochondrial phenotypes in mouse gastric mucosal tissue;
  • Figure 6 shows the detection of iron ions in gastric mucosal tissue of GES-1-134 cells and GRIM-19 -/- mice; A is the detection of Fe 2+ content in GES-1-134 cells; B is the detection of Fe 2+ in GRIM-19 -/- mice. Prussian blue staining of mouse gastric mucosa tissue (* ⁇ 0.05);
  • FIG. 7 shows the detection of lipid oxidation (MDA) in GES-1-134 cells (*** ⁇ 0.001)
  • Figure 8 shows the detection of GES-1-134 cell viability after intervention with the ferroptosis inhibitor Ferrostatin-1 (*** ⁇ 0.001);
  • Figure 9 shows the detection of ferroptosis marker protein in GES-1-134 cells and GRIM-19 -/- mouse gastric mucosal tissue after intervention with the ferroptosis inhibitor Ferrostatin-1; among them, A is the detection of ferroptosis inhibitor Ferrostatin by Western blotting -1 (0, 5 ⁇ M, 10 ⁇ M) treats the expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR) in GES-1-134 cells; B is Western blotting to detect the intervention of ferroptosis inhibitor Ferrostatin-1 in GRIM-19 - / -Expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR) in mouse gastric mucosa tissue;
  • A is the detection of ferroptosis inhibitor Ferrostatin by Western blotting -1 (0, 5 ⁇ M, 10 ⁇ M) treats the expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR)
  • Figure 10 shows the detection of expression of inflammatory factors in GES-1-134 cells and gastric mucosal tissue of GRIM-19 -/- mice after the intervention of the ferroptosis inhibitor Ferrostatin-1; among them, A is the Western blot detection of GES-1 after the intervention of Ferrostatin-1 1-134 cell IL-6, TNF- ⁇ , VEGF, GM-CSF, IL-1 ⁇ , IL-33 protein expression levels; B is the stomach of GRIM-19 -/- mice after intervention with the protein immunoblot detection agent Ferrostatin-1 IL-6, TNF- ⁇ , VEGF, GM-CSF, IL-1 ⁇ , and IL-33 protein expression levels in mucosal tissue;
  • Figure 11 shows the SPEM pathological process detection and analysis of the gastric mucosal tissue of GRIM-19 -/- mice after the intervention of the ferroptosis inhibitor Ferrostatin-1; among them, A is the detection of SPEM markers TFF2, Mist1, and Clusterin-1 in the gastric mucosal tissue by protein immunoblotting. , HE4 and MUC6 protein levels; B is immunofluorescence detection of GIF, GSII and Mist immunofluorescence three-label analysis (Bar: 50 ⁇ M) (*** ⁇ 0.001).
  • This invention uses the CRISPR/CAS9 single vector lentivirus system of Shanghai Jikai Gene Technology Co., Ltd.
  • the framework structure of GV393 lentivirus is U6-sgRNA-EIFla-Cas9-FLAG-P2A-EGFP;
  • Target gene name genetic species Gene ID GenBankID NDUFA13(GRIM-19) Human 51079 NM_015965
  • NDUFA-13-sgRNA-134-F CACCg TGCTCCAGTGCCCGTAGATC / NDUFA-13-sgRNA-134-R AAAC GATCTACGGGCACTGGAGCA C NC-sgRNA-F CACCg CGCTTCCGCGGCCCGTTCAA / NC-sgRNA-R AAAC TTGAACGGGCCGCGGAAGCG C
  • Binding Buffer:7-AAD 10:1, add 50 ⁇ L dye solution, mix, and react in the dark for 15 minutes;
  • GES-1-134 autophagy marker proteins LC3I and LC3II were detected using Western blot method. As shown in Figure 3B, GES-1-134 showed no increasing trend compared with the autophagy markers LC3I and LC3II in the control group, confirming that the slowed proliferation and reduced activity of gastric mucosal epithelial cells induced by GRIM-19 deletion was not caused by autophagy.
  • GES-1-134 cells Compared with GES-1-NC cells, GES-1-134 cells had decreased levels of glutathione peroxidase 4 (GPX4) and ferritin (FTH1), while cyclooxygenase (COX-2) and transferrin were affected. (TFR) was increased, which suggested that after GRIM-19 was knocked out in normal human gastric mucosal epithelial cells, Fe 2+ overload, lipid peroxide scavenging ability was weakened, and ferroptosis occurred.
  • GPX4 glutathione peroxidase 4
  • FTH1 ferritin
  • COX-2 cyclooxygenase
  • transferrin transferrin
  • the ferroptosis marker protein in the gastric mucosal tissue of GRIM-19 fl/- and GRIM-19 -/- mice shown in Figure 4B has the same trend as that in vitro compared with the control group, which suggests that the The mechanism by which GRIM-19 deletion in mouse gastric mucosal parietal cells induces damage to parietal cells is ferroptosis.
  • FIG. 5A Transmission electron microscopy analysis technology analysis is shown in Figure 5A.
  • the structure and number of mitochondria in GES-1-134 cells have been significantly changed: the number is reduced, the membrane density is increased, and the cristae are reduced, which are consistent with cellular iron. Morphological characteristics of death;
  • Figure 5B Compared with control mice, the mitochondrial structure of gastric mucosal tissue cells in GRIM-19 -/- mice had similar changes: mitochondrial membrane density increased, cristae decreased, and structurally disordered plate-like cristae appeared.
  • Lipid oxidation occurs when animal or plant cells undergo oxidative stress.
  • Lipid oxidation (MDA) detection kit (Beyotime) was used to analyze the lipid oxidation levels of GES-1-134 and mouse gastric mucosa tissue. The specific process is as follows:
  • the CCK-8 kit was used to analyze the changes in the proliferation activity of GES-1-134 cells affected by the ferroptosis inhibitor Ferrostatin-1. The results are shown in Figure 8. After the ferroptosis inhibitor Ferrostatin-1 intervened in GES-1-134 cells, their proliferation ability was improved and their vitality was restored.
  • ferroptosis inhibitor Ferrostatin-1 treated GES-1-134 cells, as shown in Figure 9A, the ferroptosis marker proteins GPX4 and FTH1 increased, and COX-2 and TFR decreased, indicating that Ferrostatin-1 effectively inhibited GRIM-19 knockout human cells.
  • Ferrostatin-1 was injected into GRIM-19 -/- mice.
  • GPX4, FTH1, COX-2, and TFR had the same changing trend.
  • the above in vivo and in vitro experiments confirmed that the ferroptosis inhibitor Ferrostatin-1 effectively inhibits ferroptosis.
  • GRIM-19 deficiency induces the pathological process of ferroptosis in gastric mucosal cells.
  • Example 12 Ferrostatin-1 interferes with expression analysis of inflammatory factors in GES-1-134 cells and GRIM-19 -/- mice
  • the expression of inflammatory factors in the gastric mucosa group of gastric mucosal epithelial cells and parietal cell-specific GRIM-19 knockout mice can effectively slow down the inflammatory response of gastric mucosal tissue and inhibit the pathological process of chronic atrophic gastritis.

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Abstract

一种铁死亡抑制剂在制备治疗胃炎的药物中的应用。该铁死亡抑制剂以Ferrostatin-1、或其衍生物、异构体或药学上可接受的盐为活性成分，辅以其药学上可以接受的载体、赋形剂、稀释剂、辅剂、媒介物或其组合。铁死亡抑制剂Ferrostatin-1有效抑制了慢性萎缩性胃炎病理进程，体内外实验证实铁死亡抑制剂Ferrostatin-1可用于慢性萎缩性胃炎的防治，可为慢性萎缩性胃炎药物研发和创新提供新思路。

Description

一种铁死亡抑制剂在制备治疗胃炎的药物中的应用 技术领域
本发明属于医药技术领域，具体涉及一种铁死亡抑制剂在制备治疗胃炎的药物中的应用。
背景技术
慢性萎缩性胃炎(Chronic Atrophic Gastritis,CAG)是一种“炎症→萎缩→化生(Metaplasia)→异型增生”为突出病理特征的胃癌癌前病变，与肠型胃癌(Intestinal GC)发生密切相关。其中，化生作为CAG病理进程的重要节点，是肠型胃癌早期防控关键，但形成机制并不完全清楚。因此，有关化生形成病因与机制研究，对肠型胃癌早期防控具有重要意义。
中国专利文献CN113940309A公开了课题组一种模拟度高、稳定持久、具有特定表型的、高效的慢性萎缩性胃炎病变的模拟方法：将基因编辑小鼠GRIM-19flox/flox与ATP4b-Cre基因编辑小鼠杂交，获得胃黏膜特异性壁细胞GRIM-19敲除小鼠品系(GRIM-19-/-/ATP4b-Cre，SPF级)，该基因敲除小鼠在8月龄具有慢性萎缩性胃炎肠上皮化生早期病变即自发性解痉多肽表达化生(Spasmolytic Polypeptide Expressing Metaplasia,SPEM)，且能够稳定表达，不会反复，可作为小鼠慢性萎缩性胃炎病理模型研究。
铁死亡(Ferroptosis)，又称铁依赖性死亡，作为一种不同于细胞凋亡、坏死和自噬的新型调节性细胞死亡形式，与多种疾病密切相关如神经退行性变、肿瘤、缺血再灌注损伤、肾脏损伤以及肝纤维化等。铁死亡以胞内脂质过氧化物集聚为典型特征，其中胞内过量Fe 2+经Fenton反应导致抗氧化谷胱甘肽(GSH)耗竭与谷胱甘肽过氧化物酶4(GPX4)失活，进而诱导脂质过氧化物积聚从而引起铁死亡。
Ferrostain-1是目前公认的铁死亡抑制剂，是一种人工合成的通过还原机制来防止膜脂的损伤从而抑制细胞死亡的抗氧化剂。中国专利文献CN111529518A公开了铁死亡抑制剂Ferrostatin-1明显改善了淹溺引起的小鼠肺损伤和细胞损伤，对淹溺导致的肺损伤具有显著的保护作用；中国专利文献CN113440509A公开了铁死亡抑制剂Ferrostatin-1明显提高了细胞内GPX4的含量，显著抑制了磨损颗粒刺激成骨细胞内ROS的生成，减少了成骨细胞铁死亡，且体内实验Ferrostatin-1腹腔注射对磨损颗粒所诱导的骨溶解存在显著的抑制效应。但目前尚无铁死亡抑制剂及其衍生物在慢性萎缩性胃炎方面作用中的报道。
发明内容
针对现有技术中的上述不足，本发明提供了一种铁死亡抑制剂在制备治疗胃炎的药物中的新用途，本发明首次发现了GRIM-19缺失诱导人胃黏膜上皮细胞增殖减慢、活力降低的机制为铁死亡，而非凋亡、坏死、自噬等其他死亡形式。
本发明的目的在于提供铁死亡抑制剂Ferrostatin-1可作为GRIM-19缺失为特征的慢性萎缩胃炎疾病的治疗靶点，Ferrostatin-1可通过提高胃黏膜组织GPX4水平，特异性催化谷胱甘肽将脂质过氧化物转化为类脂醇的硒蛋白，将铁死亡发生进程中的毒性产物清除，从而进一步抑制铁死亡的发生。此外，Ferrostatin-1能明显减少小鼠胃黏膜壁细胞GRIM-19缺失引起的胃黏膜组织炎症细胞和炎症因子浸润，SPEM指标降低，有效抑制慢性萎缩性胃炎的病理进程，为慢性萎缩性胃炎药物研发和创新提供新思路。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
铁死亡抑制剂在制备治疗胃炎的药物中的应用。
进一步地，铁死亡抑制剂以Ferrostatin-1为活性成分，辅以其药学上可以接受的载体、赋形剂、稀释剂、辅剂、媒介物或其组合。
进一步地，铁死亡抑制剂中的活性成分为Ferrostatin-1衍生物、异构体或药学上可接受的盐。
进一步地，胃炎为慢性萎缩性胃炎。
进一步地，慢性萎缩性胃炎为由GRIM-19基因缺陷诱导引发的慢性萎缩性胃炎。
进一步地，铁死亡抑制剂可抑制SPEM基因的表达。
进一步地，铁死亡抑制剂可提高慢性萎缩性胃炎模型中铁死亡标志蛋白谷胱甘肽过氧化物酶4(GPX4)水平。
进一步地，铁死亡抑制剂可减少慢性萎缩性胃炎模型中小鼠胃黏膜组织L-6、TNF-α、VEGF、GM-CSF、IL-1β、IL-33等炎症因子的表达。
进一步地，铁死亡抑制剂可保护胃黏膜组织。
进一步地，药物为片剂、栓剂、注射剂或胶囊剂。
进一步地，药物为注射剂，采用腹腔注射给药，给药剂量为1～1.5mg/kg。
本发明的有益效果：
本发明发现铁死亡抑制剂Ferrostatin-1体内注射可明显提高小鼠胃黏膜组织GPX4水平，抑制胃黏膜壁细胞铁死亡病理进程，从而有效减少了小鼠胃黏膜组织L-6、TNF-α、VEGF、GM-CSF、IL-1β、IL-33等炎症因子的表达，SPEM指标显著降低，有效抑制了慢性萎缩性胃炎病理进程，可用于慢性萎缩性胃炎的治疗。Ferrostatin-1体外处理GES-1-134细胞，可提高细胞GPX4水平，抑制铁死亡病理进程，从而增强细胞活力。体内外实验证实铁死亡抑制剂Ferrostatin-1可用于慢性萎缩性胃炎的治疗，可为慢性萎缩性胃炎药物研发和创新提供新思路。
附图说明
图1为GRIM-19敲除人胃黏膜上皮GES-1细胞(GES-1-134)单克隆构建图；其中，A为单克隆流式细胞仪GFP阳性检测图；B为蛋白免疫印迹检测GES-1-134细胞GRIM-19蛋白表达分析；
图2为GES-1-134细胞活力检测结果；
图3为GES-1-134细胞凋亡、坏死与自噬检测；其中，A为流式检测GES-1-134细胞凋亡与坏死情况；B为蛋白免疫印迹检测GES-1-134细胞自噬标志物LC3I/LC3II蛋白表达分析；
图4为GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织铁死亡标志蛋白检测；其中，A为蛋白免疫印迹检测GES-1-134细胞铁死亡标志蛋白(COX2、GPX4、FTH1、TFR)表达水平；B为蛋白免疫印迹检测GRIM-19 -/-小鼠胃黏膜组织铁死亡标志蛋白(COX2、GPX4、FTH1、TFR)表达水平；
图5为GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织透射电镜图；其中，A为GES-1-134细胞线粒体表型透射电镜分析；B为GRIM-19 -/-小鼠胃黏膜组织线粒体表型透射电镜分析；
图6为GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织铁离子检测；其中，A为GES-1-134细胞Fe 2+含量检测；B为GRIM-19 -/-小鼠胃黏膜组织普鲁士蓝染色(*＜0.05)；
图7为GES-1-134细胞脂质氧化(MDA)检测(***＜0.001)；
图8为铁死亡抑制剂Ferrostatin-1干预后GES-1-134细胞活力检测(***＜0.001)；
图9为铁死亡抑制剂Ferrostatin-1干预后GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织铁死亡标志物蛋白检测；其中，A为蛋白免疫印迹检测铁死亡抑制剂Ferrostatin-1(0、5μM、10μM)处理GES-1-134细胞铁死亡标志蛋白(COX2、GPX4、FTH1、TFR)表达水平；B为蛋白免疫印迹检测铁死亡抑制剂Ferrostatin-1干预GRIM-19 -/-小鼠胃黏膜组织铁死亡标志蛋白(COX2、GPX4、FTH1、TFR)表达水平；
图10为铁死亡抑制剂Ferrostatin-1干预后GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织炎症因子表达检测；其中，A为蛋白免疫印迹检测Ferrostatin-1干预后GES-1-134细胞IL-6、TNF-α、VEGF、GM-CSF、IL-1β、IL-33蛋白表达水平；B为蛋白免疫印迹检测剂Ferrostatin-1干预后GRIM-19 -/-小鼠胃黏膜组织IL-6、TNF-α、VEGF、GM-CSF、IL-1β、IL-33蛋白表达水平；
图11为铁死亡抑制剂Ferrostatin-1干预后GRIM-19 -/-小鼠胃黏膜组织SPEM病理进程检测分析；其中，A为蛋白免疫印迹检测胃黏膜组织SPEM标志物TFF2、Mist1、Clusterin-1、HE4、MUC6蛋白水平；B为免疫荧光检测GIF、GSII及Mist免疫荧光三标分析(Bar:50μM)(***＜0.001)。
具体实施例方式
下面对本发明的具体实施例方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施例方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1体外构建GES-1-134细胞模型
1、慢病毒载体系统构建
本发明采用的是上海吉凯基因技术有限公司CRISPR/CAS9单载体慢病毒系统
(1)GV393慢病毒的框架结构为U6-sgRNA-EIFla-Cas9-FLAG-P2A-EGFP；
(2)目的基因信息：
目的基因名称 基因物种 基因ID GenBankID
NDUFA13(GRIM-19) Human 51079 NM_015965
(3)目的sgRNA引物序列：
NDUFA-13-sgRNA-134-F CACCg TGCTCCAGTGCCCGTAGATC /
NDUFA-13-sgRNA-134-R AAAC GATCTACGGGCACTGGAGCA C
NC-sgRNA-F CACCg CGCTTCCGCGGCCCGTTCAA /
NC-sgRNA-R AAAC TTGAACGGGCCGCGGAAGCG C
2、GES-1(人正常胃黏膜上皮细胞)感染
(1)将GES-1细胞按照1000个细胞/孔接种96孔板；
(2)细胞生长密度达30-40％左右时感染；
①感染前，使用完全培养基1：25稀释HitransG P感染增强液，备用；
②按照MOI＝50计算病毒使用量；
③37℃、5％CO 2培养箱培养，感染8-16小时左右观察细胞形态换液；
④感染72小时，显微镜下观察感染效果。
3、流式细胞术分选细胞
应用流式细胞仪分选GFP阳性细胞，将阳性细胞稀释成单个细胞，进行单克隆增殖，即可获得获得高纯度GFP阳性细胞，如图1A所示。
4、免疫蛋白印迹检测GRIM-19表达分析，具体过程如下：
(1)用RIPA裂解液(碧云天)冰上裂解细胞30min以上；
(2)12000rpm/10min，收集上清；
(3)加入对应体积SDS-PAGE蛋白上样缓冲液，金属浴100℃/10min；
(4)SDS-PAGE电泳；
(5)室温封闭30min；
(6)敷一抗，4℃孵育摇床过夜；
(7)洗膜，TBST清洗三次，每次10min；
(8)敷二抗，室温摇床孵育1小时；
(9)洗膜，TBST清洗三次，每次10min；
(10)显影。
如图1B所示GES-1-134GRIM-19蛋白水平相比于对照明显减少，提示人正常胃黏膜细胞GES-1 GRIM-19敲除模型构建成功。
实施例2 GES-1-134细胞增值检测活力分析
利用CCK-8试剂盒(索莱宝)分析GES-1-NC和GES-1-134细胞增殖活力变化，具体过程如下：
(1)5000个细胞/孔接种于96孔板；
(2)37℃、5％CO 2分别培养24、48、72小时；
(3)检测时向每孔加入10ulCCK-8溶液；
(4)将培养板在培养箱内孵育1小时；
(5)酶标仪测定450nm处吸光度。
结果如图2所示，GES-1-134与对照相比细胞增殖速度减慢，活力降低。
实施例3 GES-1-134细胞凋亡分析
利用凋亡试剂盒分析GES-1-134凋亡，具体过程如下：
(1)收集5×10 5细胞；
(2)PBS漂洗两次(2000rpm/5min)；
(3)Binding Buffer:7-AAD＝10:1，加入50μL染液、混匀、避光反应15min；
(4)Binding Buffer：Annexin V-PE＝450:1，加入450μL染液、混匀、避光反应15min；
(5)1小时内流式细胞仪检测。
结果如图3A所示，相比于对照GES-1-NC，GES-1-134凋亡分析无明显差别，证明GRIM-19缺失诱导胃黏膜上皮细胞速度减慢，活力降低方式并非凋亡。
实施例4 GES-1-134细胞自噬分析
利用蛋白免疫印迹方法检测GES-1-134自噬标志蛋白LC3I和LC3II的表达水平。如图3B所示GES-1-134相比于对照组自噬标志物LC3I和LC3II无增加趋势，证实GRIM-19缺失诱导胃黏膜上皮细胞增殖减慢、活性降低方式并非自噬。
实施例5 GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织铁死亡标志物表达分析
利用蛋白免疫印迹检测GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织中铁死亡相关标志蛋白的表达。结果如图4A所示。
与GES-1-NC细胞相比，GES-1-134细胞谷胱甘肽过氧化物酶4(GPX4)、铁蛋白(FTH1)下降，环氧合酶(COX-2)、转铁蛋白受体(TFR)升高，这提示GRIM-19在人正常胃黏膜上皮细胞被敲除后，Fe 2+过载、脂质过氧化物清除能力减弱，铁死亡发生。同样在小鼠胃黏膜组织中，图4B所示GRIM-19 fl/-、GRIM-19 -/-小鼠胃黏膜组织相比于对照组铁死亡标志蛋白与体外有同样的趋势，这提示小鼠胃黏膜壁细胞GRIM-19缺失诱导壁细胞受损的机制为铁死亡。
实施例6 GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组织电镜分析
利用透射电镜分析技术检测GRIM-19 -/-小鼠胃黏膜组织和GES-1-134细胞线粒体结构变化，具体过程如下：
(1)将新鲜组织切成1mm 3大小，立马加入电镜固定液，4℃冰箱保存(可存放3-6个月)择期送电镜室检测；
(2)细胞消化1×10 6，1200rpm/10min，形成Ep管底部紧密的细胞团快，吸净上清液，沿管壁轻轻加入固定液(请勿破坏样品团块)，4℃冰箱保存(可存放3-6个月)择期送电镜室检测。
透射电镜分析技术分析如图5A所示，与GES-1-NC相比，GES-1-134细胞内线粒体结构和数量有明显改变：数目减少、膜密度增加、嵴减少，而这些符合细胞铁死亡形态学特征；图5B与对照鼠相比，GRIM-19 -/-小鼠胃黏膜组织细胞线粒体结构有相似改变：线粒体膜密度增加、嵴减少且出现结构紊乱的板状嵴。以上体内外透射电镜分析证实了了GRIM-19缺失诱导人胃黏膜上皮细胞和小鼠胃黏膜壁细胞线粒体发生铁死亡形态学改变。
实施例7 GES-1-134细胞亚铁离子检测分析
胞内过量Fe 2+经Fenton反应导致抗氧化谷胱甘肽(GSH)耗竭与谷胱甘肽过氧化物酶4(GPX4)失活，进而诱导脂质过氧化物积聚从而引起铁死亡。利用亚铁离子比色法试剂盒(Elabscience)检测GES-1-34细胞Fe 2+含量，具体过程如下：
(1)收集4-6×10 6个细胞；
(2)加入试剂一进行匀浆；
(3)1000g/10min,取上清备用；
(4)加入试剂二，37℃孵育10min；
(5)12000g/10min，取上清备用；
(6)酶标仪532nm测定OD值。
结果如图6A所示，GES-1-134细胞Fe 2+增多，提示GRIM-19敲除诱导胃黏膜上皮细胞Fe 2+ 增多，后续Fe 2+可经Fenton反应促进铁死亡的发生。
实施例8 GRIM-19 -/-小鼠胃黏膜组织普鲁士蓝铁离子检测分析
利用普鲁士蓝试剂盒(索莱宝)检测小鼠胃黏膜组织铁离子表达情况，具体过程如下：
(1)组织固定；
(2)常规脱水包埋；
(3)切片烤片；
(4)滴染Perls染色工作液15min；
(5)蒸馏水洗5min；
(6)滴染核固红液10min；
(7)自来水洗；
(8)常规脱水透明；
(9)中性树脂封片。
结果如图6B所示，与对照组相比GRIM-19 -/-小鼠胃黏膜组织铁离子明显增多，提示胃黏膜组织铁离子过载进一步加剧铁死亡的发生。
实施例9 GES-1-134细胞脂质氧化(MDA)检测分析
动物或植物细胞发生氧化应激(oxidative stress)时，会发生脂质氧化。利用脂质氧化(MDA)检测试剂盒(碧云天)，分析GES-1-134和小鼠胃黏膜组织脂质氧化水平，具体过程如下：
(1)取1×10 6细胞裂解；
(2)10000g/10min收集上清；
(3)加入0.1mL MDA工作液，100℃金属浴15min；
(4)1000g/10min，收集上清；
(5)酶标仪532nm测定吸光度。
结果如图7所示，与对照图相比GES-1-134细胞MDA显著上升，提示GRIM-19敲除引起人胃黏膜上皮细胞MDA增加，进一步促进铁死亡的发生。
实施例10 Ferrostatin-1干预GES-1-134细胞增殖活力检测分析
利用CCK-8试剂盒分析铁死亡抑制剂Ferrostatin-1干预GES-1-134细胞增殖活力变化。结果如图8所示，铁死亡抑制剂Ferrostatin-1干预GES-1-134细胞后增殖能力提高、活力恢复。
实施例11 Ferrostatin-1干预GES-1-134细胞和GRIM-19 -/-小鼠铁死亡标志物表达分析
利用蛋白免疫印迹检测Ferrostatin-1干预GES-1-134细胞和GRIM-19 -/-小鼠胃黏膜组 织铁死亡标志物表达分析。
铁死亡抑制剂Ferrostatin-1处理GES-1-134细胞后，如图9A所示铁死亡标志蛋白GPX4、FTH1上升、COX-2、TFR下降，提示Ferrostatin-1有效抑制了GRIM-19敲除人胃黏膜上皮GES-1细胞铁死亡病理进程。体内实验Ferrostatin-1注射GRIM-19 -/-小鼠，如图9B所示GPX4、FTH1、COX-2、TFR有相同的变化趋势，以上体内外实验证实铁铁死亡抑制剂Ferrostatin-1有效抑制了GRIM-19缺陷诱导胃黏膜细胞铁死亡病理进程。
实施例12 Ferrostatin-1干预GES-1-134细胞和GRIM-19 -/-小鼠炎症因子表达分析
利用白免疫印迹技术检测铁死亡抑制剂Ferrostatin-1干预GES-1-134细胞和GRIM-19 -/-小鼠后其炎症因子的表达情况。
结果如图10所示，铁死亡抑制剂Ferrostatin-1体外干预GES-1-134细胞后，炎症因子IL-6、TNF-α、VEGF、GM-CSF、IL-1β、IL-33显著降低；体内实验证明与PBS注射对照相比，Fer-1组炎症因子IL-6、TNF-α等也明显降低，以上提示体内外实验证实了铁死亡抑制剂Ferrostatin-1能够减少GRIM-19敲除人胃黏膜上皮细胞和壁细胞特异性GRIM-19敲除小鼠胃黏膜组炎症因子的表达，有效减缓胃黏膜组织炎症反应，抑制慢性萎缩性胃炎病理进程。
实施例13 Ferrostatin-1干预GRIM-19 -/-小鼠胃黏膜组织SPEM标志物分析
利用蛋白免疫印迹技术检测铁死亡抑制剂Ferrostatin-1腹腔注射GRIM-19 -/-小鼠后，胃黏膜组织SPEM标志物的表达情况。
结果如图11A所示，与PBS注射对照相比，铁死亡抑制剂Ferrostatin-1组，TFF2、Mist1、Clusterin-1、HE4、MUC6的表达明显减弱，这提示铁死亡抑制剂Ferrostatin-1能够有效抑制GRIM-19 -/-小鼠慢性萎缩性胃炎SPEM病理进程。
实施例14 Ferrostatin-1干预GRIM-19 -/-小鼠胃黏膜组织SPEM标志物GIF/GSII/Mist1共定位分析
运用免疫荧光技术检测铁死亡抑制剂Ferrostatin-1腹腔注射小鼠后，胃黏膜组织SPEM标志物GIF、GSII、Mist1阳性共定位表达分析，具体过程如下：
(1)OCT包埋组织；
(2)切片；
(3)4％PFA固定；
(4)血清封闭；
(5)4℃过夜孵育一抗；
(6)PBS漂洗三次；
(7)室温孵育二抗2小时；
(8)PBS漂洗三次；
(9)DAPI复染10min；
(10)PBS漂洗三次；
(11)抗荧光衰弱剂封片。
结果如图11B所示，与PBS组相比，Fer-1，GIF +、GSII +、Mist1 +组阳性细胞数明显减少，GIF +/GSⅡ +和GIF +/Mist1+双阳性、GIF +/GSII +/Mist1 +三阳性细胞数量均增少，提示铁死亡抑制剂Ferrostatin-1能够有效抑制慢性萎缩性胃炎SPEM病理进程。

Claims (8)

  1. 铁死亡抑制剂在制备治疗胃炎的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述铁死亡抑制剂以Ferrostatin-1为活性成分，辅以其药学上可以接受的载体、赋形剂、稀释剂、辅剂、媒介物或其组合。
  3. 根据权利要求2所述的应用，其特征在于，所述铁死亡抑制剂中的活性成分为Ferrostatin-1衍生物、异构体或药学上可接受的盐。
  4. 根据权利要求1～3任一项所述的应用，其特征在于，所述胃炎为慢性萎缩性胃炎。
  5. 根据权利要求4所述的应用，其特征在于，所述慢性萎缩性胃炎为由GRIM-19基因缺陷诱导引发的慢性萎缩性胃炎。
  6. 根据权利要求4所述的应用，其特征在于，所述铁死亡抑制剂可抑制SPEM基因的表达。
  7. 根据权利要求4所述的应用，其特征在于，所述药物为片剂、栓剂、注射剂或胶囊剂。
  8. 根据权利要求4所述的应用，其特征在于，所述药物为注射剂，采用腹腔注射给药，给药剂量为1～1.5mg/kg。
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