Use of promoting expression of slc2a1 in treatment of acute liver injury

Abstract

The application discloses application of SLC2A1 in treatment of paracetamol-induced acute liver injury, and the drug comprises a substance for up-regulating Slc2a1 gene or enhancing SLC2A1 protein level. SLC2A1 expression plays a key role in the disease progression of acute liver injury, and a small molecule compound AS1949490 can increase the level of SLC2A1, thereby promoting the expression of liver macrophage efferocytosis receptors, so as to activate efferocytosis. In addition, AS1949490 can also significantly reduce the increase of ALT and AST contents of the liver caused by paracetamol-induced acute liver injury, and reduce the liver injury area.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of promoting SLC2A1 expression in the treatment of acetaminophen-induced acute liver injury. Background Technology

[0002] Acetaminophen (APAP), a commonly used antipyretic and analgesic, is also one of the most common hepatotoxic drugs causing acute liver injury in clinical practice. Blindly overdosing without medical advice can increase the risk of liver failure and even death. Reports indicate that APAP-induced acute liver injury (AILI) accounts for up to 39% of acute liver failure cases. Given the high risk and serious harm of AILI, it has become a pressing clinical problem. However, currently, there are relatively few specific drugs available clinically, with only N-acetylcysteine ​​(NAC) used, but its therapeutic window is narrow. Furthermore, liver-protective drugs such as hepatoprotective enzyme-lowering drugs like Shuganning injection and reduced glutathione can temporarily reduce transaminase levels and improve symptoms, but they have no fundamental effect on liver treatment and repair. Severely ill patients may require expensive and lengthy treatments such as hemodialysis and liver transplantation. Therefore, there is an urgent need to research and develop new targets and intervention strategies for AILI to advance its clinical treatment.

[0003] AILI is mostly acute and its pathogenesis is relatively complex, mainly involving multiple mechanisms such as immune cell regulation, autophagy, oxidative stress, and inflammatory response. Increasing research shows that macrophages in the liver play an important role in hepatic immune regulation, primarily through the presence and recruitment of hepatic-resident macrophages (Kupffer cells). Kupffer cells have been reported to possess phagocytic activity, effectively clearing dead cells from the liver, preventing further necrosis, and thus promoting liver repair and mitigating liver injury (LI W, YANG Y, YANG L, et al. Monocyte-derived Kupffer cells dominate in the Kupffer cell pool during liver injury. Cell Reports, 2023, 42(10).). The process by which macrophages and other phagocytic cells clear programmed cell death is also known as cytotoxicity, a key process initiating the inflammatory repair phase. Furthermore, reports indicate that cytotoxicity plays an important role in maintaining liver homeostasis and promoting tissue repair, but the specific role and mechanism of cytotoxicity in AILI remain unclear. Therefore, further exploration of the molecular mechanisms of macrophage burial is of great clinical significance for advancing the treatment of AILI.

[0004] SLC2A1, also known as glucose transporter-1 (GLUT1), is responsible for glucose uptake and is widely distributed in the body. Studies have shown that SLC2A1 is upregulated in macrophages in response to inflammatory stimuli (CORNWELL A, ZIóŁKOWSKI H, BADIEI A. Glucose Transporter Glut1-Dependent Metabolic Reprogramming Regulates Lipopolysaccharide-Induced Inflammation in RAW264. 7 Macrophages. Biomolecules, 2023, 13(5): 770.). However, it remains unclear whether SLC2A1 in the liver regulates the Kupffer cell burial process and its role in AILI. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing or screening drugs that promote SLC2A1 expression to treat AILI, and provides the application of the small molecule compound AS1949490 as an SLC2A1 expression promoter in the treatment of acetaminophen-induced acute liver injury.

[0006] Application of promoting SLC2A1 expression in the preparation of a drug for treating AILI, said drug comprising upregulating SLC2A1 expression. Slc2a1 Genes or substances that enhance SLC2A1 protein levels.

[0007] This invention has found that promoting SLC2A1 expression plays a crucial role in the progression of acute liver injury. Kupffer-specific knockout... Slc2a1 Acetaminophen accelerates the progression of acute liver injury induced by acetaminophen, mainly manifested by elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and an increased area of ​​liver damage.

[0008] The above studies indicate that SLC2A1 expression has a negative regulatory effect on the progression of AILI, and drugs that increase SLC2A1 expression or enhance its activity can be used to treat acetaminophen-induced acute liver injury.

[0009] In this invention, the aforementioned Slc2a1 The gene has a Gene ID of 20525 on PubMed, and the corresponding amino acid sequence of the SLC2A1 protein is numbered P17809 on PubMed.

[0010] More preferably, the acute liver injury is characterized by elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the liver, and an increased area of ​​liver damage.

[0011] More preferably, the drug is a drug that reduces liver function indicators or reduces the area of ​​liver damage.

[0012] Preferably, the drug that enhances SLC2A1 protein levels is the small molecule compound AS1949490.

[0013] In this invention, the structure of the small molecule compound AS1949490 is as follows: .

[0014] More preferably, the small molecule compound AS1949490 increases the level of SLC2A1, thereby promoting the expression of the burial receptor on liver macrophages and activating the burial function.

[0015] Preferably, the small molecule compound AS1949490 reduces the ALT and AST levels in the liver, thereby reducing the area of ​​liver damage.

[0016] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0017] Preferably, the dosage form of the drug is any one of tablets, granules, solutions, emulsions, suspensions, and capsules.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a method for preparing or screening drugs to treat acute liver injury (AILI) by promoting SLC2A1 expression, and provides the application of the small molecule compound AS1949490 as an SLC2A1 expression promoter in the treatment of acetaminophen-induced acute liver injury. The small molecule compound AS1949490 can increase the level of SLC2A1, thereby promoting the expression of the burial receptor on hepatic macrophages and activating burial function. In addition, AS1949490 can significantly reduce the increase in ALT and AST levels in the liver caused by acetaminophen-induced acute liver injury, and reduce the area of ​​liver damage. Attached Figure Description

[0019] Figure 1 The images show liver pathological images of mice in the wild-type group, knockout group, wild-type model group, and knockout model group. In the image, A is a liver pathological staining image, and B is a quantitative image of the liver damage area in A.

[0020] Figure 2The graphs show the statistical analysis of serum liver function indicators in wild-type, knockout, wild-type model, and knockout model mice. A and B are the statistical graphs of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), respectively.

[0021] Figure 3 This is a statistical graph showing the level of Kupffer cell burial receptor MerTK positive cells in wild-type, knockout, wild-type model, and knockout model mice. A is the flow cytometry result of the proportion of MerTK positive cells, and B is the quantitative graph of A.

[0022] Figure 4 The image shows the Western blotting (WB) plot of the expression level of SLC2A1 by the small molecule compound AS1949490, where A is the WB image and B is the quantitative statistical plot of A.

[0023] Figure 5 The images show liver pathological images of mice in the normal control group, the drug-treated group, the APAP model group, and the APAP-treated plus model group. In the image, A is a liver pathological staining image, and B is a quantitative image of the liver damage area in A.

[0024] Figure 6 The graphs show the statistical analysis of serum liver function indicators in mice from the normal control group, the drug-treated group, the APAP model group, and the APAP-treated plus model group. In the graphs, A and B are the statistical analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), respectively.

[0025] Figure 7 The graph shows the statistical level of Kupffer cell burial receptor MerTK positive cells in mice in the APAP modeling group, APAP modeling group and drug administration group. A is the flow cytometry result of the proportion of MerTK positive cells, and B is the quantitative graph of A. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0028] Example 1: Slc2a1 Knockout aggravates acetaminophen-induced acute liver injury in mice Kupffer cell-specific recombinase system was constructed using the Loxp-Cre recombinase system. Slc2a1 Knockout Slc2a1 flox / flox Clec4f-Cre ( Slc2a1 ΔClec4fAILI model was established using wild-type mice (purchased from Jackson Laboratory) and littermate control WT mice (6-8 weeks old, male C57 / BL6, purchased from Zhejiang Vital River Co., Ltd.). The two types of mice were randomly divided into two groups of 6 mice each. The four groups were wild-type group, knockout group, wild-type model group, and knockout model group.

[0029] The AILI model was established by intraperitoneal injection of 350 mg / kg APAP (purchased from Aladdin, catalog number A305928) into the model group animals. Twenty-four hours after APAP treatment, the mice were sacrificed and blood and liver samples were collected. The collected blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 4 ℃ and 2000 g for 15 minutes. The supernatant was transferred to new EP tubes, and liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Simultaneously, liver tissue samples were collected from each group of mice, fixed in 4% formalin solution for 24 hours, and then rinsed with tap water to remove excess fixative.

[0030] The tissues were dehydrated sequentially in 70%, 80%, 90%, 95%, and 100% ethanol, 20 minutes each. After dehydration, the tissues were cleared in xylene, embedded in paraffin, and sectioned into 5 μm thick paraffin sections. The paraffin sections were then sequentially immersed in xylene I for 10 minutes, xylene II for 10 minutes, and then sequentially immersed in anhydrous ethanol, 95%, 90%, 80%, and 70% ethanol solutions for 10 minutes each. After washing with distilled water, the sections were stained with hematoxylin for 4 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed again with deionized water for 10 minutes. They were then stained with eosin for 5 minutes and rinsed with deionized water for 10 minutes. The sections were then sequentially dehydrated in ethanol solutions with concentrations of 70%, 80%, 90%, and 95%, 2 minutes each, and finally cleared with xylene. After removing the sections, add an appropriate amount of neutral resin quickly before the xylene dries, and finally seal with a coverslip. Observe the morphological changes of the liver tissue under an upright microscope.

[0031] Figure 1 The images show liver pathological images of mice in the wild-type, knockout, wild-type model, and knockout model groups. A is a liver pathological staining image, and B is a quantitative map of the liver damage area in A. As shown in the figure, the model group mice exhibited large-area liver damage, which was significantly different from the non-model mice. Kupffer cells knocked down... Slc2a1 Subsequently, the area of ​​liver damage in mice further increased.

[0032] Figure 2The figures show statistical graphs of serum liver function parameters in wild-type, knockout, wild-type model, and knockout model mice. A and B represent the statistical graphs of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), respectively. As shown in the figures, compared with the non-model group, the serum ALT and AST levels in the model group mice were significantly increased. Compared with the wild-type model group, the ALT and AST levels in the knockout model group mice were significantly decreased.

[0033] The above results indicate that in Kupffer cells Slc2a1 Knockout significantly exacerbates hepatocellular damage caused by APAP.

[0034] Example 2: Slc2a1 Regulation of macrophage demise Kupffer cell-specific recombinase system was constructed using the Loxp-Cre recombinase system. Slc2a1 Knockout Slc2a1 ΔClec4f AILI model was established using mice and littermate control WT mice (6-8 weeks old, male). Both types of mice were randomly divided into 4 groups, with 3 mice in each group. The 4 groups were wild-type group, knockout group, wild-type model group, and knockout model group.

[0035] The AILI model was established by intraperitoneal injection of 350 mg / kg APAP into the model group animals for 24 hours. DMEM high-glucose primary Kupffer cell culture medium containing 20% ​​Gibco serum and 2% penicillin-dextrose antibody was prepared. Mice were anesthetized with 0.3% sodium pentobarbital, disinfected with 75% ethanol, and placed in a clean bench. After cutting open the mouse abdomen, the inferior vena cava was located, and the syringe needle was carefully inserted into it, taking care not to puncture the blood vessel. 2 mL of PBS was carefully infused until the liver swelled. The portal vein was cut open, and approximately 10 mL of PBS was slowly infused. The mouse liver was then removed and placed in pre-cooled DMEM high-glucose culture medium.

[0036] The extracted liver was cut into small pieces and incubated in PBS containing 0.25% collagenase at 37°C for 30 minutes. The liver was slowly homogenized, and the homogenate was filtered through a 100 μm cell filter membrane. The homogenate was centrifuged at 50 g for 3 minutes at 4°C, and the supernatant was collected. The supernatant was centrifuged at 900 rpm for 7 minutes at 4°C, and the supernatant was discarded, leaving the pellet. An appropriate amount of erythrocyte lysis buffer was added to the pellet, and the mixture was incubated at room temperature for 5 minutes, followed by the addition of 10 mL of PBS to stop the incubation. The mixture was centrifuged at 900 rpm for 7 minutes at 4°C, and the supernatant was discarded, leaving the pellet. The pellet was blocked in 3% BSA for 45 minutes, then centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. A staining system was prepared by resuspending the cells in 0.2% BSA and staining at room temperature for 15 minutes, followed by the addition of PBS to stop the staining. Pacific Antibody was selected. Blue-anti-CD45 (purchased from Biolegend, catalog number 157212), PerCP / Cy5.5-anti-CD11b (purchased from Biolegend, catalog number 101228), APC-anti-F4 / 80 (purchased from Biolegend, catalog number 157306), FITC-anti-MERTK (purchased from Biolegend, catalog number 151503), CD45 detection by flow cytometry. + CD11b Middle F4 / 80 High The proportion of Kupffer cell burial receptor (MerTK) positive cells in the cell.

[0037] Figure 3 This figure shows the statistical levels of Kupffer cell burial receptor (MerTK) positive cells in wild-type, knockout, wild-type model, and knockout model mice. A represents the flow cytometry results of the percentage of MerTK positive cells, and B is the quantitative representation of A. As shown in the figure, compared with the non-model group, the APAP model group showed a significant increase in MerTK expression. In contrast, MerTK expression was significantly decreased in the knockout model group.

[0038] Experimental results suggest Slc2a1 It can mediate the macrophage demise process, thereby regulating liver damage caused by APAP.

[0039] Example 3: SLC2A1 expression promoter regulates hepatic macrophage necrolysis to alleviate AILI (1) AS1949490 can promote the expression level of SLC2A1 protein. Male C57BL / 6 mice, weighing 18–22 g, were randomly divided into two groups of three mice each: a normal control group and a treatment group. Mice were anesthetized with 0.3% sodium pentobarbital, euthanized by cervical dislocation, and thoroughly disinfected by immersion in 75% ethanol. The mice were then placed in a laminar flow hood. After cutting open the abdominal skin, the femurs were removed, and any adhering muscles were carefully removed. The femurs were then cut open at both ends to expose the medullary cavity. Cells were flushed into culture dishes using a 5 mL syringe and gently agitated with a pipette. Cells were seeded into 6-well cells according to the required amount and cultured in a 37 °C cell culture incubator with 5% CO2 for 3 days. On the fourth day, the medium was completely replaced with BMDMs, and the cells were cultured in the incubator until the seventh day. In the treatment group, AS1949490 (purchased from Tao Shu, catalog number T14327) was added to the culture medium at a final concentration of 10 μM and allowed to act for 24 hours. We took a small amount of cells into an EP tube and added 200 μL of RIPA lysis buffer. After grinding the tissue, we let it stand for 30 minutes. After centrifuging at 13500 rpm for 30 minutes, we collected the supernatant into a new EP tube and stored it at -80 °C.

[0040] A concentration gradient of protein standard solutions was prepared to construct a standard curve, along with BCA working solution (solution A:solution B = 50:1). After diluting the protein supernatant, the protein standard solutions and BCA working solution (1:100) were added to the wells of a plate and incubated at 37 °C for 30 minutes. The absorbance was measured at 562 nm to calculate the sample protein concentration. Based on a protein volume of 200 ng, an appropriate amount of protein supernatant was added, followed by 40 μL of 5× Loading buffer, and the volume was brought to a final volume of 200 μL with distilled water. The plate was boiled at 95 °C for 15 minutes and stored at -20 °C.

[0041] Clean the glass plates, ensuring the bottom edges of the long and short glass plates are aligned. Pour the separating gel into the two glass plates, followed by the stacking gel. Insert the comb and wait 20 minutes for the gel to solidify. After solidification, clamp the small glass plates together with the inner side facing inwards. Fill the tank with 1×Running (10×Running diluted with ultrapure water), remove the comb, and thoroughly vortex the protein sample. Add 10 μL of protein sample or 2.5 μL of marker to the lane. Run the protein sample to the separating gel at a constant voltage of 80 V, then adjust the voltage to a constant voltage of 120 V and continue electrophoresis. After the gel run, disconnect the power supply and pre-activate the PVDF membrane by soaking it in anhydrous ethanol. Pry off the small glass plate, gently scrape off the stacking gel, and place the gel in the following order: sponge, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, sponge. Pour in 1×Trans buffer and anhydrous ethanol, and electrophoresis at a constant current of 330 mA for 80 minutes. After transfer, prepare a 5% blocking buffer by dissolving skim milk powder in TPBS. Take 5 mL of the blocking buffer to cover the band and block at room temperature for 60 minutes. Wash three times with TPBS: the first time for 10 minutes, followed by two 5-minute washes. Dilute the primary antibody 1:1000 with TPBS and take 5 mL to cover the band. Incubate overnight at 4°C on a shaker. After primary antibody incubation, wash three times with TPBS: the first time for 15 minutes, followed by two 5-minute washes. Dilute the secondary antibody 1:1000 with blocking buffer and take 5 mL to cover the band. Incubate at room temperature on a shaker for 1 hour. Wash three times with TPBS: the first time for 15 minutes, followed by two 5-minute washes. Prepare the developing solution according to the manufacturer's instructions (A:B = 1:1), and evenly drop it onto the PVDF membrane. Place the membrane in an exposure machine for development.

[0042] Figure 4 The image shows a Western blotting (WB) plot of the effect of the small molecule compound AS1949490 on the expression level of SLC2A1. A represents the WB image, and B is the quantitative statistical plot of A. As shown in the figure, the small molecule compound AS1949490 can promote SLC2A1 protein expression and enhance its expression level.

[0043] (2) AS1949490 can alleviate acute liver injury induced by acetaminophen in mice. Male C57BL / 6 mice, weighing 18–22 g, were randomly divided into four groups of six mice each. The four groups were: normal control group, drug-treated group, APAP modeling group, and APAP-treated plus modeling group. Animals in the APAP modeling group were intraperitoneally injected with 350 mg / kg APAP to establish the AILI model. Animals in the AS1949490-treated group were intraperitoneally injected with 10 mg / kg AS1949490 30 minutes before APAP modeling. Animals were sacrificed 24 hours after APAP treatment, and blood and liver samples were collected.

[0044] Following the method in Example 1, liver tissue stained sections were prepared, and the morphological changes of the liver tissue were observed under an upright microscope.

[0045] Figure 5 The figures show liver pathological images of mice in the normal control group, the drug-treated group, the APAP model group, and the APAP-treated plus model group. In the figure, A is a liver pathological staining image, and B is a quantitative map of the liver damage area in A. As shown in the figure, the liver tissue structure of mice in the normal control group was normal. After the APAP model was established, large areas of liver tissue necrosis appeared. The liver tissue damage area of ​​mice in the AS19494901 drug-treated group was reduced compared to the model group.

[0046] Figure 6 The figures show statistical graphs of serum liver function indicators in mice from the normal control group, the drug-treated group, the APAP model group, and the APAP-treated plus model group. A and B represent the statistical graphs of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), respectively. As shown in the figures, compared with the normal control group, the serum ALT and AST levels in the APAP model group mice were significantly increased. Compared with the model group, AS19494901 significantly reduced APAP-induced ALT and AST levels.

[0047] (3) AS1949490 can regulate the macrophage burial process. Male C57BL / 6 mice, weighing 18–22 g, were randomly divided into two groups of three mice each: an APAP modeling group and an APAP modeling and drug administration group. The APAP modeling group received an intraperitoneal injection of 350 mg / kg APAP to establish the AILI model. The AS1949490 drug administration group received an intraperitoneal injection of 10 mg / kg AS1949490 30 minutes prior to APAP modeling.

[0048] Following the method in Example 2, CD45 was detected using flow cytometry. + CD11b Middle F4 / 80 High The proportion of Kupffer cell burial receptor (MerTK) positive cells in the cell.

[0049] Figure 7 This figure shows the statistical levels of Kupffer cell burial receptor MerTK positive cells in mice in the APAP model group, APAP model group, and drug-treated group. A is the flow cytometry result of the proportion of MerTK positive cells, and B is the quantitative graph of A. As shown in the figure, MerTK expression was significantly increased in the drug-treated group.

[0050] The above results demonstrate that increasing SLC2A1 expression can promote the expression of the liver macrophage burial receptor, activate burial function, and thus alleviate the progression of AILI.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of promoting SLC2A1 expression in the preparation of a drug for treating acetaminophen-induced acute liver injury, characterized in that, The drug contains upregulation Slc2a1 Genes or substances that increase the level of SLC2A1 protein.

2. The application according to claim 1, characterized in that, The acute liver injury described above is characterized by elevated levels of alanine aminotransferase and aspartate aminotransferase in the liver, and an increased area of ​​liver damage.

3. The application according to claim 2, characterized in that, The aforementioned drugs are those that lower liver function indicators or reduce the area of ​​liver damage.

4. The application according to claim 1, characterized in that, The drug that enhances SLC2A1 protein levels is the small molecule compound AS1949490.

5. The application according to claim 4, characterized in that, The small molecule compound AS1949490 increases the level of SLC2A1, thereby promoting the expression of the burial receptor in liver macrophages and activating the burial process.

6. The application according to claim 4, characterized in that, The small molecule compound AS1949490 reduces the levels of alanine aminotransferase and aspartate aminotransferase in the liver, thereby reducing the area of ​​liver damage.

7. The application according to claim 4, characterized in that, The drugs that enhance the function of the SLC2A1 protein also include pharmaceutically acceptable carriers.

8. The application according to claim 4, characterized in that, The dosage form of the drug that enhances the function of SLC2A1 protein is any one of tablets, granules, solutions, emulsions, suspensions, and capsules.

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