Application of ferroptosis inhibitor in SPEM resistance

Abstract

The invention discloses an application of a ferroptosis inhibitor in resisting SPEM, relates to the technical field of medicine, and has the technical key points that in-vivo injection of the ferroptosis inhibitor Ferrostatin-1 can obviously improve the GPX4 level of gastric mucosa tissues of mice and inhibit the pathological process of ferroptosis of gastric mucosa wall cells, and the application of the ferroptosis inhibitor Ferrostatin-1 in resisting SPEM is found. Therefore, the expression of inflammatory factors of gastric mucosa tissues of mice is effectively reduced, the SPEM index is remarkably reduced, and the pathological processes of chronic gastric ulcer and gastric cancer in the early stage are effectively inhibited; in-vivo and in-vitro experiments prove that the ferroptosis inhibitor Ferrostatin-1 can be used for treating chronic gastric ulcer and early gastric cancer, and a new thought is provided for research, development and innovation of drugs for chronic gastric ulcer and early gastric cancer.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a ferroptosis inhibitor in treating SPEM. Background Art

[0002] Spasmolytic Polypeptide-Expressing Metaplasia (SPEM) is a pathological condition of the stomach often associated with gastritis, chronic gastric ulcers, and precancerous changes in the stomach. It manifests as the transformation of normal gastric epithelial cells into abnormal cells that express spasm-inhibiting peptide (SP). SPEM is usually caused by chronic gastric acid irritation or Helicobacter pylori infection, and may also be associated with long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs). Its characteristics include hyperplasia and morphological changes of gastric epithelial cells, accompanied by the expression of spasm-inhibiting peptide. SPEM may be an adaptive response of the stomach to chronic irritation, but if left untreated, it may increase the risk of gastric cancer, particularly in the setting of H. pylori infection. Therefore, early diagnosis and treatment of SPEM are crucial to prevent cancerous development.

[0003] Ferrostain-1 is a currently recognized ferroptosis inhibitor, a synthetic antioxidant that inhibits cell death by preventing membrane lipid damage through a reduction mechanism. Current studies have demonstrated that the ferroptosis inhibitor Ferrostatin-1 significantly improves lung and cell damage in mice induced by drowning, exerting a significant protective effect against drowning-induced lung injury. Ferrostain-1 significantly increases intracellular GPX4 levels, significantly inhibits wear particle-stimulated ROS production in osteoblasts, and reduces osteoblast ferroptosis. In vivo experiments have shown that intraperitoneal injection of Ferrostatin-1 significantly inhibits wear particle-induced osteolysis. However, there are currently no reports on the effects of ferroptosis inhibitors and their derivatives in chronic gastric ulcers and precancerous conditions.

[0004] To this end, the present invention aims to provide an application of a ferroptosis inhibitor in anti-SPEM to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems and provide an application of a ferroptosis inhibitor in the treatment of SPEM. By establishing a SLC7AI1 knockout-induced parietal cell ferroptosis model, and using methods such as pathological histochemistry, immunofluorescence, and high-throughput inflammatory factor ELISA chips, the authors analyzed whether SLC7AII knockout-induced parietal cell ferroptosis can induce abnormal mucosal acid secretion, mucosal inflammation, and SPEM pathology, and clarified the important role of SLC7A11 knockout-induced parietal cell ferroptosis in the pathogenesis of SPEM.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides an application of a ferroptosis inhibitor in anti-SPEM. The ferroptosis inhibitor can be used to prepare a drug for treating gastric ulcer and gastric cancer.

[0008] The ferroptosis inhibitor uses Ferrostatin-1 as an active ingredient, supplemented by a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

[0009] The ferroptosis inhibitor can inhibit the expression of the SPEM gene.

[0010] Compared with the existing technology, this solution has the following beneficial effects:

[0011] The present invention found that in vivo injection of the ferroptosis inhibitor Ferrostatin-1 can significantly increase the GPX4 level in the gastric mucosal tissue of mice, inhibit the pathological process of ferroptosis of gastric mucosal parietal cells, thereby effectively reducing the expression of inflammatory factors such as NLRP3, ASC, Casapse-1, IL-1β, IL-33 in the gastric mucosal tissue of mice, and significantly reducing the SPEM index, effectively inhibiting the pathological process of chronic gastric ulcers and precancerous lesions. In vitro and in vivo experiments confirmed that the ferroptosis inhibitor Ferrostatin-1 can be used to treat chronic gastric ulcers and precancerous lesions, providing new ideas for the research and development and innovation of drugs for chronic gastric ulcers and precancerous lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the synthesis process of the SLC7A11- / - mouse model in an embodiment of the present invention;

[0013] Figure 2 Schematic diagram of Western blotting for detecting the expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR) in gastric mucosal tissue of SLC7A11- / - mice by the intervention of ferroptosis inhibitor Ferrostatin-1 in the embodiment of the present invention;

[0014] Figure 3 Schematic diagram of the expression levels of NLRP3, ASC, Casapse-1, IL-1β, and IL-33 proteins in gastric mucosal tissue of SLC7A11- / - mice after intervention with the protein immunoblotting detection agent Ferrostatin-1 in an embodiment of the present invention;

[0015] Figure 43. Schematic diagram of the SPEM pathological process detection and analysis of gastric mucosal tissue in SLC7A11- / - mice after intervention with the ferroptosis inhibitor Ferrostatin-1 in an embodiment of the present invention; wherein, A is protein immunoblotting to detect the protein levels of SPEM markers TFF2, Mist 1, Clusterin-1, and HE4 in gastric mucosal tissue; B is immunofluorescence detection of GIF and GSII immunofluorescence double-labeling analysis (Bar: 50 μM) (***<0.001). DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0017] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0018] Example 1:

[0019] 1. Establishment of parietal cell-specific SLC7A11 knockout mice;

[0020] Using Cre-loxp technology, SLC7A11 flox / flox mice (SLC7A11 flox / flox) were crossed with ATP4b-Cre mice to generate gastric mucosal-specific parietal cell SLC7A11 knockout mice (SLC7A11- / - / ATP4b-Cre) (6-8 mice / group) and SLC7A11 flox / flox mice (6-8 mice / group). These mice were housed in a SPF environment for subsequent experiments. Gastric epithelial tissues were obtained from these mice and analyzed for changes in ferroptosis characteristics, including mitochondrial phenotype, MDA content, and tissue iron deposition in gastric mucosal parietal cells using histopathological histochemistry and electron microscopy (methods as before). I confocal microscopy and immunoblotting were used to analyze changes in the expression of ferroptosis-related molecules, such as SLC7A11 and GPX4, in parietal cells (methods as before). This study aimed to determine whether parietal cell-specific SLC7A11 knockout could induce parietal cell-specific ferroptosis.

[0021] 2. Determine the effects of parietal cell-specific knockout of SLC7A11 on mucosal inflammation and SPEM pathogenesis;

[0022] Gastric mucosal epithelial tissues of the above-mentioned mice were obtained, and the gastric acid pH, gastric acid secretion, pepsin activity, parietal cell marker protein and acid secretion functional protein expression of the gastric mucosal epithelium of the mice before and after SLC7A11 knockout were detected (the method is the same as before), to clarify whether the knockout of parietal cell SLC7A11 can induce mucosal acid secretion atrophy: then, immunoblotting, IF confocal microscopy analysis and high-throughput inflammatory factor ELISA chip were used to detect changes in NLRP3 pathway-related molecules, tissue inflammatory factors, the ratio of MI and M2 macrophages, and the number and ratio of gastric mucosal epithelial cell populations (the method is the same as before), to clarify whether the specific knockout of parietal cell SLC711 can induce gastric mucosal inflammation and SPEM pathological formation.

[0023] Example 2:

[0024] Analysis of the expression of ferroptosis markers in SLC7A11- / - mice after ferrostatin-1 intervention

[0025] Western blotting was used to detect the expression levels of ferroptosis marker proteins (COX2, GPX4, FTH1, TFR) in the gastric mucosal tissue of SLC7A11- / - mice after Ferrostatin-1 intervention.

[0026] In vivo experiments, Ferrostatin-1 was injected into SLC7A11- / - mice, and GPX4, FTH1, COX-2, and TFR showed the same change trend. The above in vivo and in vitro experiments confirmed that the ferroptosis inhibitor Ferrostatin-1 effectively inhibited the pathological process of ferroptosis of gastric mucosal cells induced by GRIM-19 deficiency.

[0027] Analysis of inflammatory factor expression in SLC7A11- / - mice treated with Ferrostatin-1

[0028] Immunoblotting was used to examine the expression of NLRP3, ASC, Casapse-1, IL-1β, and IL-33 proteins in the gastric mucosa of SLC7A11- / - mice following treatment with the ferroptosis inhibitor Ferrostatin-1. In vivo experiments demonstrated that inflammatory factors such as NLRP3 and IL-1β were significantly reduced in the Ferrostatin-1 group compared to the PBS-injected control group.

[0029] Analysis of SPEM markers in gastric mucosal tissue of SLC7A11- / - mice treated with Ferrostatin-1

[0030] Western blotting was used to detect the pathological progression of SPEM after intraperitoneal injection of the ferroptosis inhibitor Ferrostatin-1 into SLC7A11- / - mice.

[0031] Compared with the PBS injection control, the expression of TFF2, Mist1, Clusterin-1, HE4, and MUC6 in the ferroptosis inhibitor Ferrostatin-1 group was significantly weakened, suggesting that the ferroptosis inhibitor Ferrostatin-1 can effectively inhibit the pathological process of chronic atrophic gastritis SPEM in SLC7A11- / - mice.

[0032] Ferrostatin-1 treatment and co-localization analysis of SPEM markers GIF / GSII in gastric mucosal tissue of SLC7A11- / - mice

[0033] Immunofluorescence was used to detect the co-localization of SPEM markers GIF and GSII in gastric mucosal tissue after intraperitoneal injection of the ferroptosis inhibitor Ferrostatin-1 into mice. The specific process is as follows:

[0034] (1) OCT-embedded tissue;

[0035] (2) Slicing;

[0036] (3) 4% PFA fixation;

[0037] (4) serum blocking;

[0038] (5)4③ Overnight incubation with primary antibody;

[0039] (6) Rinse three times with PBS;

[0040] (7) Incubate with secondary antibody at room temperature for 2 hours;

[0041] (8) Rinse three times with PBS;

[0042] (9) DAPI counterstaining for 10 min;

[0043] (10) Rinse three times with PBS;

[0044] (11) Seal the slides with anti-fluorescence attenuation agent.

[0045] Compared with the PBS group, Fer-1, GIF + GSII + The number of positive cells in the GIF group was significantly reduced. + / GSⅡ + Double positive, GIF + / GSII + The numbers of triple-positive cells were all reduced, suggesting that the ferroptosis inhibitor Ferrostatin-1 can effectively inhibit the pathological process of chronic atrophic gastritis SPEM.

[0046] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A use of a ferroptosis inhibitor in treating SPEM, characterized by: The ferroptosis inhibitor can be used to prepare medicines for treating gastric ulcer and gastric cancer.

2. Use of a ferroptosis inhibitor in treating SPEM according to claim 1, characterized in that: The ferroptosis inhibitor uses Ferrostatin-1 as an active ingredient, supplemented by a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

3. Use of a ferroptosis inhibitor in treating SPEM according to claim 1, characterized in that: The ferroptosis inhibitor can inhibit the expression of NLRP3, ASC, Casapse-1, IL-1β, and IL-33 inflammatory factors, thereby inhibiting the expression of the SPEM gene.

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