Application of gentiopicroside in preparation of medicine for protecting liver ischemia-reperfusion injury

Abstract

The invention discloses an application of gentiopicroside in preparation of a medicine for protecting liver ischemia-reperfusion injury, discusses the protection effect of gentiopicroside on mouse liver ischemia-reperfusion injury, and preliminarily reveals a ferroptosis related molecular mechanism. The invention discloses that gentiopicroside can play a role in protecting HIRI by inhibiting ferroptosis, oxidative stress and inflammatory response, further indicates that gentiopicroside can be further used for preparing a medicine for protecting liver ischemia-reperfusion injury, and meanwhile, the invention discloses the effect of ferroptosis in HIRI, discovers a new therapeutic target, and has a good application prospect. A new thought and a new direction are provided for treatment of liver diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of new pharmaceutical applications of gentiopicroside, specifically relating to the application of gentiopicroside in the preparation of drugs to protect against hepatic ischemia-reperfusion injury. Background Technology

[0002] Hepatic ischemia-reperfusion injury (HIRI) is a type of liver injury caused by the restoration of blood flow after interruption. It is commonly seen in clinical situations such as liver surgery and liver transplantation, and seriously affects the prognosis of patients (Li Zexin, Wang Xiao, Wang Ying, et al. Effect of fisetin on hepatocyte apoptosis in mice with hepatic ischemia-reperfusion injury [J]. Journal of Xinxiang Medical College, 2021, 38(02): 118-123). Existing studies have shown that HIRI not only causes early organ failure after liver transplantation, but is also closely related to acute and chronic tissue rejection and organ damage. This pathological change not only significantly narrows the scope of liver resection and reduces the utilization rate of marginal liver donors, but also seriously hinders the clinical promotion and treatment effect of liver transplantation (Liu R, Cao H, Zhang S, et al. ZBP1-mediated apoptosis and inflammation exacerbate steatotic liver ischemia / reperfusion injury[J]. J Clin Invest,2024,134(13):e180451). Ferroptosis is characterized by the accumulation of intracellular iron ions and the peroxidation of polyunsaturated fatty acids, which leads to the inability of cells to maintain normal redox balance, ultimately causing cell death (Liang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol Cell,2022,82(12):2215-2227).In recent years, ferroptosis, as a novel type of cell death distinct from apoptosis, has been shown to participate in various pathological processes, covering areas such as tumors, ischemia-reperfusion injury, and neurodegenerative diseases (Ru Q, Li Y, Chen L, et al. Iron homeostasis and ferroptosis in human diseases: mechanisms and therapeutic prospects[J]. Signal Transduct TargetTher,2024,9(1):271; Jin X, Tang J, Qiu X, et al. Ferroptosis: Emerging mechanisms, biological function, and therapeutic potential in cancer and inflammation[J]. Cell Death Discov,2024,10(1):45). In HIRI, ferroptosis may serve as an important cell death mechanism, but its specific role and regulatory mechanisms require further investigation. Gentipicroside (GPS) is a natural compound with various pharmacological effects extracted from the traditional Chinese medicine gentian. Previous studies have shown that it has a certain protective effect on the liver (Li Shenghao, Li Junyi, Wu Kunli, et al. Research progress on the pharmacological effects and molecular mechanisms of gentiopicroside [J]. Journal of Kunming Medical University, 2020, 41(01):158-162). However, there is limited research on the protective effect of gentiopicroside on HIRI and whether it exerts its effect through influencing ferroptosis mechanisms; the specific mechanisms are unclear. Therefore, this invention aims to explore the protective effect of gentiopicroside on HIRI in mice and to preliminarily reveal its potential molecular mechanisms, especially its influence on the ferroptosis pathway, providing new strategies and theoretical basis for the clinical treatment of HIRI.

[0003] Patent document CN202510731695.9 discloses the application of gentiopicroside in the preparation of drugs to improve hepatic steatosis and ferroptosis in type 2 diabetes mellitus. Using a high-fat diet combined with streptozotocin-induced T2DM model in C57BL / 6J mice as the research subject, gentiopicroside was administered orally by gavage for 8 weeks. Metabolic indicators such as blood glucose, body weight, food intake, and water intake were dynamically monitored. After sacrifice, serum and liver tissue were collected. The pharmacological effects of gentiopicroside on T2DM were systematically evaluated through liver pathological staining, detection of oxidative stress indicators in serum and liver tissue, and analysis of the PI3K / AKT / Nrf2 signaling pathway and ferroptosis-related protein expression. This study is the first to clearly demonstrate that gentiopicroside has a regulatory effect on hepatic steatosis and ferroptosis in T2DM, filling the research gap on the mechanism of action of gentiopicroside in liver complications of T2DM and providing experimental evidence for the development of novel drugs to improve hepatic steatosis and ferroptosis in T2DM. However, the patent document does not contain any record of gentiopicroside exerting a protective effect against hepatic ischemia-reperfusion injury by inhibiting ferroptosis, oxidative stress, and inflammatory response, and thus being used to prepare drugs that protect against hepatic ischemia-reperfusion injury. Summary of the Invention

[0004] The purpose of this invention is to provide the application of gentiopicroside in the preparation of drugs that protect against hepatic ischemia-reperfusion injury, to explore the protective effect of gentiopicroside on hepatic ischemia-reperfusion injury in mice, and to preliminarily reveal the molecular mechanism related to ferroptosis.

[0005] To achieve the above objectives, the present invention employs the following technical solution: the application of gentiopicroside in the preparation of drugs that protect against hepatic ischemia-reperfusion injury.

[0006] Furthermore, the gentiopicrin exerts a protective effect against hepatic ischemia-reperfusion injury by inhibiting ferroptosis, oxidative stress, and inflammatory responses.

[0007] Furthermore, the gentiopicroside exerts a protective effect against hepatic ischemia-reperfusion injury by regulating the expression of elevated ferroptosis-related proteins.

[0008] Furthermore, the related proteins are GPX4 protein, FTH1 protein, and TFR1 protein.

[0009] Furthermore, the gentiopicroside exerts a protective effect against hepatic ischemia-reperfusion injury by reducing the levels of tumor necrosis factor α and interleukin 1β.

[0010] Furthermore, the gentiopicrin exerts a protective effect against liver ischemia-reperfusion injury by reducing the content of iron ions and malondialdehyde in liver tissue.

[0011] Furthermore, the gentiopicrin exerts a protective effect against hepatic ischemia-reperfusion injury by inhibiting apoptosis in cells affected by hepatic ischemia-reperfusion injury.

[0012] Furthermore, the application of gentiopicroside in the preparation of drugs for ferroptosis caused by hepatic ischemia-reperfusion injury.

[0013] The experimental method of this invention is as follows: Sixty male C57BL / 6 mice were randomly divided into six groups: sham-operated group (Sham + Saline), HIRI + Saline group, HIRI + low-dose gentiopicroside (GPL) group, HIRI + high-dose gentiopicroside (GPH) group, and HIRI + diammonium glycyrrhizinate (DG) group. After one week of continuous gavage, surgical modeling was performed. HE staining was used to observe the morphological changes in liver tissue of each group of mice. TUNEL assay was used to detect hepatocyte apoptosis. ELISA was used to measure the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in serum samples, as well as the expression levels of ferroptosis-related indicators and related proteins. Spectrophotometer was used to measure the level of malondialdehyde (MDA) in liver tissue, and atomic absorption spectrometry was used to measure the level of unbound iron ions (Fe) in liver tissue. 2+ The concentration of glutathione (GSH) and its oxidized form (GSSG) in liver tissue was determined by colorimetric method, and the expression levels of proteins such as glutathione peroxidase 4 (GPX4), iron transporter 1 (FTH1), and transferrin receptor 1 (TFR1) in liver tissue were detected by Western blot.

[0014] The experimental conclusions of this invention are as follows: In the Sham+Saline group, hepatocytes were arranged regularly and orderly, with no abnormalities in the lobular structure; in the HIRI+Saline group, hepatocytes were disordered, with scattered vacuolated cells and significant inflammatory cell infiltration; in the HIRI+GPH and HIRI+DG groups, hepatocyte vacuolar degeneration was significantly reduced, and cell arrangement was closer to normal; the pathological changes in hepatocytes in the HIRI+GPL group were intermediate between those in the HIRI+Saline and HIRI+GPH groups. Compared with the Sham+Saline group, the apoptosis rate, ALT, AST, TNF-α, IL-1β, iron ion content, and MDA were significantly increased in the HIRI+Saline, HIRI+GPL, HIRI+GPH, and HIRI+DG groups, while the expression of GSH / GSSG, GPX4, FTH1, and TFR1 proteins was significantly decreased. P <0.05); Compared with the HIRI+Saline group, the apoptosis rate, ALT, AST, TNF-α, IL-1β, iron ion content, and MDA were significantly decreased in the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group, while the expression of GSH / GSSG, GPX4, FTH1, and TFR1 proteins were significantly increased. P<0.05); Compared with the HIRI+GPL group, the HIRI+GPH group and the HIRI+DG group showed significantly decreased apoptosis rate, ALT, AST, TNF-α, IL-1β, iron ion content, and MDA, while the expression of GSH / GSSG, GPX4, FTH1, and TFR1 proteins was significantly increased. P <0.05).

[0015] The present invention has the following advantages and beneficial effects: The present invention reveals that gentiopicroside can exert a protective effect against HIRI by inhibiting ferroptosis, oxidative stress and inflammatory response, thus indicating that gentiopicroside can be further used to prepare drugs that protect against hepatic ischemia-reperfusion injury. At the same time, the present invention reveals the role of ferroptosis in HIRI, discovers new therapeutic targets, and provides new ideas and directions for the treatment of liver diseases. Attached Figure Description

[0016] Figure 1 The histopathological changes of liver tissue in each group (HE staining, ×200) are shown below: A: Sham+Saline group; B: HIRI+Saline group; C: HIRI+GPL group; D: HIRI+GPH group; E: HIRI+DG group.

[0017] Figure 2 For comparison of liver tissue cell apoptosis in each group, compared with the Sham+Saline group, a P <0.05; compared with the HIRI+Saline group, b P <0.05; compared with the HIRI+GPL group, c P <0.05.

[0018] Figure 3 For each group of liver tissue cells, apoptosis was indicated by TUNEL staining (×400). A: Sham+Saline group; B: HIRI+Saline group; C: HIRI+GPL group; D: HIRI+GPH group; E: HIRI+DG group.

[0019] Figure 4 For comparison of serum liver function indicators in each group of mice, A: Comparison of serum ALT levels in mice; B: Comparison of serum AST levels in mice compared to the Sham+Saline group. a P <0.05; compared with the HIRI+Saline group, b P <0.05; compared with the HIRI+GPL group, c P <0.05.

[0020] Figure 5 For comparison of serum inflammatory factor levels in each group of mice, A: Comparison of serum TNF-α levels in mice; B: Comparison of serum IL-1β levels in mice compared to the Sham+Saline group. a P <0.05; compared with the HIRI+Saline group, b P <0.05; compared with the HIRI+GPL group, c P <0.05.

[0021] Figure 6 For comparison of ferroptosis-related indicators in liver tissue for each group: A: Comparison of iron ion content in liver tissue; B: Comparison of MDA in liver tissue; C: Comparison of GSH / GSSG ratio in liver tissue, compared with the Sham+Saline group. a P <0.05; compared with the HIRI+Saline group, b P <0.05; compared with the HIRI+GPL group, c P <0.05.

[0022] Figure 7 For comparison of ferroptosis-related protein expression in liver tissue of each group, A: GPX4 protein expression in liver tissue; B: FTH1 protein expression in liver tissue; C: TFR1 protein expression in liver tissue, compared with the Sham+Saline group. a P <0.05; compared with the HIRI+Saline group, b P <0.05; compared with the HIRI+GPL group, c P <0.05.

[0023] Figure 8 Comparison of ferroptosis-related protein expression in liver tissue for each group. Detailed Implementation

[0024] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Example

[0025] 1. Materials and Methods 1.1 Animals and Reagents Male C57BL / 6 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., License No.: SYXK(Su)2023-0036), physiological saline (Sichuan Kelun Pharmaceutical Co., Ltd.), gentiopicrin (purity >98%, Shanghai Boke Biotechnology Co., Ltd.), diammonium glycyrrhizate (Chia Tai Tianqing Pharmaceutical Group Co., Ltd.), and 1% sodium pentobarbital (Shanghai Jianglai Biotechnology Co., Ltd.).

[0026] 1.2 Establishing a mouse model Sixty 8-week-old male C57BL / 6 mice (weighing approximately 24g) were randomly divided into six groups: sham surgery + saline, ischemia-reperfusion injury (HIRI) + Saline, HIRI + low-dose gentiopicroside (GPL), HIRI + high-dose gentiopicroside (GPH), and HIRI + diammonium glycyrrhizinate (DG).

[0027] The Sham+Saline and HIRI+Saline groups were administered Saline, the HIRI+GPL group was administered GPS (100 mg / kg), the HIRI+GPH group was administered GPS (200 mg / kg), and the HIRI+DG group was administered DG (200 mg / kg). All mice were administered Saline via gavage for one week. Afterward, mice were anesthetized by intraperitoneal injection of 1 wt% sodium pentobarbital. The abdominal wall tissue was then incised along the midline, and the tissue was dissected layer by layer during the procedure. All blood flow to the liver was completely blocked for 30 minutes using a non-invasive vascular clamp. The mice were then transferred to an incubator, and liver blood supply was restored for 6 hours. The Sham+Saline group only underwent the abdominal opening and closing procedure.

[0028] 1.3 Collect blood and liver tissue from mice After successful establishment of the HIRI model, mice were anesthetized by intraperitoneal injection of 1 wt% sodium pentobarbital, and sutures were removed after disinfection. Approximately 0.5–1 mL of blood was drawn from the inferior vena cava of the mice using a 1 mL sterile syringe. After removing the needle, the blood was transferred to a clean EP tube and left at room temperature for several hours until serum precipitated. The serum was then separated by high-speed centrifugation at 4°C. The collected serum was aliquoted into new EP tubes, labeled with the group and serum volume, and stored at -80°C for later use. After removing the liver tissue, it was first cleaned with 0.9 wt% sodium chloride solution, then cut according to the liver lobes using sterile ophthalmic scissors. An appropriate amount of left liver lobe tissue was selected and fixed in fixative. The remaining liver tissue was stored at -80°C.

[0029] 1.4 Relevant Indicator Testing 1.4.1 Histomorphological observation: After fixation, liver tissue was dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to prepare slides for observation under a microscope.

[0030] 1.4.2 Detection of hepatocyte apoptosis: The TUNEL assay was used to detect hepatocyte apoptosis. Liver tissue sections were dewaxed, hydrated, and treated with PBS to increase permeability before the TUNEL reaction. The reaction results were then detected and analyzed.

[0031] 1.4.3 Liver function assessment: The expression levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected using a fully automated biochemical analyzer to assess the degree of liver damage.

[0032] 1.4.4 Assessment of inflammatory response: The levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in serum samples were detected by ELISA to assess the intensity of the inflammatory response.

[0033] 1.4.5 Detection of Ferrocytosis-Related Indicators: The level of malondialdehyde (MDA) in liver tissue was measured using a spectrophotometer to assess the degree of lipid peroxidation; unbound iron ions (Fe2+) in liver tissue were measured using atomic absorption spectrometry. 2+ The concentration of glutathione (GSH) and its oxidized form (GSSG) was determined by colorimetric assay to assess the redox status of cells.

[0034] 1.4.6 Expression of ferroptosis-related proteins: Western blot was used to detect the expression levels of key proteins such as glutathione peroxidase 4 (GPX4), iron transporter 1 (FTH1), and transferrin receptor 1 (TFR1) in liver tissue to explore the regulatory mechanism of ferroptosis.

[0035] 1.5 Statistical Methods Statistical analysis was performed using SPSS 26.0 software. For normally distributed measurement data, the mean ± standard deviation was used. `x ± s The data is represented as follows: First, a homogeneity of variance test is performed. If the homogeneity of variance test is met, one-way ANOVA is used for data analysis; further pairwise comparisons are performed using LSD-... t test, P <0.05 indicates a statistically significant difference.

[0036] 2 Results 2.1 Morphological observation of liver tissue in each group In the Sham+Saline group, hepatocytes were arranged regularly and orderly, and the lobular structure of the liver was normal. In the HIRI+Saline group, hepatocytes were arranged disordered, vacuolated cells were scattered, and inflammatory cell infiltration was obvious. In the HIRI+GPH and HIRI+DG groups, hepatocyte vacuolar degeneration was significantly reduced, and cell arrangement was closer to normal. The pathological changes of hepatocytes in the HIRI+GPL group were between those in the HIRI+Saline and HIRI+GPH groups. See Figure 1 .

[0037] 2.2 Comparison of apoptosis in liver tissue cells among different groups Compared with the Sham+Saline group, the apoptosis rate of liver tissue cells in the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group was significantly increased. P <0.05); Compared with the HIRI+Saline group, the apoptosis rate of liver tissue cells in the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group was significantly decreased ( P <0.05); compared with the HIRI+GPL group, the apoptosis rate of liver tissue cells in both the HIRI+GPH group and the HIRI+DG group was significantly reduced ( P <0.05). See Figure 2 , Figure 3 .

[0038] 2.3 Comparison of serum liver function indicators among different groups of mice Compared with the Sham+Saline group, ALT and AST were significantly elevated in the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group. P <0.05); Compared with the HIRI+Saline group, ALT and AST were significantly lower in the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group ( P <0.05); Compared with the HIRI+GPL group, ALT and AST were significantly lower in both the HIRI+GPH group and the HIRI+DG group ( P <0.05). See Figure 4 .

[0039] 2.4 Comparison of serum inflammatory factor levels in mice of different groups Compared with the Sham+Saline group, the levels of TNF-α and IL-1β were significantly increased in the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group. P<0.05); Compared with the HIRI+Saline group, TNF-α and IL-1β were significantly reduced in the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group ( P <0.05); Compared with the HIRI+GPL group, the TNF-α and IL-1β levels in the HIRI+GPH group and the HIRI+DG group were significantly reduced ( P <0.05). See Figure 5 .

[0040] 2.5 Comparison of ferroptosis-related indicators in liver tissue among different groups Compared with the Sham+Saline group, the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group all showed significantly increased liver tissue iron content and MDA, and significantly decreased GSH / GSSG ratio. P <0.05); Compared with the HIRI+Saline group, the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group all showed significantly decreased liver tissue iron content and MDA, and significantly increased GSH / GSSG ratio. P <0.05); Compared with the HIRI+GPL group, the HIRI+GPH group and the HIRI+DG group showed significantly decreased liver tissue iron content and MDA, and significantly increased GSH / GSSG ratio ( P <0.05). See Figure 6 .

[0041] 2.6 Expression of ferroptosis-related proteins in liver tissue of each group Compared with the Sham+Saline group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue was significantly reduced in the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group. P <0.05); Compared with the HIRI+Saline group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue of the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group was significantly increased ( P <0.05); Compared with the HIRI+GPL group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue of the HIRI+GPH and HIRI+DG groups was significantly increased ( P <0.05). This indicates that gentiopicroside exerts its protective effect by regulating the expression of key molecules GPX4, FTH1, and TFR1 in the ferroptosis signaling pathway. See Figure 7 , Figure 8 .

[0042] Hepatic ischemia-reperfusion injury refers to the phenomenon in which, after the liver has experienced ischemia, hepatocytes undergo metabolic dysfunction when blood supply is restored, leading to pathological changes in liver physiological function and tissue structure (Zhang Y, Wei H, Wang M, et al. Dexmedetomidine alleviates ferroptosis following hepaticischemia-reperfusion injury by upregulating Nrf2 / GPx4-dependent antioxidant responses[J]. Biomed Pharmacother,2023 Dec 31;169:115915). HIRI is often unavoidable in liver transplantation, hepatectomy, abdominal trauma, and hemorrhagic shock, and is one of the key factors inducing acute hepatitis and liver failure (Liu J, Man K. Mechanistic Insight and Clinical Implications of Ischemia / Reperfusion Injury Post Liver Transplantation[J]. Cell Mol Gastroenterol Hepatol,2023,15(6):1463-1474). Related studies have shown that during the occurrence of HIRI, the body undergoes a series of pathophysiological changes, including activation of oxidative stress, initiation of inflammatory cascades, and activation of apoptosis programs. These are all identified as important mechanisms by which HIRI causes damage to the body (George J, Lu Y, Tsuchishima M, et al. Cellular and molecular mechanisms of hepatic ischemia-reperfusion injury: The role of oxidative stress and therapeutic approaches[J]. RedoxBiol,2024,75:103258). Therefore, regulating oxidative stress and clearing inflammatory mediators can be an effective strategy to delay or alleviate the pathological process of HIRI. Ferroptosis, as a new non-apoptotic cell death mechanism, is essentially caused by the accumulation of reactive oxygen species due to intracellular redox imbalance under the catalysis of iron ions, leading to cell death (Li J, Cao F, Yin HL, et al. Ferroptosis: past, present and future[J]. Cell Death Dis,2020,11(2):88).Therefore, this invention aims to explore the protective effect of gentiopicroside on liver disease relapse (HIRI) in mice and its potential ferroptosis mechanism. By establishing a mouse HIRI model, the effects of gentiopicroside on liver function indicators, inflammatory factors, apoptosis and ferroptosis-related indicators will be evaluated. This will help to deepen the understanding of the molecular mechanism of HIRI, provide new intervention strategies for the clinical treatment of liver diseases, and thus improve the success rate of surgery and patient prognosis.

[0043] In this invention, the hepatocytes in the HIRI+Saline group exhibited disordered arrangement, vacuolated and scattered distribution, and significant inflammatory cell infiltration, with significantly elevated ALT and AST levels. After treatment with gentiopicroside, the vacuolar degeneration of hepatocytes was significantly reduced, the cell arrangement became more orderly, and the ALT and AST levels were significantly decreased. This reveals that gentiopicroside pretreatment can alleviate liver tissue damage in HIRI mice. Cell apoptosis in liver tissue is also an important characteristic of HIRI, and this process is regulated by factors such as oxidative stress and inflammatory response (Wang Lijiang, Zhuang Likun, Yang Tongwang, et al. Study on the protective effect and mechanism of trehalose on liver ischemia-reperfusion injury [J]. Chinese Journal of Organ Transplantation, 2021, 42(2): 109-115). In this invention, the apoptosis rate of hepatocytes in HIRI mice was significantly increased, while the pretreatment with gentiopicroside could reduce the apoptosis rate of hepatocytes, indicating that gentiopicroside can inhibit cell apoptosis in HIRI and thus exert a protective effect. Furthermore, inflammatory cell infiltration plays a crucial role in the mechanism of liver injury. Under pathological conditions such as ischemia, hypoxia, and reperfusion, inflammatory cells are activated and release cytokines such as TNF-α and IL-1β. The continuous production of these factors can further promote the cascade progression of the inflammatory response (Taru V, Szabo G, Mehal W, et al. Inflammasomesin chronic liver disease: Hepatic injury, fibrosis progression and systemic inflammation[J]. J Hepatol,2024,81(5):895-910). In this invention, the levels of TNF-α and IL-1β in HIRI mice were significantly increased, while the pretreatment with gentiopicroside significantly reduced the levels of TNF-α and IL-1β, indicating that gentiopicroside can inhibit the inflammatory response induced by HIRI. The molecular mechanisms of ferroptosis involve multiple metabolic pathways, including redox homeostasis, iron metabolism, and mitochondrial activity (Dixon SJ, Olzmann JA. The cell biology of ferroptosis[J]. Nat RevMol Cell Biol,2024,25(6):424-442). Under normal physiological conditions, transferrin transports extracellular ferric ions (Fe3+). 3+ ) enters the cell, Fe3+ It is reduced to ferrous ions (Fe) inside the cell. 2+ Fe 2+ It can not only bind to ferritin (FTH1 / FTL) to form intracellular iron storage, but also undergo oxidation to convert it into Fe. 3+ These Fe 3+It is released into the extracellular space via transferrin to participate in the iron cycle in the body, thereby maintaining iron homeostasis (Singh LP, Yumnamcha T, DeviTS. Mitophagy, Ferritinophagy and Ferroptosis in Retinal Pigment Epithelial Cells Under High Glucose Conditions: Implications for Diabetic Retinopathy and Age-Related Retinal Diseases[J]. JOJ Ophthalmol,2021,8(5):77-85). Glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) are core regulatory molecules of intracellular redox balance. Their dynamic conversion and ratio directly affect the signal transduction of cellular antioxidant capacity and metabolic homeostasis (Averill-BatesDA. The antioxidant glutathione[J]. Vitam Horm,2023,121:109-141). During HIRI, cell damage caused by oxygen free radicals is most significant. GSH plays a key role in the scavenging of oxygen free radicals, directly neutralizing reactive oxygen species and blocking the lipid peroxidation chain reaction (Rochette L, Dogon G, Rigal E, et al. Lipid Peroxidation and Iron Metabolism: Two Corner Stones in the Homeostasis Control of Ferroptosis[J]. Int J Mol Sci,2022,24(1):449). MDA, on the other hand, is a stable metabolite of lipid peroxidation and is highly sensitive to oxidative damage mediated by oxygen free radicals (Deng Y, Chu X, LiQ, et al. Xanthohumol ameliorates drug-induced hepatic ferroptosis via activating Nrf2 / xCT / GPX4 signaling pathway[J]. Phytomedicine,2024,126:155458). In this invention, the iron and MDA contents in the liver tissue of HIRI mice were significantly increased, while the pretreatment with gentiopicrin could reduce the iron and MDA contents in the liver tissue, indicating that the pretreatment with gentiopicrin can inhibit HIRI-induced oxidative stress and ferroptosis.GPX4 is an intracellular antioxidant enzyme that can scavenge membrane lipid peroxides and resist damage caused by oxidative stress. It is a core regulator of ferroptosis (Li J, Cao F, Yin HL, et al. Ferroptosis: past, present and future[J]. Cell Death Dis,2020,11(2):88). Previous studies have indicated that intracellular Fe... 2+ It can catalyze the conversion of oxygen free radicals into peroxy free radicals, causing cell membrane damage and promoting ferroptosis; enhancing GPX4 activity can reduce the accumulation of intracellular oxygen free radicals and peroxy free radicals by oxidizing GSH, thereby inhibiting the ferroptosis process (Hu S, Sechi M, Singh PK, et al. A Novel Redox Modulator Induces a GPX4-Mediated CellDeath That Is Dependent on Iron and Reactive Oxygen Species[J]. J Med Chem,2020,63(17):9838-9855). FTH1 is a key component of ferritin, and as a storage iron protein, it can store up to about 4500 Fe atoms. 2+ It is oxidized to Fe through an oxygen-dependent mechanism. 3+ (Lei P, Bai T, Sun Y. Mechanisms of Ferroptosis and Relations with Regulated Cell Death: A Review[J]. Front Physiol,2019,10:139). TFR1 is a key regulator of cellular iron uptake; its expression level on the cell membrane is upregulated when cells are stimulated, leading to increased iron uptake. 3+ After entering the cell by binding to TFR1, it is reduced to Fe. 2+ This leads to ferroptosis (Guo W, Zhao Y, Li H, et al. NCOA4-mediated ferritinophagy promoted inflammatory responses in periodontitis[J]. J Periodontal Res,2021,56(3):523-534). In this invention, compared with the Sham+Saline group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue of the HIRI+Saline group, HIRI+GPL group, HIRI+GPH group, and HIRI+DG group was significantly reduced. P<0.05); Compared with the HIRI+Saline group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue of the HIRI+GPL group, HIRI+GPH group, and HIRI+DG group was significantly increased ( P <0.05); Compared with the HIRI+GPL group, the expression of GPX4, FTH1, and TFR1 proteins in liver tissue of the HIRI+GPH and HIRI+DG groups was significantly increased ( P <0.05). This further illustrates that gentiopicroside exerts a protective effect against HIRI by regulating ferroptosis-related proteins.

[0044] In summary, gentiopicroside can exert a protective effect against HIRI by inhibiting ferroptosis, oxidative stress, and inflammatory response. This study reveals the role of ferroptosis in HIRI, discovers a new therapeutic target, and provides new ideas and directions for the treatment of liver diseases.

[0045] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. Application of gentiopicroside in the preparation of drugs to protect against hepatic ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that: Gentianoside exerts a protective effect against hepatic ischemia-reperfusion injury by inhibiting ferroptosis, oxidative stress, and inflammatory response.

3. The application according to claim 2, characterized in that: Gentianoside exerts a protective effect against hepatic ischemia-reperfusion injury by regulating the expression of elevated ferroptosis-related proteins.

4. The application according to claim 3, characterized in that: The relevant proteins are GPX4 protein, FTH1 protein, and TFR1 protein.

5. The application according to claim 2, characterized in that: Gentianoside exerts a protective effect against liver ischemia-reperfusion injury by reducing the levels of tumor necrosis factor α and interleukin 1β.

6. The application according to claim 2, characterized in that: Gentianoside exerts a protective effect against liver ischemia-reperfusion injury by reducing the content of iron ions and malondialdehyde in liver tissue.

7. The application according to claim 2, characterized in that: The gentiopicrin exerts a protective effect against hepatic ischemia-reperfusion injury by inhibiting apoptosis in cells affected by hepatic ischemia-reperfusion injury.

8. The application according to claim 1, characterized in that: The application of gentiopicroside in the preparation of drugs for ferroptosis caused by liver ischemia-reperfusion injury.

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