Application of SLC1A4 as a drug target in the preparation of drugs for the prevention and treatment of hepatic ischemia-reperfusion injury

Abstract

This invention discloses the application of SLC1A4 as a drug target in the preparation of drugs for the prevention and treatment of hepatic ischemia-reperfusion injury, belonging to the field of biomedical technology. The drug is a substance capable of downregulating the expression of SLC1A4 in myeloid cells. This invention is the first to discover a novel function of myeloid SLC1A4 in hepatic ischemia-reperfusion injury, which can serve as a new target for the preparation of drugs to treat hepatic ischemia-reperfusion injury, providing a new target for the clinical prevention and treatment of hepatic ischemia-reperfusion injury and the development of related drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of SLC1A4 as a drug target in the preparation of drugs for the prevention and treatment of liver ischemia-reperfusion injury. Background Technology

[0002] Hepatic ischemia-reperfusion injury (HIRI) is an unavoidable pathophysiological phenomenon during clinical procedures such as liver transplantation, hepatectomy, and traumatic shock. It refers to the situation where, after a period of interruption of blood flow to the liver, the restoration of blood flow not only fails to alleviate the damage but also exacerbates tissue damage and organ dysfunction. HIRI is a significant risk factor for primary graft nonfunction after liver transplantation, acute and chronic rejection, and liver failure after hepatectomy, severely limiting the applicability of hepatectomy and the clinical application of marginal donor livers. Currently, the exact mechanism of HIRI is not fully understood, and effective prevention and treatment strategies and drugs are lacking in clinical practice.

[0003] The pathological mechanisms of hemorrhagic inflammatory resection (HIRI) are complex, mainly involving multiple stages such as energy metabolism disorders, ATP depletion, and ion homeostasis imbalance during ischemia, and oxidative stress bursts, mitochondrial dysfunction, excessive activation of inflammatory responses, and cell death during reperfusion. Macrophages (including hepatic intrinsic macrophages Kupffer cells and infiltrating monocyte-derived macrophages) play a central role in the initiation, progression, and repair of HIRI. Macrophages are highly plastic and can polarize into either the classically activated pro-inflammatory M1 type or the alternatively activated anti-inflammatory M2 type under different microenvironmental stimuli. In the early stages of HIRI, M1 macrophages dominate, releasing large amounts of pro-inflammatory factors (such as TNF-α and IL-1β), exacerbating inflammatory damage; while in the later stages of HIRI, M2 macrophages promote tissue repair and regeneration by secreting anti-inflammatory and repair factors such as IL-10 and TGF-β. Therefore, targeting and regulating macrophage polarization to promote its conversion from M1 to M2 has become an important strategy for treating HIRI. Although some existing small molecule drugs or protein drugs have been shown to regulate macrophage polarization, they still have drawbacks such as narrow effective and safe dose windows, high production costs, and poor tolerability in specific patient groups. At present, there is no ideal clinical treatment option.

[0004] SLC1A4 (member 4 of solute carrier family 1), also known as ASCT1, is a sodium-dependent neutral amino acid transporter that primarily mediates the transmembrane transport of neutral amino acids such as L-serine, alanine, and cysteine. Current research on SLC1A4 mainly focuses on its role in nervous system development and tumorigenesis. For example, loss-of-function mutations in SLC1A4 can lead to severe nervous system developmental disorders; and in various tumors, high expression of SLC1A4 is closely associated with tumor cell proliferation, drug resistance, and poor prognosis. However, the specific role and regulatory mechanisms of SLC1A4 in liver diseases, especially in HIRI (acute aseptic inflammation), are not yet reported domestically or internationally. Summary of the Invention

[0005] To address the technical challenges of existing technologies in preventing and treating liver ischemia-reperfusion injury (LIDI), such as the lack of effective targets, poor targeting, significant side effects, and failure to precisely regulate macrophage polarization, this invention provides the application of SLC1A4 as a drug target in the preparation of drugs for the prevention and treatment of LIDI. This application involves inhibiting the gene expression level and / or protein biological activity of SLC1A4, specifically delivering SLC1A4 inhibitors to myeloid cells via a targeting vector, and regulating macrophage polarization from the pro-inflammatory M1 type to the anti-inflammatory M2 type. This reduces liver inflammation, oxidative stress, and hepatocyte damage, thereby achieving the prevention and treatment of LIDI.

[0006] To achieve the above objectives, the present invention provides the application of SLC1A4 as a drug target in the preparation of a drug for preventing and treating liver ischemia-reperfusion injury. The drug inhibits the gene expression level and / or protein biological activity of SLC1A4, suppresses pro-inflammatory M1 polarization, and promotes anti-inflammatory M2 polarization, thereby reducing liver inflammation, oxidative stress, and hepatocyte damage.

[0007] Furthermore, the drug is a substance capable of downregulating the mRNA expression level and protein expression level of SLC1A4, and / or inhibiting the transport activity and biological function of SLC1A4.

[0008] Furthermore, the substance is selected from one or more of the following: small molecule inhibitors targeting SLC1A4, small interfering RNA, short hairpin RNA, antisense oligonucleotides, monoclonal antibodies, bispecific antibodies, nanobodies, aptamers, and CRISPR / Cas9 gene editing reagents.

[0009] Furthermore, the route of administration of the drug includes any one of oral administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, and local injection into the liver.

[0010] Furthermore, the dosage form of the drug includes either an intestinal-targeted formulation or a macrophage-targeted formulation.

[0011] Furthermore, the intestinal-targeting formulation is selected from any one of colon-coated tablets, colon-coated capsules, intestinal-targeting microspheres, and intestinal-targeting nanoparticles; the macrophage-targeting formulation is selected from any one of macrophage-targeting liposomes, macrophage-targeting polymer nanoparticles, and viral vectors with myeloid-specific promoters.

[0012] Furthermore, a pharmaceutical composition for preventing and treating hepatic ischemia-reperfusion injury is provided, the pharmaceutical composition having an inhibitor of SLC1A4 expression and / or activity as the active ingredient, and further comprising a pharmaceutically acceptable targeting vector capable of delivery to myeloid cells.

[0013] Furthermore, the inhibitor is selected from one or more of the following: small molecule inhibitors targeting SLC1A4, small interfering RNA, short hairpin RNA, antisense oligonucleotides, monoclonal antibodies, bispecific antibodies, nanobodies, aptamers, and CRISPR / Cas9 gene editing reagents; the myeloid cell targeting vector is selected from any one of the following: lipid nanoparticles (LNPs), viral vectors with myeloid-specific promoters, and polymer nanoparticles.

[0014] Furthermore, the dosage form of the pharmaceutical composition includes any one of liposome injection, polymer nanoparticle lyophilized powder injection, exosome injection, and antibody-drug conjugate lyophilized powder injection.

[0015] Furthermore, the route of administration of the pharmaceutical composition includes any one of intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, and local injection into the liver.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: 1. First discovery of a new target: This invention reveals for the first time the function and mechanism of action of SLC1A4 in liver ischemia-reperfusion injury, breaking through the limitations of existing research on SLC1A4 and providing a novel, previously unreported molecular target for the prevention and treatment of HIRI.

[0017] 2. Cell-Specific Targeting Advantage: Using a cell-specific knockout mouse model, the functional cellular origin of SLC1A4 was precisely identified as myeloid macrophages, rather than hepatocytes. This provides an important theoretical basis for the future development of myeloid cell-targeted drug delivery systems, potentially enabling precision treatment and reducing potential side effects on hepatocytes.

[0018] 3. Significant preventive and therapeutic effects: In animal models, myeloid-specific knockout of SLC1A4 can significantly reduce serum transaminase levels, reduce hepatocyte necrosis and apoptosis, and alleviate liver pathological damage, demonstrating strong HIRI prevention and treatment potential.

[0019] 4. Broad application prospects: Based on this target, various forms of drugs can be developed, including: small molecule inhibitors targeting SLC1A4, RNA interference drugs targeting SLC1A4 (small interfering RNA, such as siRNA and shRNA), and targeted formulations that can deliver these inhibitors to macrophages, which have extremely high clinical translational value and commercial development prospects. Attached Figure Description

[0020] Figure 1 This is a diagram showing the results of systemic SLC1A4 knockout in Example 1 of this invention, which reduced liver ischemia-reperfusion injury in mice; (A) H&E staining results of representative mouse liver injury after HIRI modeling, I / R is the abbreviation for ischemia-reperfusion injury, scale bar size is 400 μm; (B) Serum detection of aspartate aminotransferase (AST); (C) Serum detection of alanine aminotransferase (AST); (D) Serum detection of lactate dehydrogenase (LDH). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=4-9.

[0021] Figure 2 This is a graph showing the results of increased SLC1A4 expression levels under in vitro inflammatory environmental stimulation in Example 2 of one embodiment of the present invention; (A) The expression of SLC1A4 in mouse BMDM was determined by Western blot experiment after 24 hours of LPS stimulation; (B) The expression of SLC1A4 in mouse BMDM was determined by Western blot experiment after 0, 6, 24 and 48 hours of LPS stimulation; (C) The relative quantitative analysis of SLC1A4 protein expression level in Figure B.

[0022] Figure 3 This is a diagram showing the results of Example 3 of the present invention, in which the absence of SLC1A4 in hepatocytes had no significant protective effect against liver function damage during the HIRI process; (A) Representative SLC1A4 cells after HIRI modeling. fl / fl and SLC1A4 LKO H&E staining results of mouse liver injury, scale bar size 400 μm; (B) Serum detection of aspartate aminotransferase (AST); (C) Serum detection of alanine aminotransferase (AST); (D) Detection of SLC1A4 by ELISA. fl / fl and SLC1A4 LKOSerum TNF-α levels in mice; (E) SLC1A4 levels were detected by ELISA. fl / fl and SLC1A4 LKO Serum levels of the inflammatory factor IL-1β in mice; (F) Serum detection of lactate dehydrogenase (LDH). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 4 This is a diagram showing the HIRI results improved by myeloid-specific SLC1A4 deficiency rather than hepatocyte deficiency in Example 3 of one embodiment of the present invention; (A) Representative SLC1A4 after HIRI modeling. fl / fl and SLC1A4 MKO (A) H&E staining results of mouse liver injury, scale bar size 400 μm; (B) Serum detection of aspartate aminotransferase (AST); (C) Serum detection of alanine aminotransferase (AST); (D) Detection of SLC1A4 by ELISA. fl / fl and SLC1A4 MKO Serum TNF-α levels in mice; (E) SLC1A4 levels were detected by ELISA. fl / fl and SLC1A4 MKO (F) Serum IL-1β levels in mice; (G) Relative MDA content in mouse liver tissue after HIRI; (H) Relative SOD content in mouse liver tissue after HIRI; (F) DHE staining of liver tissue sections after HIRI modeling, with red fluorescence indicating DHE probe labeled at 610 nm and blue fluorescence indicating cell nuclei stained with DAPI fluorescent dye labeled at 360 nm. Scale bar size: 150 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 5 This is a diagram showing the results of Example 4 of this invention, where myeloid SLC1A4 knockout inhibits M1-type macrophage polarization and promotes M2-type polarization; (AD) RT-qPCR detection of iNOS, TGF-β, IL-10, and CD206 gene expression in HIRI mouse liver tissue; (E) Immunofluorescence staining of liver tissue sections after HIRI modeling to detect CD11B, F4-80, CD86, and CD206, with a scale bar of 20 μm; (FG) Relative quantitative analysis of CD86 and CD206 in Figure E. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0025] Figure 6This is a diagram showing the protective effect of tail vein injection of the SLC1A4 inhibitor L-(+)-α-phenylglycine on liver ischemia-reperfusion injury in mice according to Example 5 of one embodiment of the present invention; (A) Representative SLC1A4 after HIRI modeling. fl / fl H&E staining results of liver damage in mice injected with SLC1A4 inhibitor, scale bar size 400 μm, L-PG is abbreviation for L-(+)-α-phenylglycine; (B) Serum detection of aspartate aminotransferase (ALT). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0029] Laboratory animals: This experiment used the SLC1A4 conditional knockout tool mouse (SLC1A4). fl / fl Systemic SLC1A4 knockout mice (SLC1A4) KO SLC1A4 myeloid-specific knockout mice (SLC1A4) MKO ) and SLC1A4 hepatocyte-specific knockout mice (SLC1A4 LKO Mice, all of strain C57BL / 6, including SLC1A4. fl / fl Mice and the construction of SLC1A4 KO SLC1A4 MKO The tool mice (EIIA-Cre, Lyz-Cre) were purchased from Cyagen Biosciences (Suzhou, China), USA, and SLC1A4 was constructed.LKO The tool mouse (Alb-Cre) was purchased from Beijing Weishang Lide Biotechnology Co., Ltd. It was used with the LoxP system's SLC1A4... fl / fl Mice were crossbred with various tool mice to produce the laboratory mice used in this research institute. SLC1A4 fl / fl Mice were used as a control group, and SLC1A4 was used. KO SLC1A4 MKO SLC1A4 LKO All mice used in the experiments were male mice aged 6-8 weeks.

[0030] Experimental instruments and reagents: Water bath (Shanghai ZHICHENG Instruments Co., Ltd.), Nano Drop micro spectrophotometer (Thermo Fisher Scientific, USA), inverted fluorescence microscope (Olympus Life Sciences, Japan), high-throughput tissue homogenizer (Ningbo Xinzhi Technology Co., Ltd.), ambient temperature centrifuge (Eppendorf, Germany), multifunctional temperature-controlled microplate reader (Shanghai Meigu Molecular Instruments Co., Ltd.), paraffin tissue embedding machine (Thermo Fisher Scientific, USA), CO2 incubator (Thermo Fisher Scientific, USA), three-gas oxygen-controlled temperature-controlled incubator (Jiangsu Xinchunlan Instruments Co., Ltd.), cryostat (Eppendorf AG, Germany), live cell workstation (Thermo Fisher Scientific, USA), trinocular electron biological microscope (Leica Instruments GmbH, Germany), upright fluorescence microscope (Olympus Life Sciences, Japan), etc.

[0031] H&E staining solution (hematoxylin / eosin) (Nanchang Yulu Co., Ltd. / 571-28), DHE dye (MedChemExpress / PD-MY003), Malondialdehyde (MDA) detection kit (Beijing Solarbio Science & Technology Co., Ltd. / BC6415), Trizol total RNA extraction reagent (Shanghai Yisheng Biotechnology Co., Ltd. / 19202), BeyoRT cDNA synthesis kit (Shanghai Beyotime Biotechnology Co., Ltd. / D7170), rapid protein quantification kit (BCA method) (Beijing Solarbio Science & Technology Co., Ltd. / PC0020), mouse tumor necrosis factor α (TNF-α) ELISA kit (Xiamen Lunchangshuo Biotechnology Co., Ltd.), mouse interleukin 1β (IL-1β) ELISA kit (Xiamen Lunchangshuo Biotechnology Co., Ltd.), four-label five-color multiplex immunofluorescence staining kit (Hunan Aifang Biotechnology Co., Ltd. / AFIHC025), L-(+)-α-phenylglycine (Merck Group, Germany / 237647), etc.

[0032] Example 1: Systemic knockout of SLC1A4 reduces HIRI in mice 1. Gene knockout mouse construction: SLC1A4 systemic knockout mice (SLC1A4) KO () is a conditional knockout mouse using SLC1A4 (SLC1A4) fl / fl It was obtained by mating with Cre (EIIA-Cre) that expresses Cre throughout the body.

[0033] 2. HIRI model construction: 6-8 week old male SLC1A4 samples were used. fl / fl SLC1A4 KO A 70% hepatic ischemia-reperfusion model was established. Mice were anesthetized and underwent laparotomy. The portal vein and hepatic artery branches in the left and middle lobes of the liver were clamped using non-invasive vascular clamps. Blood flow was restored after 60 minutes of ischemia. In the sham-operated group (Sham mice), only the laparostomy was performed to separate the blood vessels without clamping. Mice were sacrificed 12 hours after reperfusion, and serum and liver tissue samples were collected.

[0034] 3. Liver tissues from each group of mice were stained with Hematoxylin and eosin (H&E) to analyze their pathological morphology. Next, serum liver function indicators AST and ALT were measured. The steps for H&E staining and liver function indicator detection are as follows: (1) The experimental methods for paraffin-embedded sections and H&E staining are as follows: 1) Dehydration, embedding, and sectioning of liver tissue ① The mice were sacrificed, the liver tissue was removed, a 2cm×2cm tissue block was cut off, and it was fixed in 4% PFA (paraformaldehyde) overnight; ② Remove the liver from the paraformaldehyde and wash it in 1XPBS; ③ Remove the liver from the PBS, dry it, and place it in a tissue embedding cassette, marking it with a pencil; ④ Rinse the embedding cassette in clean water for 15 minutes; ⑤ Place the tissue into an automatic dehydrator and set the program as follows: 70% ethanol for 15 min; 80% ethanol for 15 min; 95% ethanol for 30 min; 95% ethanol for 30 min; 100% ethanol for 30 min; 100% ethanol for 20 min; ½ ethanol + ½ xylene for 10 min; xylene for 3 min; xylene for 3 min; ½ xylene + ½ paraffin for 30 min; paraffin for 90 min; paraffin for 90 min; ⑥ Embed the tissue blocks in a paraffin embedding machine, and store them at 4°C after the paraffin has cooled. ⑦ Slice the liver tissue (4μm) and store it at room temperature.

[0035] 2) Hematoxylin-eosin (H&E) staining: ①Immerse paraffin slices in xylene for 10 minutes twice; ② 100% ethanol for 2 minutes, twice; ③ 1 min each of 95% ethanol, 90% ethanol, 85% ethanol, 75% ethanol, and 50% ethanol; ④ Dry thoroughly, immerse in hematoxylin for 5 minutes, then rinse with tap water for 1 minute; ⑤ Differentiate hydrochloric acid with ethanol for 5 seconds, then rinse under tap water for 1 minute; ⑥ Eosin 1s, rinse under tap water for 1min; ⑦ 95% ethanol for 1 min; ⑧ 100% ethanol for 1 minute, twice; ⑨ Xylene, 2 min, twice; ⑩ Mount the slide with neutral resin and observe it under a microscope.

[0036] (2) Detection of liver function indicators ALT, AST and LDH ① Anesthetize mice with isoflurane. Gently stabilize the mouse's trunk with your left thumb and forefinger, and use your other hand to slightly lift the mouse's head. Using sterile surgical scissors, trim the mouse's whiskers, remove the eyeballs, and collect blood. Ensure the blood drips directly into the tube, avoiding contact with the mouse's fur (to prevent contamination). After blood collection, refrigerate at 4°C until coagulation occurs. Let it stand for 12 hours. Once clear separation of red blood cells from the upper layer of clear serum is observed, centrifuge at 4°C (12000 rpm, 15 min) to separate the serum.

[0037] ② Take the upper layer of serum into an EP tube, freeze it with liquid nitrogen, store it on dry ice, and send it to Celler Company. Analyze the indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), using a biochemical analyzer and analyze the data.

[0038] ③ The serum LDH detection procedure is as follows: Mouse serum is diluted 10 times with physiological saline and placed on ice for testing. Following the LDH detection kit instructions, matrix buffer, accelerator, etc. are added sequentially, mixed well, and incubated. The absorbance at 440 nm is then measured using an ELISA reader.

[0039] The results are as follows Figure 1 As shown: Compared to the control group, SLC1A4 KO -The area of ​​liver tissue necrosis in mice in the HIRI group was significantly improved. Figure 1 (A), and serum ALT, AST, LDH ( Figure 1 The levels of B, C, and D were significantly reduced, indicating that systemic knockout of SLC1A4 can effectively improve liver tissue damage and liver function impairment caused by ischemia-reperfusion injury in mice.

[0040] Example 2: Increased SLC1A4 expression levels due to in vitro inflammatory environment stimulation Constructing an in vitro stimulation model: Extracting SLC1A4fl / fl Primary mouse bone marrow macrophages (BMDM) were treated with 1 μg / mL LPS for 0, 6, 12, 24, and 48 hours, respectively.

[0041] Expression detection: Western blotting was used to detect the protein expression level of SLC1A4 in BMDM.

[0042] The results are as follows Figure 2 As shown: SLC1A4 protein expression level increased under LPS treatment ( Figure 2 (A), and exhibits time dependence ( Figure 2 (Figures B and C) indicate that SLC1A4 expression is regulated by the inflammatory microenvironment and may play a role in the inflammatory response of macrophages.

[0043] Example 3: Myeloid-specific SLC1A4 deficiency, rather than hepatocyte deficiency, improves HIRI 1. Construction of gene knockout mice: SLC1A4 hepatocyte-specific knockout mice (SLC1A4) LKO (This is SLC1A4) fl / fl SLC1A4 myeloid-specific knockout mice (SLC1A4) were obtained by mating with hepatocyte-specific Cre (Alb-Cre). MKO (This is SLC1A4) fl / fl Obtained by mating with myeloid cell-specific Cre (Lyz-Cre).

[0044] 2. HIRI model construction: 6-8 week old male SLC1A4 samples were used. fl / fl SLC1A4 MKO SLC1A4 LKO Mice were used to establish a HIRI model according to the method in Example 1.

[0045] 3. Liver tissues from each group of mice were stained with Hematoxylin and eosin (H&E) to analyze their pathological morphology. Next, serum liver function indicators AST, ALT, and LDH were measured. Finally, serum levels of relevant inflammatory factors (TNF-α and IL-1β) were detected using ELISA. H&E staining and liver function indicator detection were performed according to Example 2, with the ELISA detection steps as follows: Equilibrate the kit to room temperature and prepare the standards and washing buffer. Add the standards and serum samples to be tested to the ELISA plate, seal the plate, and incubate at room temperature for 2 hours. Shake off the liquid, wash 5 times, add biotin antibody working solution, and incubate at room temperature for 1 hour. Wash again, add HRP enzyme working solution, and incubate at room temperature for 20 minutes. Wash 7 times, add chromogenic solution, incubate in the dark for 20 minutes, and finally add stop solution. Measure the OD value at 450 nm and calculate the sample concentration according to the standard curve.

[0046] The results are as follows Figures 3-4 As shown: In SLC1A4 liver knockout mice, the area of ​​liver necrosis was not significantly improved ( Figure 3 (A), and the liver function-related indicators of mice, ALT and AST ( Figure 3 In groups B and C, there were no significant differences in serum inflammatory TNFα and IL-1β levels. Although serum LDH levels decreased significantly, there was no statistically significant difference. Figure 3 (D, E, F) This indicates that SLC1A4 knockout in hepatocytes has no effect on hepatic ischemia-reperfusion injury. However, in SLC1A4 myeloid-specific knockout mice, the area of ​​liver necrosis was significantly improved compared to the control group. Figure 4 In the middle A, liver function-related indicators ALT and AST decreased significantly ( Figure 4 In addition to B and C), the expression of inflammatory factors in serum was also significantly improved. Figure 4 D, E).

[0047] 4. Determination of MDA content in mouse liver tissue: Weigh 0.05g of sample, add 500μL of extraction buffer, homogenize in an ice bath, then centrifuge at 8000g for 10min at 4℃. Collect the supernatant to prepare a sample homogenate. The homogenate needs to be used for BCA protein quantification to assist in quantitative analysis. Prepare the test tubes and blank tubes according to the kit instructions, mix well, incubate in a 100℃ metal bath for 1h, cool, centrifuge, collect the supernatant, and measure the absorbance at 532nm and 600nm.

[0048] The results showed that in SLC1A4 myeloid-specific knockout mice, the content of malondialdehyde, a lipid peroxidation product, was significantly decreased compared to the control group. Figure 4 (Middle F).

[0049] 5. SOD assay in mouse liver tissue: Weigh 0.05g of sample, add 450μL of physiological saline, homogenize on ice, centrifuge at 4000g for 10min at 4℃, and collect the supernatant to prepare a 10% sample homogenate. Dilute the 10% sample homogenate 100-fold with PBS and place on ice for testing. Set up the assay wells and control wells according to the SOD kit instructions, gently shake the plate to mix, incubate at 37℃ for 20min, and read the value at 450nm using a microplate reader.

[0050] The results showed that SOD activity was significantly decreased in SLC1A4 myeloid-specific knockout mice compared to the control group. Figure 4 (G).

[0051] 6. DHE fluorescent probe staining: (1) Frozen embedding sections: ① The mice were sacrificed, the liver tissue was removed, a 2cm×2cm tissue block was cut off, and it was fixed in 4% PFA (paraformaldehyde) overnight; ② Remove the liver from the paraformaldehyde and wash it in 1XPBS; ③ The tissues were sequentially dehydrated in 15% and 30% sucrose solutions; ④ After settling to the bottom, remove the sample and embed it with OCT embedding solution. After embedding, place it in a -80℃ freezer. ⑤ The liver tissue was sectioned (4μm) and stored at -20℃.

[0052] (2) DHE staining: ① Remove frozen sections from -20℃ and thaw at room temperature for 1 hour, then wash with PBS for 5 minutes twice; ②Permeabilize with 0.1% Triton for 10 min, wash with PBS for 5 min, repeat 3 times; ③ Draw circles on tissue, add 50 μm DHE working solution to each tissue, incubate at 37°C in the dark for 30 min, wash with PBS for 5 min, twice; ④ Mount the slide with DAPI and observe it under a fluorescence microscope.

[0053] The results showed that in SLC1A4 myeloid-specific knockout mice, the production of superoxide anions in the liver tissue was significantly inhibited compared to the control group. Figure 4 (H).

[0054] Example 4: Myeloid SLC1A4 knockout can inhibit macrophage M1 polarization and promote M2 polarization. 1. qPCR detection: RNA was extracted from liver tissue to detect the mRNA expression of M1 (iNOS) and M2 (TGF-B, IL-10, CD206) related genes.

[0055] 2. Immunofluorescence staining: Frozen sections of mouse liver tissue from each group in Example 3 were subjected to immunofluorescence staining with F4 / 80 (macrophage marker), CD11b (macrophage marker), CD86 (M1 marker), and CD206 (M2 marker). The steps for the immunofluorescence staining experiment are as follows: ① After removing the frozen sections from -20℃, allow them to thaw at room temperature for 1 hour. Fix for 20 minutes, wash with PBS for 5 minutes, repeat 3 times, add 0.3% Triton-X100 membrane permeabilization buffer for 20 minutes, wash with PBS for 5 minutes, repeat 3 times.

[0056] ② Blocking endogenous peroxidase: Add 3% hydrogen peroxide solution to the slices, incubate at room temperature in the dark for 15 min, wash with PBS for 5 min, repeat 3 times.

[0057] ③ Non-specific target blocking: After the section is dried, draw a circle around the tissue with a histochemical pen, add goat serum to the section, and block at room temperature for 30 minutes.

[0058] ④ Add primary antibody: Discard the blocking solution, add primary antibody, and place the slide in a dark box for overnight incubation at 4°C.

[0059] ⑤ Add HRP secondary antibody: Wash sections with PBS for 5 min, 3 times. Add secondary antibody and incubate at room temperature in the dark for 30 min, then wash with PBS for 5 min, 3 times.

[0060] ⑥TSA fluorescent dye reaction solution: Add fluorescent dye dropwise and incubate at room temperature for 8 min, then wash with PBS for 5 min, 3 times.

[0061] ⑦ Antibody elution: Add antibody elution buffer and incubate at 37°C for 20 min, then wash with PBS for 5 min, repeat 3 times.

[0062] ⑧ Repeat steps ②-⑦ (using a different fluorescent dye) to label with the second antibody.

[0063] ⑨ Repeat steps ②-⑦ (using a different fluorescent dye) to label with the third antibody.

[0064] ⑩ Repeat steps ②-⑦ (using a different fluorescent dye) to label with the fourth antibody.

[0065] Counterstaining cell nuclei with DAPI: Wash with PBS for 5 min, 3 times. After slightly drying the sections, add DAPI mounting medium and incubate at room temperature for 5 min.

[0066] Observe and photograph under a microscope.

[0067] The results are as follows Figure 5 As shown: Markers of macrophage M1 and M2 were detected in liver tissue after modeling using qPCR, including SLC1A4. MKO The expression level of the mouse M1 marker iNOS was significantly suppressed. Figure 5 In the middle A), the expression levels of M2 markers TGF-B, IL-10, and CD206 were significantly upregulated ( Figure 5 (B, C, D). Further immunofluorescence detection of macrophage M1 and M2 markers ( Figure 5 (E), SLC1A4 after modeling MKO More prone to polarization into M2 macrophages labeled with CD206 ( Figure 5 The control group, on the other hand, tended to polarize into M1 macrophages labeled with CD86. Figure 5 (Middle F).

[0068] Example 5: Protective effect of tail vein injection of SLC1A4 inhibitor L-(+)-α-phenylglycine on hepatic ischemia-reperfusion injury in mice. 1. Drug administration and model establishment: Male SLC1A4 cells aged 6-8 weeks were used. fl / fl L-(+)-α-phenylglycine or an equal volume of physiological saline was administered via tail vein injection at doses of 10 mg / kg and 50 mg / kg, respectively; HIRI model was established 12 h after administration, following the method in Example 1.

[0069] 2. The liver tissues of mice in each group were stained with H&E according to the method in Example 1 to analyze the pathological morphology of the liver tissues; then, the liver function index ALT in the mouse serum was detected.

[0070] The results are as follows Figure 6 As shown: Compared with the control group, the area of ​​liver tissue necrosis in mice in the 50 mg / kg inhibitor model group was significantly improved. Figure 6 In the middle A group, the pathological damage was significantly milder than that in the low-dose group, and serum ALT ( Figure 6 The level of B in the middle decreased.

[0071] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. Application of L-(+)-α-phenylglycine in the preparation of drugs for the prevention of hepatic ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that, The drug can be administered via any one of the following routes: oral administration, intravenous injection, or local injection into the liver.

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