Application of itaconic acid in sepsis liver injury

Abstract

The invention discloses application of itaconic acid in sepsis liver injury, and relates to the field of medicine. The invention discloses application of itaconic acid or derivatives thereof in preparation of drugs for treating sepsis liver injury. The itaconic acid derivative is 4-octyl itaconic acid (4-OI). The action mechanism is to down-regulate liver tissue LC3-II protein expression and / or up-regulate P62 protein expression. The compound is used for reducing the levels of ALT and AST in serum or inhibiting the activity of TNF-alpha, IL-6 and MPO in liver tissue. In lipopolysaccharide-induced mouse liver injury, LPS excessively activates autophagy by down-regulating P62 and up-regulating LC3-II, so that inflammatory disorder and liver injury are caused, and the immune response gene 1 / itaconic acid can achieve the effect of protecting the liver by inhibiting excessive autophagy and down-regulating autophagy flux.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of itaconic acid in treating septic liver injury. Background Art

[0002] Sepsis is a syndrome characterized by a dysregulated host response to infection, which leads to organ dysfunction and can cause damage and dysfunction to multiple organs. During sepsis, the liver plays a central role in regulating immune balance. At the same time, it is also one of the targets of sepsis-related organ damage, leading to further immune dysregulation, worsening of the disease, and even death in patients with sepsis. The mechanisms of sepsis-related liver injury are complex and are currently believed to be related to inflammation, oxidative stress, hepatic circulatory and metabolic disorders, mitochondrial metabolic dysfunction, and autophagy. Its treatment is comprehensive, including control of the source of infection, immunomodulatory therapy targeting the inflammatory response, early targeted hemodynamic support, and treatment to protect liver cells.

[0003] However, targeted treatments are still very limited, so the development of new and more effective therapeutic drugs is urgent.

[0004] Therefore, the present invention proposes the use of itaconic acid in treating septic liver injury. Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the application of itaconic acid in septic liver injury.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of itaconic acid in treating septic liver injury, and application of itaconic acid or its derivatives in preparing drugs for treating septic liver injury.

[0008] Preferably, the itaconic acid derivative is 4-octylitaconic acid (4-OI).

[0009] Preferably, its mechanism of action is down-regulating the expression of LC3-II protein in liver tissue and / or up-regulating the expression of P62 protein.

[0010] Preferably: used to reduce serum ALT and AST levels, or inhibit liver tissue TNF-α, IL-6, and MPO activities.

[0011] The beneficial effects of the present invention are:

[0012] 1. In the present invention, in lipopolysaccharide-induced liver injury in mice, LPS over-activates autophagy by down-regulating P62 and up-regulating LC3-Ⅱ, thereby causing inflammatory disorders and liver damage, while immune response gene 1 / itaconate can protect the liver by inhibiting excessive autophagy and down-regulating autophagic flux. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram showing that Irg1 knockout aggravates LPS-induced liver injury in mice according to the present invention, wherein: (A) Liver histological HE staining, magnified 200x; (B) (C) Detection of serum ALT and AST levels; (D) Liver injury score (WT, wild-type mice; Irg1- / -, Irg1 knockout mice; NS, normal saline; LPS, lipopolysaccharide; n = 4 per group; mean ± SD; ns, P > 0.05; **, P < 0.01);

[0014] Figure 2 Schematic diagram showing that Irg1 knockout exacerbates lipopolysaccharide-induced inflammatory response in the liver of mice according to the present invention, wherein: (A, B, C) TNF-α, IL-6, and MPO levels in liver tissue were measured; (WT, wild-type mice; Irg1- / -, Irg1 knockout mice; LPS, lipopolysaccharide; n = 4 per group; mean ± SD, ns, P > 0.05, **P < 0.01);

[0015] Figure 3 Schematic diagram of the present invention showing that 4-OI supplementation alleviates lipopolysaccharide-induced liver injury in mice, wherein: (A) Liver histology HE staining, magnified 200x; (B) (C) Detection of serum ALT and AST levels; (D) Liver injury score (WT, wild-type mice; NS., normal saline; LPS, lipopolysaccharide; 4-OI, 4-octylitaconic acid; n = 4 per group; mean ± SD; ***, P < 0.001);

[0016] Figure 4 Schematic diagram of the present invention showing that 4-OI supplementation alleviates lipopolysaccharide-induced inflammatory responses in mouse livers. (A, B, C) TNF-α, IL-6, and MPO levels in liver tissue were measured. (WT, wild-type mice; LPS, lipopolysaccharide; 4-OI, 4-octylitaconate; n = 4 per group; mean ± SD; **, P < 0.01).

[0017] Figure 5Schematic diagram of the inhibition of lipopolysaccharide-induced autophagy in mouse liver by supplementation with 4-OI according to the present invention, wherein: (A) Western blot images of autophagy-related proteins in the liver; (B) Relative expression of P62 protein in the liver; (C) Relative expression of LC3II protein in the liver; (LPS, lipopolysaccharide; 4-OI, 4-octylitaconic acid; β-actin, protein internal control; n = 4 per group; mean ± SD; *, P < 0.05; **, P < 0.01);

[0018] Figure 6 Schematic diagram of the present invention showing that Irg1 knockout promotes LPS-induced autophagy in the liver of mice, (A) western blot images of autophagy-related proteins in the liver; (B) relative expression of P62 protein in the liver; (C) relative expression of LC3II protein in the liver; (WT, wild-type mice; Irg1- / -, Irg1 knockout mice; LPS, lipopolysaccharide; 4-OI, 4-octylitaconic acid; β-actin, protein internal control; n = 4 per group; mean ± SD; ns, P > 0.05; *, P < 0.05; **, P < 0.01). DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be further described in detail below in conjunction with specific implementation methods.

[0020] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "connected," "connected," and "disposed" should be understood in a broad sense. For example, they may refer to fixed connection or disposition, detachable connection or disposition, or integral connection or disposition. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0021] Example 1 (Immune response gene 1 / itaconic acid has a protective effect on lipopolysaccharide-induced liver damage in mice):

[0022] 1. Materials and Methods

[0023] 1.1. Materials

[0024] 1.1.1. Animals

[0025] Wild-type male C57BL / 6J mice were purchased from the Experimental Animal Center of Chongqing Medical University. Irg1 knockout (Irg1- / -) mice were purchased from Guangzhou Saiye Biotechnology Co., Ltd. and include relevant animal quarantine certificates from the purchasing company, as well as the toe numbers and genotype identification results of the breeding mice. All animal husbandry and experimental procedures complied with the requirements of the Ethics Committee of Chongqing Medical University. All mice were 7-8 weeks old and weighed 18-22 g. All mice were acclimated to laboratory conditions for one week before the experiment. The laboratory provided mice with sufficient and clean drinking water and food, and mice had free access to food and water. The temperature in the laboratory was maintained at 20-25°C, the humidity was controlled at 50% ± 5%, and a 12-hour dark and 12-hour light cycle was maintained. All animal-related experimental projects were approved by the Ethics Committee of Chongqing Medical University.

[0026] Reagents

[0027]

[0028] 1.1.3. Instruments

[0029]

[0030]

[0031] 1.2. Methods

[0032] 1.2.1. Animal grouping and modeling

[0033] 1. Weighing: Weigh the mouse and record its weight

[0034] 2. Classification: Based on whether the mouse immune response gene 1 is knocked out, it is divided into two categories: WT mice (wild-type mice) and Irg1- / - mice (Irg1 knockout mice)

[0035] 3. Grouping: Sixteen WT mice were divided into four groups: saline control group, 4-OI control group, LPS model group, and 4-OI + LPS intervention group, with four mice in each group. Eight Irg1- / - mice were divided into two groups: saline control group and LPS model group, with four mice in each group.

[0036] 4. Modeling:

[0037] WT mice: Normal saline control group: Normal saline was injected intraperitoneally at a dose of 0.01 ml / g based on the mouse body weight; 4-OI control group: 100 mg of 4-OI was first dissolved in 200 μl of DMSO and mixed thoroughly, then 5% of 4-OI was mixed with 95% of olive oil to prepare a 4-OI solution, which was then injected intraperitoneally into the mice at a dose of 50 mg / kg; LPS model group: LPS solution was prepared by dissolving 1 mg of LPS in 1 ml of normal saline and injected intraperitoneally into the mice at a dose of 15 mg / kg; 4-OI+LPS intervention group: 4-OI was first injected intraperitoneally into the mice at a dose of 50 mg / kg, followed by an intraperitoneal injection of LPS at a dose of 15 mg / kg half an hour later.

[0038] Irg1- / - mice: Normal saline control group: saline was injected intraperitoneally at a dose of 0.01 ml / g based on the mouse body weight; LPS model group: LPS solution was prepared at a ratio of 1 mg LPS to 1 ml saline and injected intraperitoneally at a dose of 15 mg / kg into the mice;

[0039] 1.2.2. Animal sacrifice and sample collection

[0040] 18 hours after the intraperitoneal injection of LPS into the mice, anesthetize the mice with an intraperitoneal injection of 1% sodium pentobarbital at a dose of 0.01 ml / g and then sacrifice by cervical dislocation. Collect eye blood from the mice into heparinized centrifuge tubes, centrifuge at 8000 rpm for 10 minutes at 4°C, and store the supernatant in a -80°C freezer until use. A portion of the collected mouse liver tissue was fixed in an EP tube containing 4% paraformaldehyde and stored sealed at room temperature. The remaining liver tissue was placed in another EP tube and stored at -80°C.

[0041] 1.2.3. Determination of plasma transaminases (ALT, AST)

[0042] 1. Remove the mouse serum sample from the -80°C freezer and thaw on ice. Centrifuge the thawed serum again (4°C, 2500 rpm, 10 minutes) and collect the supernatant for testing.

[0043] 2. Remove the kit from the 4°C refrigerator and equilibrate to room temperature. Preheat the matrix solution in a 37°C constant temperature water bath.

[0044] 3. Prepare the corresponding consumables according to the kit instructions, and prepare 0.4 mol / l NaOH stop solution according to the instructions, and mix the required amount.

[0045] 4. The sample dosage operation is as follows:

[0046]

[0047] 5. Preheat the microplate reader and measure the absorbance at 505 nm. Calculate the activity value based on the standard curve.

[0048] 1.2.4. HE staining

[0049] 1. Send the previously fixed tissue to Chongqing Zhuying Biotechnology Co., Ltd. for slide preparation and staining.

[0050] 2. Observe and photograph the stained sections under an upright fluorescence microscope at a magnification of 200×. Score the liver tissue for damage.

[0051] 1.2.5.ELISA

[0052] 1.2.5.1. Preparation of total protein

[0053] 1. Weigh 20 mg of each liver tissue using a microbalance and place it into a homogenate tube.

[0054] 2. Add 200 μl of RIPA lysis buffer and 4 μl of protease inhibitor (PMSF) to every 20 mg of liver tissue and add grinding beads.

[0055] 3. Place the homogenate tube containing the sample into a biological sample homogenizer, set the parameters to 3950 rpm, 8 cycles, 23 seconds / cycle, and perform lysis and homogenization at 4°C.

[0056] 4. After homogenization, place the homogenate into a low-temperature high-speed centrifuge, centrifuge at 4°C, 12000 rpm, for 10 minutes, and collect the supernatant, which is the extracted total protein.

[0057] 1.2.5.2. Determination of protein concentration

[0058] 1. Prepare consumables and reagents according to the BCA kit instructions.

[0059] 2. Dilute the sample: dilute the extracted homogenate supernatant 40 times with double distilled water (2 μl sample + 78 μl pure water).

[0060] 3. Prepare standard solution and create standard curve:

[0061] 1) Dissolve the standard solution: Add 1.2 ml of protein standard solution to the protein standard (30 mg BSA) according to the instructions and mix thoroughly to obtain a 25 mg / ml standard. Then, add 20 μl of the 25 mg / ml protein standard solution to 980 μl of diluent to prepare a standard solution with a final concentration of 0.5 mg / ml.

[0062] 2) Serial dilution of standard solution: Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of standard solution to the standard wells of a 96-well plate. Add standard diluent to make up to 20 μl, which is equivalent to protein concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml of standard solution. Number and label.

[0063] 4. Prepare BCA working solution:

[0064] Calculate and prepare BCA working solution according to the number of samples to be tested and the ratio of solution A to solution B = 50:1. Mix thoroughly and keep away from light for later use.

[0065] 5. Use a pipette to add 20 μl of diluted protein homogenate sample to the 96-well plate, along with the serially diluted standard solution, and add 200 μl of BCA working solution to each well. After gently shaking to mix, place the plate in a 37°C constant temperature water incubator and incubate in the dark for 30 minutes.

[0066] 6. Turn on the microplate reader and measure the OD value of each well at a wavelength of 562 nm.

[0067] 1.2.5.3. Determination of inflammatory factors (TNF-α, IL-6)

[0068] 1. Prepare relevant reagents and consumables according to the kit instructions.

[0069] 2. Take out the TNF-α and IL-6 test kits from the 4°C refrigerator and place them flat at room temperature for equilibrium.

[0070] 3. Prepare standard solution:

[0071] 1) IL-6: Centrifuge the lyophilized powder of the standard at 1000 rpm for 1 minute before opening. Then, add 1 ml of the Universal Diluent for Standards and Specimens to the standard. Mix thoroughly and incubate at room temperature for 15 minutes to obtain a standard solution with a concentration of 1000 pg / ml. Finally, perform a serial dilution with double-distilled water to the following concentrations: 0, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 pg / ml.

[0072] 2) TNF-α: Centrifuge the lyophilized powder of the standard at 1000 rpm for 1 minute before opening. Then, add 0.6 ml of the Universal Diluent for Standards and Specimens to the standard. Mix thoroughly and incubate at room temperature for 15 minutes to obtain a standard solution with a concentration of 2000 pg / ml. Finally, perform a serial dilution with double-distilled water to the following concentrations: 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 pg / ml.

[0073] 4. Dilute the sample 80 times

[0074] 5. Add 100 μl / well of the diluted sample to be tested to a 96-well plate, seal the wells with sealing tape, and incubate in a 37°C water bath in the dark for 90 minutes.

[0075] 6. Prepare washing solution: Dilute the 20X concentrated washing solution 20 times with double distilled water to make the washing working solution according to the required amount.

[0076] 7. Prepare biotinylated antibody working solution: Start preparing 20 minutes in advance and dilute the 30× concentrated biotinylated antibody 30 times with biotinylated antibody diluent to make biotinylated antibody working solution.

[0077] 8. Washing: After incubating in the dark for 90 minutes, pat dry the liquid in the wells on filter paper, add 350 μl of washing solution to the reaction wells, let it stand for 30 seconds, and pat dry again. Repeat this process 5 times.

[0078] 9. Add 100 μl of biotinylated antibody working solution to each well, seal the reaction wells with sealing tape, and incubate in a 37°C water bath in the dark for 60 minutes.

[0079] 10. Prepare enzyme conjugate working solution: Start preparation 20 minutes in advance and dilute the 30× concentrated enzyme conjugate 30 times with enzyme conjugate diluent to make enzyme conjugate working solution.

[0080] 11. Incubate in the dark for 60 minutes and wash the plate five times.

[0081] 12. Add 100 μl of enzyme conjugate working solution to each well, seal the reaction wells with sealing tape, and incubate in a 37°C water bath in the dark for 30 minutes.

[0082] 13. Incubate in the dark for 30 minutes and then wash the plate 5 times.

[0083] 14. Add 100 μl of chromogenic substrate to each well and incubate in a 37°C incubator in the dark for 15 minutes.

[0084] 15. Open the microplate reader in advance to preheat and adjust the detection program.

[0085] 16. Add 100 μl of reaction stop solution to each well, shake gently to mix, and immediately place in a microplate reader to measure the OD value at a wavelength of 450 nm.

[0086] 17. Calculate the levels of TNF-α and IL-6 based on the standard curve and dilution factor.

[0087] 1.2.6. Determination of MPO activity in liver tissue

[0088] 1. Prepare the consumables according to the MPO kit instructions and dissolve reagents 1, 2, 3, and 5. Mix reagent 1 and reagent 5 to make the color developer and store in a dark place.

[0089] 2. Use a microbalance to weigh 20 mg of each liver tissue and place it into a homogenate tube.

[0090] 3. Add 400 μl of Reagent 2 to every 20 mg of liver tissue and add grinding beads.

[0091] 4. Place the homogenate tube containing the sample into a biological sample homogenizer, set the parameters to 3950 rpm, 8 cycles, 23 seconds / cycle, and perform lysis and homogenization at 4°C.

[0092] 5. After homogenization, obtain a 5% liver tissue homogenate. Without centrifugation, proceed directly to the following steps:

[0093]

[0094] 6. Preheat the microplate reader in advance and measure the OD value at 460nm immediately after the water bath ends.

[0095] 7. Calculate the MPO activity in liver tissue according to the calculation formula in the instructions.

[0096] Statistical analysis

[0097] Data were analyzed using GraphPad Prism 8 and expressed as mean ± standard deviation (SD). Comparisons between multiple groups were performed using one-way analysis of variance with Tukey's multiple comparison test. Differences were considered statistically significant when P < 0.05.

[0098] 2. Results

[0099] Irg1 knockout aggravates lipopolysaccharide-induced liver injury in mice

[0100] In order to explore whether endogenous itaconic acid encoded by the Irg1 gene has protective significance for lipopolysaccharide-induced liver damage in mice, the present invention conducted experiments in WT mice and Irg1- / - mice at the same time. The results showed that in the normal saline control group, there was no obvious pathological damage in the liver tissue of Irg1- / - mice and WT mice, and the serum transaminase levels of the two groups of mice were low and there was no significant difference. However, in the LPS model group, the present invention found that the pathological changes in the liver tissue of Irg1- / - mice were more significant than those of WT mice, and the serum transaminase levels of Irg1- / - mice were higher than those of WT mice. This shows that in lipopolysaccharide-induced liver damage in mice, knocking out Irg1 will cause more severe liver damage, and it also shows that endogenous itaconic acid has a protective effect on the mouse liver.

[0101] Irg1 knockout exacerbates LPS-induced inflammatory response in mouse liver

[0102] Furthermore, the present invention also found that LPS induced elevated levels of inflammatory cytokines TNF-α and IL-6, as well as increased MPO activity in the liver, in both WT and Irg1 knockout mice. However, in the same LPS-induced liver injury, the inflammatory cytokine levels and MPO activity were significantly higher in Irg1- / - mice than in WT mice. These results suggest that Irg1 knockout exacerbates the inflammatory response in LPS-induced liver injury, further aggravating the damage.

[0103] 4-OI supplementation alleviates lipopolysaccharide-induced liver damage in mice

[0104] To further explore the medicinal value of itaconic acid, the present invention used a synthetic itaconic acid derivative, 4-OI, to pretreat mice 0.5 hours before initiating LPS modeling to observe its effects on the mouse liver. The results are as follows: First, compared with the saline control group, the LPS model group showed significantly elevated serum transaminase (ALT) and AST levels. Liver tissue HE sections also revealed significant pathological abnormalities: disorganized hepatic lobule structure, loose hepatocyte cytoplasm with ballooning, inflammatory cell infiltration, and increased liver tissue injury scores. This suggests that lipopolysaccharide (LPS) can induce liver injury in mice. Second, compared with the LPS model group, the 4-OI pretreatment group showed significantly decreased serum transaminase (ALT) and AST levels. Liver tissue HE sections revealed reduced liver tissue abnormalities: fewer hepatocytes with ballooning, fewer inflammatory cell infiltrates, mildly disorganized hepatic lobule structure, and lower liver tissue injury scores. This suggests that 4-OI supplementation can alleviate LPS-induced liver injury in mice. Finally, compared with the saline control group, there were no significant differences in serum transaminase (ALT) and AST levels in mice treated with 4-OI alone, and no significant pathological changes were observed in hepatic tissue HE sections. This suggests that 4-OI, a synthetic itaconic acid derivative, has no significant toxic side effects on the mouse liver. Therefore, it is concluded that 4-OI, an itaconic acid derivative, can alleviate LPS-induced liver damage in mice.

[0105] 4-OI supplementation alleviates LPS-induced inflammatory responses in mouse liver

[0106] In addition, the present invention further detected the inflammatory response-related indicators TNF-α, IL-6 and MPO in the liver of WT mice. The results showed that compared with the normal saline control group, the levels of inflammatory cytokines TNF-α, IL-6 and MPO in the liver of mice in the LPS model group were increased. In addition, compared with the LPS model group, the levels of TNF-α, IL-6 and MPO in the liver of mice pre-intervened with 4-OI decreased. Of course, the administration of 4-OI alone did not cause significant changes in the levels of TNF-α, IL-6 and MPO in the liver. These results all indicate that 4-OI can reduce the inflammatory response in the liver of mice induced by lipopolysaccharide.

[0107] 3. Discussion

[0108] In recent years, research has intensively explored the role of metabolic enzymes and metabolites in inflammatory diseases, particularly IRG1 / itaconate in sepsis. Previous studies have shown that the Irg1 gene is upregulated in LPS-activated macrophages, and its translated product, IRG1, catalyzes the decarboxylation of aconitic acid to produce itaconic acid. Numerous studies have demonstrated the immunomodulatory activity of itaconic acid, and more recently, its protective effects against fulminant liver injury and ischemia-reperfusion liver injury have been found. However, research on itaconic acid in sepsis-related liver injury is limited. As this research deepens, research has begun to explore synthetic itaconic acid derivatives for their potential medicinal applications. The most representative of these is 4-OI, which not only readily penetrates cell membranes and is transported into cells, but also hydrolyzes it intracellularly to produce a large amount of free itaconic acid. Therefore, its biological function more closely reflects the role of itaconic acid in vivo.

[0109] The present study used intraperitoneal injection of lipopolysaccharide to establish a mouse model of sepsis-related liver injury. Lipopolysaccharide, a major component of the cell wall of Gram-negative bacteria, is absorbed into the bloodstream via intraperitoneal injection, creating a clinical model of endotoxemia associated with sepsis. The present research group has consistently used intraperitoneal injection of lipopolysaccharide to establish a relatively stable mouse sepsis model, which has led to extensive research on sepsis-related pathologies.

[0110] When liver cells are damaged, the integrity of the cell membrane is impaired, and the transaminases ALT and AST are released from the liver cells into the blood, leading to increased serum ALT and AST activity. Therefore, serum ALT and AST levels are basic biochemical indicators for determining the severity of liver lesions. HE-stained sections of liver tissue can more intuitively reflect the pathological changes of liver damage. The research results of the present invention show that under the stimulation of LPS, both Irg1 knockout mice (Irg1- / -) and wild-type mice (WT) have different increases in serum ALT and AST levels, and liver tissue also undergoes varying degrees of pathological changes. This shows that the present invention successfully established a mouse sepsis-related liver injury model by intraperitoneal injection of lipopolysaccharide. Further comparison found that under the same LPS modeling conditions, the serum transaminases ALT and AST levels of Irg1 knockout mice (Irg1- / -) were higher, and the liver tissue pathology was also more abnormal. This shows that the endogenous itaconic acid encoded and catalyzed by Irg1 has a significant protective effect on lipopolysaccharide-induced mouse liver damage, which is consistent with the results reported by Kerui et al.

[0111] The release of large amounts of inflammatory factors in the liver is a key pathogenesis of sepsis-related liver injury. Studies have found that lipopolysaccharide (LPS) stimulates Kupffer cells in the liver and activates the intracellular MyD88 / NF-κB pathway to produce a large number of inflammatory cytokines, such as TNF-α and IL-6. TNF-α has cytotoxic effects, directly damaging hepatocytes. Furthermore, TNF-α can reactivate Kupffer cells and hepatocytes to produce other inflammatory cytokines, such as IL-6 and IL-1. IL-6 can further cause hepatocyte damage by inducing the production of acute phase proteins. Furthermore, MPO activity in liver tissue is directly proportional to the degree of neutrophil and macrophage infiltration, and elevated MPO activity can lead to early generation of reactive oxygen species (ROS). The data presented here demonstrate that, under the same LPS model, Irg1- / - mice exhibit higher levels of TNF-α, IL-6, and MPO in the liver than WT mice. HE also demonstrates greater inflammatory cell infiltration and liver damage, suggesting that endogenous itaconic acid, encoded and catalyzed by Irg1, plays a role in reducing hepatic inflammatory responses. Secondly, compared with the LPS model group, pre-administration of 4-OI can significantly reduce the levels of TNF-α, IL-6, and MPO in liver tissue. Significantly reduced inflammatory cell infiltration and hepatocyte damage can also be seen on the HE sections of liver tissue, indicating that supplementation of 4-OI can reduce the inflammatory response in the liver induced by LPS.

[0112] These results indicate that immune response gene 1 / itaconate has a significant protective effect against lipopolysaccharide-induced liver damage.

[0113] Example 2 (Immune response gene 1 / itaconate protects mice from lipopolysaccharide-induced liver damage by downregulating autophagic flux):

[0114] 2. Materials and Methods

[0115] Materials

[0116] 2.1.1. Animals

[0117] Same as Example 1.

[0118] Reagents

[0119]

[0120]

[0121] The remaining reagents are the same as in Example 1.

[0122] 2.1.3. Instruments

[0123]

[0124] The remaining instruments are the same as in Example 1.

[0125] 2.1.4. Preparation of other reagents

[0126] 1.1× TBST buffer (1 L)

[0127]

[0128] 2.1× electrophoresis buffer (1 L)

[0129]

[0130]

[0131] 3.1× transfer buffer (1 L)

[0132]

[0133] Methods

[0134] 2.2.1. Animal grouping

[0135] Same as 1.2.1 in Example 1.

[0136] 2.2.2. Animal sacrifice and sample collection

[0137] Same as 1.2.2 in Example 1.

[0138] 2.2.3.Western blotting

[0139] 2.2.3.1. Preparation of total protein

[0140] Same as 1.2.5.1 in Example 1.

[0141] Determination of protein concentration

[0142] Same as 1.2.5.2 in Example 1.

[0143] Protein denaturation

[0144] Aliquot the extracted liver tissue homogenate into 10 μl aliquots per tube. Add 2.5 μl of 5× SDS protein loading buffer and vortex to mix thoroughly, achieving a 4:1 protein to loading buffer ratio. Boil in a water bath for 10 minutes, then remove and cool to room temperature. Reserve a portion of the protein for the next experiment and store the remainder at -80°C.

[0145] 2.2.3.4. Glue making

[0146] Prepare 1.15% separating gel: Follow the instructions of the 15% separating gel preparation kit to add 5.4 ml of lower gel solution, 5.4 ml of lower gel buffer, and 120 μl of modified ammonium persulfate to an EP tube. Mix thoroughly by pipetting, minimizing the creation of bubbles. Immediately inject the mixture between two glass separating gel plates. Finally, press the gel with anhydrous ethanol. Allow to solidify on a horizontal surface for approximately 30 minutes. Pour off the anhydrous ethanol and absorb any excess liquid with filter paper.

[0147] 2.1 Preparation of 2.5% Resolving Gel: Following the instructions of the 12.5% ​​separating gel preparation kit, add 5.4 ml of lower gel solution, 5.4 ml of lower gel buffer, and 120 μl of modified ammonium persulfate to an EP tube. Mix thoroughly by pipetting, minimizing the creation of bubbles. Immediately inject the mixture between two glass separating gel plates. Finally, press the gel with anhydrous ethanol. Allow to solidify on a horizontal surface for approximately 30 minutes. Pour off the anhydrous ethanol and absorb any excess liquid with filter paper.

[0148] 3. Prepare 7.5% separating gel: Follow the instructions of the 7.5% separating gel preparation kit to add 5.4 ml of lower gel solution, 5.4 ml of lower gel buffer, and 120 μl of modified ammonium persulfate to an EP tube. Mix thoroughly by pipetting, minimizing the creation of bubbles. Immediately inject the mixture between two glass separating gel plates. Finally, press the gel with anhydrous ethanol. Allow to solidify on a horizontal surface for approximately 30 minutes. Pour off the anhydrous ethanol and absorb any excess liquid with filter paper.

[0149] 4. Prepare the stacking gel: Follow the instructions in the gel preparation kit to add 1.5 ml of upper gel solution, 1.5 ml of upper gel buffer, and 30 μl of modified ammonium persulfate to an EP tube. Mix thoroughly by pipetting, avoiding bubbles. Immediately after mixing, inject the solution between two glass gel preparation plates, above the solidified separating gel. Insert a well comb and allow to solidify on a horizontal surface for approximately 15 minutes.

[0150] 2.2.3.5. Electrophoresis

[0151] 1. Place the prepared electrophoresis gel on the electrophoresis holder and secure it. Pour 1x electrophoresis buffer into the designated position of the tank. Ensure there is no obvious leakage.

[0152] 2. Gently pull out the comb hole, add markers to the holes at both ends as marks, and add the denatured protein samples to the remaining holes in sequence according to the plan to identify the order and direction.

[0153] 3. Close the cylinder cover, turn on the power supply, and adjust the electrophoresis voltage to 150V for 60 minutes. During this period, pay attention to the straightness of the bromophenol blue line. Adjust the electrophoresis clamp and voltage if necessary to prevent obvious distortion. Stop electrophoresis when the bromophenol blue line approaches the bottom.

[0154] 2.2.3.6. Transfer

[0155] 1. Soak the electroporation clip, sponge, and filter paper in 1× transfer buffer in advance.

[0156] 2. Soak the PVDF membrane in methanol for 1-2 minutes in advance.

[0157] 3. Pry open the electrophoresis glass plate, remove the concentrated gel and place the separation gel onto the filter paper in the transfer solution.

[0158] 4. Place the sandwich clip into the transfer chuck marked with positive and negative electrodes. Place the chuck in the following order: black sponge → filter paper → PVDF membrane → adhesive strip → filter paper → black sponge. The transparent side of the chuck faces the positive (red) electrode of the electrotransfer tank, and the black side faces the negative (black) electrode of the electrotransfer tank. Pour 1x transfer buffer into the designated area of ​​the tank.

[0159] 5. Cover the cylinder lid, place the electric cylinder on ice, turn on the power, 100v, 100min.

[0160] 2.2.3.7. Closure

[0161] Prepare 5% skim milk in advance. After transfer, immerse the PVDF membrane in the milk and slowly shake it on a shaker at room temperature for 2 hours to block it.

[0162] 2.2.3.8. Incubation with primary antibody

[0163] Cut out the bands corresponding to the internal control and target proteins based on the molecular weight of the protein indicated by the marker. Use primary antibody diluent to dilute β-actin, p62, and LC3A / B 5000-fold, 2000-fold, and 1000-fold, respectively, for later use. Immerse the bands in each dilution and incubate overnight at 4°C on a shaker.

[0164] 2.2.3.9. Incubation with secondary antibody

[0165] 1. Wash the membrane: Immerse the above strips in 1×TBST and shake gently on a shaker at room temperature for 5 minutes to wash away the unbound primary antibody. Then replace with 1×TBST and repeat 5 times.

[0166] 2. Secondary Antibody Reaction: Dilute the goat anti-rabbit IgG secondary antibody 5000-fold in secondary antibody diluent. Immerse the strip in the secondary antibody diluent and gently shake on a shaker at room temperature for 1 hour.

[0167] 2.2.3.10. Development

[0168] 1. Wash the membrane: Immerse the strips in 1× TBST again and wash the membrane 5 times as before.

[0169] 2. Exposure: In a dark environment, prepare the developer solution. Mix Solution A and Solution B from the ECL kit in a 1:1 ratio. Place the strip on the development plate of the gel / luminescence image analysis system. Pipette an appropriate amount of developer solution onto the strip, and photograph the development. Export the data and analyze it using Image J software.

[0170] 2.2.4. Statistical analysis method is the same as 1.2.6 in Example 1.

[0171] 3. Results

[0172] 3.1. 4-OI Supplementation Inhibits LPS-Induced Autophagy in Mouse Liver

[0173] After confirming that 4-OI has a protective effect on lipopolysaccharide-induced liver damage in mice, in order to explore its mechanism of action, the present invention detected its relationship with autophagy in WT mice. Western Blot results showed that the relative expression of LC3II / β-actin in the liver of the LPS model group was higher than that of the normal saline control group. At the same time, the relative expression of P62 / β-actin in the liver of the LPS model group was lower than that of the normal saline control group. This indicates that autophagy in the liver of mice with liver damage established by intraperitoneal injection of lipopolysaccharide was activated and the autophagic flux was upregulated. After 4-OI intervention, the relative expression of LC3 II / β-actin in the liver was significantly downregulated compared with the LPS model group, and the relative expression of P62 / β-actin was upregulated, indicating that 4-OI downregulated autophagy and inhibited autophagy in the liver.

[0174] Irg1 knockout promotes lipopolysaccharide-induced autophagy in mouse liver

[0175] Next, in order to study the regulatory effect of endogenous itaconic acid encoded by the Irg1 gene on lipopolysaccharide-induced mouse liver autophagy, the present invention detected and compared the changes in autophagy-related proteins in the liver of Irg1- / - mice and WT mice. Western Blot results showed that in the model group established by intraperitoneal injection of LPS, the relative expression of LC3II / β-actin in the liver of Irg1- / - mice was increased compared with that of WT mice. In addition, the relative expression of P62 / β-actin in the liver of the Irg1- / - mouse LPS model group was decreased compared with the WT mouse LPS model group. This shows that the deletion of the Irg1 gene has a promoting effect on lipopolysaccharide-induced mouse liver autophagy.

[0176] 4. Discussion

[0177] The results of the first part of the study have revealed that itaconic acid has anti-inflammatory protective effects against lipopolysaccharide-induced liver injury. After reviewing previous research, the present inventors have summarized that itaconic acid's anti-inflammatory process primarily involves the following pathways: 1. Activation of NRF2 / KEAP1, 2. Activation of ATF3, 3. Regulation of type I IFN expression, 4. Inhibition of TET DNA dioxygenase, 5. Inhibition of NLRP3 inflammasome activation, 6. Inhibition of GSDMD activity, 7. Regulation of JAK1-STAT6 pathway activity, 8. Regulation of TFEB and lysosome biogenesis, 9. Regulation of key glycolytic enzymes and inhibition of glycolysis (GAPDH, ALDOA, LDHA), and 10. Inhibition of SDH to reduce ROS production. The present inventors' research group previously discovered that lipopolysaccharide-induced acute lung injury is associated with autophagy, and that itaconic acid can reduce lung injury by promoting autophagy. Others have found that inhibiting autophagy can reduce the inflammatory storm and vascular leakage in sepsis. This led the present inventors to investigate whether itaconic acid could protect mice from lipopolysaccharide-induced liver injury by regulating autophagy.

[0178] Physiological levels of autophagy maintain homogeneous cellular metabolism in response to environmental stimuli, but excessive or insufficient autophagy can cause disease. In sepsis, the liver exhibits the highest levels of autophagy induction, followed by the heart and spleen. Other studies have found increased autophagic activity and increased autophagic vacuoles in hepatocytes of patients who died from sepsis. Some researchers have hypothesized that in sepsis, hepatocytes enhance autophagy to degrade damaged organelles and pathogens, thereby mitigating the progression of organ damage. Consequently, many studies have shown that promoting autophagy can protect the liver. However, some studies suggest that excessive autophagy can lead to uncontrolled inflammation and liver failure. Appropriately inhibiting autophagy and reducing autophagic flux can alleviate inflammation and protect the liver, consistent with the findings of the present study. Elevated LC3-II levels are a key indicator of autophagy. P62, a protein degraded by autophagy and a ubiquitin-binding autophagy receptor, connects the Nrf2 pathway to autophagy and accumulates when autophagy is impaired. The experiments of the present invention found that in WT mice, the level of P62 in the liver decreased under LPS stimulation, while the level of LC3-II increased, indicating that autophagy was activated and autophagic flux increased under LPS stimulation. Supplementation with 4-OI reduced the level of LC3-II and increased the expression of P62, indicating that autophagy was inhibited and the autophagic flux was downregulated. After knocking out Irg1, the level of LC3-II in the liver was higher and the level of P62 was lower under LPS stimulation, which means that the lack of Irg1 will further promote LPS-induced liver autophagy and increase LPS damage to the liver. The results of this study show that itaconic acid has a significant protective effect on the liver in the LPS-induced liver injury disease model. The immune response gene 1 / itaconate can inhibit excessive liver autophagy by downregulating the autophagic flux, thereby protecting the liver. This coincides with the autophagy regulatory role of itaconic acid in autoimmune hepatitis and renal fibrosis disease models studied by others. The present invention speculates that itaconic acid increases the content of P62 by inhibiting excessive autophagy in the liver, thereby promoting the dissociation of Keap1 and Nrf2, and then Nrf2 is transported to the cell nucleus to stimulate downstream anti-inflammatory and antioxidant pathways.

[0179] 4-OI's regulation of autophagy marker expression suggests that it inhibits the initiation of autophagy. Qiyu Zhang et al.'s research suggests that itaconic acid also inhibits the fusion of autophagosomes and lysosomes. Previous studies have found that itaconic acid can also alkylate KEAP1, thereby activating NRF2 and reducing the production of inflammatory factors and ROS. Reduced ROS, in turn, inhibits the initiation of autophagy. Inhibiting autophagy can moderately increase P62 levels, further exerting anti-inflammatory and antioxidant immunomodulatory effects through the P62-KEAP1-NRF2 pathway.

[0180] Comparison of English and Chinese abbreviations and nouns

[0181]

[0182]

[0183] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. The use of itaconic acid in treating septic liver injury, characterized in that: Use of itaconic acid or its derivatives in preparing a drug for treating septic liver injury.

2. The use of itaconic acid in septic liver injury according to claim 1, characterized in that: The itaconic acid derivative is 4-octylitaconic acid (4-OI).

3. The use of itaconic acid in septic liver injury according to claim 1, characterized in that: Its mechanism of action is to downregulate the expression of LC3-II protein in liver tissue and / or upregulate the expression of P62 protein.

4. The use of itaconic acid in septic liver injury according to claim 1, characterized in that: Used to lower serum ALT and AST levels, or inhibit TNF-α, IL-6, and MPO activity in liver tissue.

Images