A drug targeting histone lactylation to regulate ferroptosis of intestinal epithelial cells and application thereof

Abstract

This invention discloses a drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells and its application, belonging to the field of animal intestinal health regulation and veterinary biological agents. The drug comprises a lactate dehydrogenase A inhibitor, GSK2837808A. Animal and cell experiments have demonstrated that oxidative stress promotes ferroptosis in intestinal epithelial cells by inducing lactate accumulation, upregulating histone H3K18 lactation levels, and activating the H3K18la / HMOX1 signaling axis. GSK2837808A effectively alleviates intestinal oxidative damage by inhibiting lactate production, downregulating pan-lactic acidification and H3K18 lactation levels, blocking the H3K18la / HMOX1 signaling pathway, improving mitochondrial function, and inhibiting ferroptosis. This drug is particularly suitable for treating intestinal oxidative damage, especially in weaned piglets. This invention has advantages such as strong targeting, multi-dimensional relief of intestinal damage, and high safety.

Description

Technical Field

[0001] This invention belongs to the field of animal intestinal health regulation and veterinary biological agents, specifically relating to a drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells and its application. Background Technology

[0002] In large-scale livestock and poultry farming, intestinal oxidative stress is one of the core problems causing diarrhea, growth retardation, decreased immunity, and increased mortality in animals. Early weaning of piglets, environmental stress, and toxic irritation are particularly likely to induce intestinal oxidative stress, causing significant economic losses to the livestock industry. The intestine is a vital organ for nutrient digestion and absorption and immune defense in animals; the homeostasis of intestinal epithelial cells directly determines the intestinal barrier function. When oxidative stress occurs, large amounts of reactive oxygen species (ROS) accumulate in cells, disrupting the balance between the oxidative and antioxidant systems, damaging intestinal villi structure, reducing tight junction protein expression, and causing intestinal barrier damage.

[0003] Ferroprelation is an iron-dependent programmed cell death characterized by the massive accumulation of lipid peroxides, and it has been confirmed as a key pathway mediating oxidative damage in the gut. Current research indicates that oxidative stress drives cellular glucose metabolism reprogramming, leading to abnormal activation of the glycolysis pathway and the excessive production and accumulation of lactate. Lactic acid not only exacerbates cellular metabolic disorders as a metabolite but also acts as a substrate to mediate histone lactation, a novel post-translational modification of histones. Histone lactation can participate in various pathological processes such as oxidative stress and cell death by regulating gene transcription. However, the regulatory mechanisms among histone lactation, lactate accumulation, and ferroptosis in the gut system remain unclear, and there is a lack of targeted intervention products developed based on this pathway.

[0004] Currently, most products on the market for alleviating oxidative damage in the animal gut are common antioxidants, probiotics, and herbal extracts. These have broad targets and cannot precisely block the cascade of oxidative stress, lactate accumulation, histone lactation, and ferroptosis, resulting in limited intervention effects. Furthermore, existing technologies have not developed targeted regulatory strategies for the H3K18 lactation site specific to intestinal epithelial cells, the downstream target gene HMOX1, and the upstream regulatory enzyme AARS2. This makes it difficult to inhibit ferroptosis in intestinal epithelial cells at the molecular level. Therefore, there is an urgent need to develop novel regulatory drugs and application strategies targeting this signaling pathway. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells and its application. The drug can inhibit lactate production, downregulate H3K18 lactation levels, block the H3K18la / HMOX1 signaling axis, inhibit ferroptosis in intestinal epithelial cells, and effectively alleviate oxidative damage in the animal intestine. This invention also provides specific applications of this drug in alleviating oxidative damage in the animal intestine.

[0006] To achieve the above objectives, the first aspect of the present invention provides a drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells, the drug comprising a lactate dehydrogenase A inhibitor, GSK2837808A. The lactate dehydrogenase A inhibitor, GSK2837808A, is the active ingredient of the drug.

[0007] A second aspect of the present invention provides the use of the above-mentioned drug in the preparation of an agent to alleviate oxidative damage to the animal intestinal tract.

[0008] Furthermore, the animal in question is a weaned piglet.

[0009] Furthermore, the intestinal oxidative damage is induced by at least one of Diquat, hydrogen peroxide (H2O2), weaning stress, and environmental stress.

[0010] The third aspect of the present invention provides a method for inhibiting ferroptosis of intestinal epithelial cells by targeting histone lactation. The method involves administering the above-mentioned drug to animals with intestinal oxidative damage; or treating intestinal epithelial cells under oxidative stress with GSK2837808A at the cellular level to inhibit lactate production and downregulate pan-lactic acidification and H3K18 lactation levels.

[0011] Furthermore, during cell treatment, the working concentration of GSK2837808A is 4-6 μM, preferably 5 μM; the pretreatment time is 18 h.

[0012] The fourth aspect of this invention provides the application of the HMOX1 gene or protein as a drug target in screening drugs against oxidative damage in the animal intestine, wherein the drug works by inhibiting HMOX1 expression and blocking HMOX1 lactation.

[0013] Furthermore, the lactation site of the HMOX1 protein is the K35 site, and regulating lactation at this site can inhibit ferroptosis in intestinal epithelial cells.

[0014] This invention, through animal and cell experiments, demonstrates that oxidative stress promotes ferroptosis in intestinal epithelial cells by inducing lactate accumulation, upregulating histone H3K18 lactation levels, and activating the H3K18la / HMOX1 signaling axis. GSK2837808A effectively alleviates intestinal oxidative damage in animals by inhibiting lactate production, downregulating pan-lactic acidification and H3K18 lactation levels, blocking the H3K18la / HMOX1 signaling pathway, improving mitochondrial function, and inhibiting ferroptosis. The drug of this invention is used to alleviate intestinal oxidative damage induced by diquat, hydrogen peroxide, weaning stress, or environmental stress, and is particularly suitable for weaned piglets. This invention also provides the application of HMOX1 as a drug target in screening drugs against intestinal oxidative damage in animals. This invention has the advantages of strong targeting, multi-dimensional relief of intestinal damage, and high safety.

[0015] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0016] 1. The drug containing the main active ingredient GSK2837808A in this invention can effectively inhibit lactate accumulation induced by oxidative stress and downregulate the modification level of the core histone lactation site H3K18; it can improve mitochondrial dysfunction caused by oxidative stress and block the conditions for ferroptosis from upstream; it can effectively alleviate toxin-induced intestinal oxidative damage in animals.

[0017] 2. This invention is the first to target lactic acid, H3K18 histone lactation, and the HMOX1 signaling axis as intervention points, which is different from traditional broad-spectrum antioxidant products. By using GSK2837808A, it blocks the cascade transmission of oxidative stress to ferroptosis from the molecular pathway root, which is highly targeted.

[0018] 3. The drug containing the main active ingredient GSK2837808A in this invention has a comprehensive effect. The drug not only inhibits lactic acid production and histone lactation, but also repairs mitochondrial function, restores the intestinal barrier, inhibits ferroptosis, and alleviates oxidative damage to the animal intestine in multiple dimensions.

[0019] 4. The drug containing the main active ingredient GSK2837808A in this invention has high safety. The core active ingredient GSK2837808A is non-toxic to normal cells and animals at effective concentrations, does not affect the normal physiological metabolism of animals, and meets the safety requirements for veterinary preparations. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The diagram shows the detection results of high-concentration lactic acid and Diquat-induced intestinal oxidative stress in mice in this embodiment of the invention; where A: animal experiment flowchart; B: mouse weight record; C: mouse food intake record; D: mouse intestinal HE pathological section; E: intestinal villus height statistics; F: intestinal crypt depth statistics; G: intestinal villus height / crypt depth diagram.

[0022] Figure 2 The figures shown are from embodiments of the present invention and represent the detection results of Diquat and high lactate-induced intestinal iron homeostasis imbalance and mitochondrial dynamics disorder in mice. A: Mouse serum lactate concentration; B: Porcine jejunum lactate concentration; C: Mouse jejunum lactate concentration; D: Western blot (WB) detection results of H3K18la, NCOA4, LC3b, DRP1, and MFN1 protein expression levels; E: H3K18la expression level and statistical results; F: NCOA4 protein expression level and statistical results; G: LC3b protein expression level and statistical results; H: DRP1 protein expression level and statistical results; I: MFN1 protein expression level and statistical results; J: Jejunal tissue MDA content; K: Mouse jejunal tissue total SOD activity.

[0023] Figure 3 The figures shown are the results of hydrogen peroxide-induced cell viability reduction and ROS accumulation in this embodiment of the invention; where A: CCK8 cell viability measurement results; B: cell ROS measurement results.

[0024] Figure 4 This is a diagram showing the results of H2O2 disrupting the intestinal epithelial barrier structure and inducing ferroptosis in an embodiment of the present invention; wherein: A, F: expression level and statistics of Nrf2 protein in cells; A, G: expression level and statistics of GPX4 protein in cells; A, H: expression level and statistics of ZO-1 protein in cells; A, I: expression level and statistics of Claudin-1 protein in cells; B, J: IF and statistics of Claudin-1 protein in cells; CE: lipid peroxide level and statistics of cells (magnification: 630×).

[0025] Figure 5 The diagram shows the results of H2O2-induced mitochondrial dysfunction in the embodiments of the present invention; wherein: A, B: DRP1 protein expression level and statistical results; A, C: PGC-1 protein expression level and statistical results; DG: mitochondrial membrane potential level results.

[0026] Figure 6The following diagrams show the results of H2O2-induced upregulation of enzymes related to cellular lactate production and lactate accumulation in embodiments of the present invention; wherein: A: lactate content detection results; B: LDHA mRNA level detection results; C: PFKFB2 mRNA level detection results; D: HK2 mRNA level detection results; E: MCT1 mRNA level detection results.

[0027] Figure 7 The figures shown are the results of high-concentration lactate-induced cellular oxidative stress in the embodiments of the present invention; wherein, A, B: Nrf2 protein expression level and statistical results; A, C: PGC-1α protein expression level and statistical results; A, D: GPX4 protein expression level and statistical results; E, F: ROS level and statistical results (100×).

[0028] Figure 8 The image shows the results of GSK2837808A reversing oxidative stress-induced lactate accumulation in this embodiment of the invention; where A: cell viability results detected by CCK8 assay; B, C: lactate level detection results.

[0029] Figure 9 This diagram illustrates the results of inhibiting lactate production and alleviating H2O2-induced ferroptosis in an embodiment of the present invention. A, B: ZO-1 protein expression level and statistical results; A, C: NCOA4 protein expression level and statistical results; A, D: GPX4 protein expression level and statistical results; A, E: FTH1 protein expression level and statistical results; A, F: LC3 expression level detection and statistical results; G: FPN mRNA level detection results; H: FTH1 mRNA level detection results; I: LC3B mRNA level detection results; J: Claudin-1 mRNA level detection results; K: Occludin mRNA level detection results; L: ZO-1 mRNA level detection results; M: Total iron ion level detection results; N: CCK8 cell viability detection results; O, P: Lipid peroxide detection and statistical results.

[0030] Figure 10 This is a graph showing the results of inhibiting lactate production and alleviating H2O2-induced mitochondrial dysfunction in an embodiment of the present invention; wherein, A, B: PGC-1α protein expression level and statistical results; A, C: DRP1 protein expression level and statistical results; A, D: MFN1 protein expression level and statistical results; E, G: Mito-Tracker mitochondrial number and statistical results; F, H: JC-1 mitochondrial membrane potential level detection and statistical results.

[0031] Figure 11The diagram shows the results of H2O2-induced upregulation of cellular lactase in an embodiment of the present invention; wherein, A, B: P300 protein expression level and statistical results; A, C: GCN5 / KAT2A protein expression level and statistical results; A, D: HDAC2 protein expression level and statistical results; A, E: LDHA protein expression level and statistical results.

[0032] Figure 12 The figures shown are the results of H2O2-induced increase in cellular lactation levels in this embodiment of the invention; where A: Pan Kla expression level detection results; B, F: Pan Kla expression level detection and statistical results; C, G: Pan Kla expression level detection and statistical results; D, H: Pan Kla protein expression level IF detection and statistical results (630 ×); E, I: Pan Kla expression level detection and statistical results after inhibiting lactate production.

[0033] Figure 13 These are the results of screening histone lactation sites in embodiments of the present invention; wherein, A, F: H3K18la expression level detection and statistical results; A, G: H4K12la expression level detection and statistical results; A, H: H3K9la expression level detection and statistical results; A, I: H3K14la expression level detection and statistical results; A, J: H2BK16la expression level detection and statistical results; A, K: H4K5la expression level detection and statistical results; B: H3K18la expression level detection results after cell stimulation with gradient concentrations of H2O2; C: H3K18la expression level detection results after cell stimulation with gradient concentrations of GSK2837808A; D, L: H3K18la expression level detection results after cell co-stimulation with GSK2837808A and H2O2; E, M: H3K18la expression level detection results after high lactate stimulation.

[0034] Figure 14 This diagram shows the results of oxidative stress promoting HMOX1 expression via lactate-H3K18la in an embodiment of the present invention. A and B represent HMOX1 protein expression levels and statistical results; C represents HMOX1 mRNA level detection results; D and E represent HMOX1 protein expression levels and statistical results after lactate inhibition; F represents HMOX1 mRNA level detection results after lactate inhibition; G and H represent the results of lactate-promoted HMOX1 protein expression; and H and I represent STEAP3 and VDAC2 mRNA level detection results. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0037] Currently, most products on the market for alleviating oxidative damage in the animal gut are common antioxidants, probiotics, and herbal extracts. These have broad targets and cannot precisely block the cascade of oxidative stress, lactate accumulation, histone lactation, and ferroptosis, resulting in limited intervention effects. Furthermore, existing technologies do not target the H3K18 lactation site specific to intestinal epithelial cells, the downstream target gene HMOX1, or the upstream regulatory enzyme AARS2, making it difficult to inhibit ferroptosis in intestinal epithelial cells at the molecular level. Therefore, there is an urgent need to develop novel regulatory drugs and application strategies targeting this signaling pathway. Based on the above shortcomings, this invention provides a drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells and its specific application in alleviating oxidative damage in the animal gut.

[0038] The general test materials and methods used in the examples are as follows:

[0039] Experimental cells: porcine intestinal epithelial cells IPEC-J2; Experimental animals: 8-week-old C57BL / 6 mice and weaned piglets; Core reagents: GSK2837808A (LDHA selective inhibitor), hydrogen peroxide (H2O2), Diquat, sodium lactate, and a universal lactate assay kit (Wuhan Yilairuit Biotechnology Co., Ltd., product code: E-BC-K044-M); Detection technologies: CCK-8 cell viability assay, ROS / lipid peroxide fluorescent probe detection, Western blotting, qPCR, HE staining, co-immunoprecipitation (Co-IP), liquid chromatography-mass spectrometry (LC-MS), CUT & Tag, transcriptome sequencing, etc.

[0040] To investigate the effects of oxidative stress and high concentrations of lactate on intestinal oxidative damage, mitochondrial dysfunction, and ferroptosis, this invention randomly divided 30 eight-week-old C57BL / 6 mice into a Control group, a Diquat group, and a high-lactate group; IPEC-J2 cells were divided into a Control group and an H2O2 group. HE staining, Western blotting, and fluorescent probe methods were used to analyze the jejunal structure, oxidative damage-related markers, mitochondrial dynamics, and ferroptosis-related protein expression levels in mice.

[0041] Example 1: High concentrations of lactic acid and Diquat promote oxidative stress in the mouse intestine.

[0042] like Figure 1 As shown, this example investigates the relationship between lactic acid and oxidative damage in the mouse intestine. Animal experiments were conducted as follows: Figure 1 As shown in Figure A, the food intake and body weight of the mice were recorded daily. At the end of the experiment, intestinal tissues were collected from different groups of mice for paraffin sectioning, HE staining, and protein extraction. The results showed that there was no significant difference in food intake among the three groups of mice. Figure 1 C), however, both Diquat and high-concentration lactate stimulation led to a significant decrease in body weight in mice (C). Figure 2 B); In addition, HE staining results showed that both Diquat and high lactate stimulation led to villus atrophy, intestinal crypt deepening, and a decrease in the ratio of villus height to crypt depth in mice. Figure 1 The above results indicate that Diquat and high lactic acid stimulation cause intestinal damage in mice, disrupting their intestinal structure and digestive and absorptive functions, which in turn leads to a decrease in mouse body weight.

[0043] The jejunum is the main site of iron absorption in the body, a process that requires a significant amount of energy provided by the mitochondria. Therefore, iron metabolism homeostasis and mitochondrial metabolic homeostasis are crucial for maintaining normal intestinal function. Oxidative stress leads to abnormal lactic acid accumulation in animals. To investigate whether accumulated lactic acid affects iron metabolism and mitochondrial function and its potential mechanisms, this study used colorimetric methods and Western blotting to detect lactic acid, oxidative indicators, iron metabolism, and mitochondrial dynamics-related indicators. The results are as follows: Figure 2 As shown, oxidative stress significantly upregulated lactate levels and intestinal iron levels in the body. Figure 2 AC); Diquat and high lactate both induce the intestinal autophagy-related protein NCOA4 (AC ...). Figure 2 D, F) and LC3b expression were significantly increased ( Figure 2 D,G; expression of mitochondrial splitting protein DRP1 was significantly increased ( Figure 2 D, H), while the expression of the mitochondrial fusion protein MFN1 was significantly reduced (D, H), Figure 2D, I); Regarding oxidative indicators, the MDA content in the intestinal tissue of the Diquat and high lactate groups was significantly upregulated (D, I); Figure 2 J), while T-SOD activity was significantly downregulated (J). Figure 2 K); in addition, both Diquat and high lactate levels significantly increased histone H3K18 lactation levels (K); Figure 2 (D, E). The above results indicate that Diquat and high lactate stimulation induced mitochondrial dynamics disorder and promoted intestinal ferrophagy, suggesting that oxidative stress-induced lactate may promote intestinal oxidative damage by regulating cellular ferroptosis through histone lactation.

[0044] Example 2: H2O2 induces oxidative stress and ferroptosis in IPEC-J2 cells.

[0045] To further investigate the mechanisms of oxidative damage and ferroptosis in the mouse intestine, this example uses hydrogen peroxide (H2O2). An intestinal oxidative stress cell model was established by stimulating IPEC-J2 cells. To screen the effective concentration of H2O2, IPEC-J2 cells were stimulated for 6 h with gradient concentrations (0, 150, 300, 450, 600, and 750 μM). Cell viability was measured using the CCK8 assay, and ROS content was determined using a total ROS fluorescent probe. The results showed that at a concentration of 300 μM, cell viability decreased by approximately 25%, a significant difference, and total ROS levels increased significantly. Figure 3 Therefore, this concentration was chosen for subsequent experiments.

[0046] Oxidative stress involves multiple forms of cell death, among which ferroptosis and oxidative stress are mutually influential and inseparable, jointly causing cell death and disruption of the intestinal epithelial barrier. In this study, the levels of tight junction proteins, GPX4, and lipid peroxides (LPO) in H2O2-induced oxidative stress in IPEC-J2 cells were detected by Western blotting, immunofluorescence (IF), and the BODIPY 581 / 591 C11 fluorescent probe method. Results are as follows... Figure 4 As shown: Oxidative stress significantly reduced the expression of tight junction proteins ZO-1 and Claudin-1 in IPEC-J2 cells, and significantly reduced the expression of antioxidant Nrf2 protein and ferroptosis inhibitor GPX4 protein. Figure 4 A, B); In addition, H2O2 induced a significant increase in the level of lipid peroxides, a biomarker of ferroptosis (A, B); Figure 4 (CE). The above results indicate that H2O2 disrupts the intestinal epithelial barrier structure and induces ferroptosis in IPEC-J2 cells.

[0047] Mitochondria are not only one of the most important organelles in cells, but also a major source of reactive oxygen species (ROS) and are highly susceptible to ROS attack, playing a crucial role in cellular redox regulation. To investigate the effects of H2O2-induced oxidative stress on mitochondrial function, this study examined mitochondrial dynamics-related proteins and mitochondrial membrane potential. The results are as follows: Figure 5 As shown: Oxidative stress leads to a significant increase in DRP1 protein expression ( Figure 5 A, B), the expression of mitochondrial biosynthetic protein PGC-1 protein was significantly decreased ( Figure 5 A, C), and significantly reduced mitochondrial membrane potential ( Figure 5 The DG indicates that H2O2-induced oxidative stress induces mitochondrial dynamics disorder and membrane potential decrease, leading to mitochondrial dysfunction and potentially promoting the release of mitochondrial ROS, thus accelerating ferroptosis.

[0048] The regulation of lactation levels depends on lactate-related enzymes in cells regulating lactate levels. In this example, animal experiments showed that oxidative stress promotes serum and intestinal lactate accumulation and lactation. To further investigate the effect of H2O2-induced cellular oxidative stress on lactate production, this example used colorimetric assays, quantitative real-time PCR (qPCR), and Western blotting to detect the levels of lactate, lactate-related enzymes, transport-related proteins, and histone lactylases (Note: qPCR was normalized after three replicates). The results are as follows... Figure 6 As shown: Oxidative stress leads to increased mRNA levels of HK2 and PFKFB2 during glycolysis, increased mRNA levels of the monocarboxylic acid transporter MCT1, and no significant change in LDHA expression. Figure 6 BE), cell lactate content increased significantly ( Figure 6 A). The above results indicate that oxidative stress induces cellular glucose metabolism reprogramming, promotes cellular glycolysis and lactate transport, and ultimately leads to the accumulation of intracellular lactate.

[0049] This embodiment has confirmed that oxidative stress induces lactate accumulation in intestinal epithelial cells, promoting ferroptosis and thus exacerbating oxidative damage to the intestine. To further verify this result, this embodiment stimulated cells with different concentrations of sodium lactate and detected the expression of mitochondrial biosynthetic protein PGC-1α, cellular oxidative stress-related proteins Nrf2 and GPX4, and the total ROS content of cells using Western blotting and fluorescent probes. The results are as follows: Figure 7 As shown, high lactate levels (greater than 30 mM) downregulated the expression of Nrf2, GPX4, and PGC-1α proteins. Figure 7 AD), upregulated the level of total ROS in cells ( Figure 7(E, F). In summary, the role of high lactate in promoting intestinal ferroptosis in animals has been further validated in cells, suggesting that high lactate may promote intestinal oxidative damage by inducing mitochondrial dynamics disorder and ferroptosis in intestinal epithelial cells.

[0050] Example 3: GSK2837808A inhibited lactate and reversed H2O2-induced ferroptosis and mitochondrial dysfunction.

[0051] Oxidative stress induces lactate accumulation in IPEC-J2 cells and animal organisms. High concentrations of lactate exacerbate oxidative stress and ferroptosis. To further explore the molecular mechanism by which lactate regulates intestinal ferroptosis and mitochondrial function in animals, this study reverses oxidative stress-induced lactate accumulation using a small molecule compound. GSK2837808A is a chemically synthesized, potent, and selective LDHA inhibitor that efficiently inhibits lactate production during glycolysis. The CCK8 assay confirmed that GSK2837808A at concentrations up to 20 μM had no negative impact on cell viability. Figure 8 A), and 5 μM GSK2837808A can significantly inhibit lactic acid production (A). Figure 8 B), therefore, 5 μM GSK2837808A was selected for the next step of the experiment; subsequent experiments showed that GSK2837808A significantly reversed the hydrogen peroxide-induced increase in lactate content in IPEC-J2 cells (B). Figure 8 C). The above results indicate that GSK2837808A can effectively inhibit cellular lactate accumulation without affecting cell viability.

[0052] To further investigate the regulatory role of lactate on ferroptosis, this study used Western blotting, qPCR, colorimetric assays, and the BODIPY581 / 591 C11 fluorescent probe method to detect ferroptosis-related indicators in IPEC-J2 cells after lactate inhibition. These indicators included total iron ion concentration and the expression levels of mRNA and protein of ferroptosis-related genes such as GPX4, NCOA4, FPN, FTH, LC3, and tight junction proteins (Note: qPCR was normalized after three replicates). The results are as follows: Figure 9 As shown: H2O2 induced ferroptosis, while GSK2837808A reversed H2O2-induced ferroptosis, as evidenced by the significant upregulation of the expression of the ferrophagy protein NCOA4 by H2O2-induced oxidative stress. Figure 9 A, C), promoted the autophagy marker LC3 To LC3 The transformation ( Figure 9 A, F, M), upregulated LC3 mRNA levels ( Figure 9 I) to promote ferrophagy to release free iron and significantly downregulated the mRNA level of the membrane iron transporter FPN1, leading to cellular iron accumulation. Figure 9 G); In addition, oxidative stress also significantly reduced the level of GPX4 protein (G); Figure 9 A, D) and the expression of ferritin heavy chain FTH1 protein and mRNA ( Figure 9 A, E, H) and downregulated protein and mRNA levels of some tight junction proteins ( Figure 9 A, B, JL); Finally, H2O2 induced a significant increase in cell LPO (A, B, JL). Figure 9 O, P). However, GSK2837808A reversed the changes in the above-mentioned ferroptosis-related indicators induced by H2O2, significantly reducing iron ion accumulation and LPO generation to alleviate ferroptosis (O, P). Figure 9 (N, O, P). The above results suggest that H2O2-induced oxidative stress promotes cellular iron accumulation by promoting ferrophagy and inhibiting iron efflux, leading to increased ROS, GPX4 protein depletion, and promoting ferroptosis. Inhibiting H2O2-induced lactate can alleviate ferroptosis in IPEC-J2 cells.

[0053] Mitochondria are the main source of reactive oxygen species in cells. Their abundant lipids, metals, and redox-related enzymes determine their crucial role in regulating oxidative stress and ferroptosis. Mitochondrial metabolism not only regulates the processes of oxidative stress and ferroptosis, but mitochondrial homeostasis is also affected by these processes. To further investigate the regulatory role of lactate on mitochondrial function in IPEC-J2 cells, this study used Western blotting, Mito-Tracker Green assay, and JC-1 fluorescent probe method to detect the expression of mitochondrial dynamics-related proteins such as PGC-1α, DRP1, and MFN1, as well as changes in mitochondrial number and mitochondrial membrane potential. The results are as follows: Figure 10 As shown: H2O2-induced oxidative stress significantly upregulated the expression of mitochondrial division-related protein DRP1 ( Figure 10 A, C), reduced the expression of mitochondrial biosynthesis-related protein PGC-1α ( Figure 10 A, B), reduced MFN1 protein expression and significantly reduced the number of mitochondria ( Figure 10 E, G), and significantly reduced mitochondrial membrane potential ( Figure 10 (F, H); However, GSK2837808A significantly reversed the changes in the above-mentioned mitochondrial dynamics-related indicators by inhibiting lactate, restoring mitochondrial function. Excessive mitochondrial division releases a large amount of ROS, and previous studies have shown that increased DRP1 promotes ferroptosis. Therefore, this embodiment suggests that H2O2-induced oxidative stress may promote mitochondrial dynamic disorder and decreased membrane potential through lactate accumulation, thereby inducing ferroptosis. Inhibiting lactate during oxidative stress has a positive effect on maintaining normal mitochondrial function.

[0054] Example 4: Oxidative stress upregulated the level of cellular lactation and promoted H3K18 lactation.

[0055] Oxidative stress promotes intestinal lactate accumulation by regulating the expression of enzymes related to lactate production and transport. To further investigate whether oxidative stress upregulates cellular lactation levels by promoting lactate production, this study used Western blotting (WB) and inductively coupled plasma (IF) to detect the expression of cellular lactation-related enzymes and pan-lactated proteins. The results are as follows: Figure 11 As shown, H2O2-induced oxidative stress significantly promoted the expression of two histone lactases, P300 and KAT2A (GCN5), without affecting the expression of LDHA and delactase HDAC2. Figure 11 The results indicate that H2O2-induced oxidative stress significantly increased the level of lactase in IPEC-J2 cells and may promote the lactation of cellular proteins.

[0056] To further confirm whether oxidative stress-induced upregulation of lactate and lactase promotes intestinal lactation, this study detected the lactation level of total cellular protein using Western blotting and inductively coupled plasma (IF) assays, and used high lactate and GSK2837808A stimulation of cells for negative and positive comparisons. Results are as follows: Figure 12 As shown, H2O2 upregulates protein pan-lactation levels in a dose-dependent manner ( Figure 12 A), and both histone lactation and non-histone lactation levels were upregulated, especially with a significant increase in histone lactation signaling in the nucleus (A). Figure 12 B, D), and the results of H2O2-induced lactation and high lactate stimulation are similar ( Figure 12 C), and inhibiting lactate production can reverse oxidative stress-induced protein lactation (C). Figure 12 E). The above results indicate that oxidative stress promotes lactation of histones and non-histones in IPEC-J2 cells by inducing lactate accumulation, and that the level of lactation is regulated by the level of lactate.

[0057] This study has demonstrated that oxidative stress promotes pan-lactation of cellular proteins by increasing lactate accumulation and upregulating lactase levels. However, numerous sites on chromatin are involved in histone lactation. To further screen histone lactation sites that may be involved in the regulation of oxidative stress, this embodiment utilizes antibodies against some typical histone lactation modification sites (H3K18la, H3K9la, H3K14la, H2BK16la, H4K5la, and H4K12la), and screens them using Western blotting (WB). Negative and positive controls were performed using high lactate levels and GSK2837808A stimulation of cells. Results are as follows: Figure 13 The results showed that oxidative stress induced significant upregulation of lactation at multiple histone sites, including H3K18la, H3K9la, and H4K12la, while the expression of H3K14la, H2K16la, and H4K5la did not differ significantly. Figure 13 A, FK). Among them, H3K18la was upregulated most significantly, and different concentrations of hydrogen peroxide stimulation of cells all increased the level of H3K18la to varying degrees. Figure 13 B), and is regulated by cellular lactate levels ( Figure 13 E, M), inhibiting lactate production can reverse oxidative stress-induced H3K18la ( Figure 13 (C, D, L), therefore, H3K18la was chosen as a target for later research. The above results indicate that hydrogen peroxide-induced oxidative stress upregulates the level of lactation at multiple histone sites by inducing lactate accumulation in IPEC-J2 cells, and the potential histone lactation site H3K18la involved in the regulation of intestinal oxidative stress was screened.

[0058] Based on the combined CUT&Tag and RNA-seq sequencing results, this embodiment uses qPCR and WB to verify the screening results, as shown in the figure. Figure 14 As shown: Oxidative stress did indeed significantly increase the levels of HMOX1 mRNA and protein in cells. Figure 14 AC, inhibiting lactate downregulates HMOX1 protein and mRNA expression ( Figure 14 DF), and lactate promotes HMOX1 protein expression in a dose-dependent manner ( Figure 14 GSK2837808A significantly reversed oxidative stress-induced HMOX1 protein expression by inhibiting lactate (lactic acid and oxidative stress had no significant effect on the expression of genes such as VDAC2). Figure 14 H, I).

[0059] Example 5 Preparation of Drug (Powder)

[0060] This embodiment provides a powdered drug for animal feed mixing. The formula, by weight, is: 1 part GSK2837808A, 90 parts glucose, and 9 parts corn starch. The preparation method is as follows: the raw materials are thoroughly mixed, passed through an 80-mesh sieve, and aseptically packaged to obtain the feed mixing powder.

[0061] Example 6 Preparation of Drug (Oral Liquid)

[0062] This embodiment provides an oral liquid drug for animal drinking water. The formula, by volume, is: GSK2837808A 5 μmol / L, and sterile physiological saline to a final volume of 1 L. The preparation method is as follows: GSK2837808A is dissolved in sterile physiological saline, thoroughly mixed, sterilized by a 0.22 μm filter membrane, and then filled into an oral liquid.

[0063] Example 7: Animal husbandry application: The effect of the drug on alleviating intestinal damage in weaned piglets

[0064] Sixty 21-day-old weaned piglets were randomly divided into a control group, a weaning stress model group, and a drug-mixed feed group (fed with the powder from Example 5 for 21 consecutive days). Results: The weaning stress group showed increased diarrhea rate and decreased daily weight gain, with oxidative damage and ferroptosis in the intestinal tissue. After adding the drug of this invention, the diarrhea rate significantly decreased, growth performance improved, and intestinal oxidative stress, lactic acid accumulation, and ferroptosis were effectively alleviated. Conclusion: The drug of this invention is suitable for the prevention and control of intestinal damage in weaned piglets in large-scale farming, with excellent application effects.

[0065] This invention uses mouse and cell models to analyze the effects of oxidative stress on the expression levels of lactate, glycolysis-related enzymes, and lactylating enzymes. The effects of a lactate dehydrogenase A (LDHA) inhibitor (GSK2837808A) on mitochondrial function and ferroptosis were also analyzed. Results showed that oxidative stress significantly increased serum lactate, cellular glycolysis-related enzymes (PFKFB2 and HK2, etc.), and lactylating enzymes (p300 and KAT2A, etc.) levels in mice. It also increased the levels of pan-lactation (Pan Kla) and histone site lactation (H3K18la, H3K9la, H4K12Bla) in cellular proteins, with particularly pronounced changes in H3K18. This indicates that oxidative stress promotes lactate accumulation and protein lactation in IPEC-J2 cells. Further research revealed that GSK2837808A significantly reduced H2O2-induced Pan Kla and H3K18la levels by inhibiting lactate, and partially reversed oxidative stress and high lactate-induced mitochondrial dysfunction and ferroptosis. This indicates that oxidative stress induces lactate accumulation and protein lactation, while inhibiting lactation improves mitochondrial function and resists ferroptosis.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A drug that targets histone lactation to regulate ferroptosis in intestinal epithelial cells, characterized in that: The drug includes the lactate dehydrogenase A inhibitor GSK2837808A.

2. The use of the drug according to claim 1 in the preparation of an agent to alleviate oxidative damage to the animal intestine.

3. The application according to claim 2, characterized in that: The animal in question is a weaned piglet.

4. A method for inhibiting ferroptosis in intestinal epithelial cells by targeting histone lactation, characterized in that: Administer the drug of claim 1 to animals with intestinal oxidative damage; or treat intestinal epithelial cells under oxidative stress with GSK2837808A at the cellular level.

5. The method according to claim 4, characterized in that, During cell treatment, the working concentration of GSK2837808A was 4-6 μM, and the pretreatment time was 18 h.

6. Application of HMOX1 gene or HMOX1 protein as drug targets in screening drugs against oxidative damage in animal intestines.

7. The application of the HMOX1 gene or HMOX1 protein as a drug target in screening drugs against oxidative damage in the animal intestinal tract according to claim 6, characterized in that: The drug works by inhibiting HMOX1 expression and blocking HMOX1 lactation.

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