Application of bile acid derivatives in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury

Abstract

The present invention proposes the use of a bile acid derivative in the preparation of a drug for preventing and treating liver ischemia-reperfusion injury. The bile acid derivative is glycine-β-muricholic acid, which mediates the inflammation-inhibiting effect in liver ischemia-reperfusion injury through glycine-β-muricholic acid, and confirms its molecular mechanism of inhibiting macrophage pyroptosis, providing a new therapeutic target and strategy for the clinical prevention and alleviation of liver ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of bile acid derivatives in preparing drugs for preventing and treating liver ischemia-reperfusion injury. Background Art

[0002] Orthotopic liver transplantation is a commonly used surgical procedure for treating end-stage liver disease and malignant tumors. Common indications include viral hepatitis cirrhosis, alcoholic liver disease, nonalcoholic steatohepatitis, liver cancer, and other metabolic liver diseases. Hepatic ischemia-reperfusion injury (IRI), a sterile inflammatory response driven by innate immunity, is a significant risk factor in liver transplantation and a major cause of early graft dysfunction and acute or chronic graft rejection, significantly compromising liver transplant outcomes.

[0003] Currently, there are four main approaches to treat reperfusion injury during liver transplantation: 1. Inhibiting reactive oxygen species (ROS) during ischemia and reperfusion; 2. Modulating cytokines; 3. Inhibiting immune activation; and 4. Improving organ preservation. However, the use of ROS scavengers to mitigate reperfusion injury has been clinically ineffective, as ROS-activated immune cells are crucial for repairing damaged tissue. Recent advances in improving organ preservation have included ex vivo perfusion, which can mitigate reperfusion injury to some extent. However, innovative strategies known as ischemia-free transplantation are complex, require expensive center-level adaptation, and remain difficult to popularize in the current setting. Given the crucial role of infiltrating immune cells in injury repair and restoring homeostasis during reperfusion, targeted immune cell modulation—specifically, regulating cytokines and immune function—to mitigate reperfusion injury is a promising therapeutic approach. Pyroptosis, a recently discovered unique mode of macrophage death, leads to rapid lysis and the release of proinflammatory cytokines, and has been shown to play a significant role in reperfusion injury. Pyroptosis activates Caspase1 and GSDMD to produce CASP1-p20 and GSDMD-N. GSDMD-N then creates pores in the cell membrane, leading to cell lytic death and the release of IL-1β and LDH, thereby exacerbating inflammation.

[0004] Bile acids are metabolites of cholesterol and are synthesized in the liver through a complex multi-step process. Recent studies have shown that bile acid-mediated signaling pathways are involved in regulating the progression of diseases such as cholestatic and metabolic liver diseases, and related bile acids have been developed for clinical treatment. Glyco-β-muricholic acid (Gly-β-MCA) is a novel synthetic bile acid that has been shown to prevent and treat insulin resistance and hepatic steatosis when administered orally and is safe and tolerable in animals. However, the regulatory effects of Gly-β-MCA on acute hepatic inflammation have not yet been studied. Summary of the Invention

[0005] The present invention proposes the use of a bile acid derivative glycine-β-muricholic acid in the prevention and treatment of liver ischemia-reperfusion injury. By confirming its molecular mechanism of inhibiting macrophage pyroptosis, it provides a new therapeutic target and strategy for the clinical prevention and alleviation of liver ischemia-reperfusion injury.

[0006] In one aspect, the present invention provides the use of a bile acid derivative in the preparation of a medicament for preventing and / or treating liver ischemia-reperfusion injury; the bile acid derivative is glycine-β-muricholic acid Gly-β-MCA.

[0007] The CAS number of Gly-β-MCA is 66225-78-3. The molecular formula is C 26 H 43 NO6 has the following structural formula:

[0008]

[0009] In the present invention, the liver ischemia-reperfusion injury is liver ischemia-reperfusion injury, which can be caused by various reasons, such as organ transplantation, partial or complete tissue resection, vascular embolism leading to tissue ischemia. The triggering factors of liver ischemia-reperfusion injury and related diseases include, but are not limited to, one or more of liver cysts, liver transplantation, thrombolytic therapy, portal occlusion, and hepatic coma.

[0010] In the above application of the present invention, the drug is used to inhibit the pro-inflammatory macrophage bile acid receptor S1PR2.

[0011] Preferably, the drug includes at least one of the following uses:

[0012] a) Inhibit macrophage pyroptosis;

[0013] b) inhibit the release of the pro-inflammatory cytokine IL-1β;

[0014] c) Reduce lactate dehydrogenase.

[0015] Furthermore, in the above application, the drug is used to reduce the release of alanine aminotransferase and aspartate aminotransferase, and reduce the area of ​​liver tissue necrosis.

[0016] Furthermore, in the above applications, the drug also includes a pharmaceutically acceptable carrier, and / or excipient, and / or diluent. The carrier, excipient, or diluent used is well known to those skilled in the pharmaceutical field and can be appropriately added to the drug administration route or dosage form based on the drug concentration, dosage, and therapeutic efficacy. Preferably, the drug is administered in an injectable carrier for intravenous injection.

[0017] Furthermore, the drug may be in the form of an oral dosage form, such as a tablet, capsule, pill, powder, granule, suspension, syrup, etc.; or in the form of an injectable dosage form, such as an injection solution, powder injection, etc., administered intravenously, intraperitoneally, subcutaneously, or intramuscularly. All dosage forms used are well known to those skilled in the art of pharmacy; the present invention preferably uses an injectable solution for intravenous administration.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention verifies the role of glycine-β-muricholic acid in mediating inflammation inhibition in liver ischemia-reperfusion injury and confirms its molecular mechanism of inhibiting macrophage pyroptosis. It can be used for the prevention and treatment of liver ischemia-reperfusion injury, providing a new therapeutic target and strategy for the clinical prevention and alleviation of liver ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the result of the inhibition of pyroptosis of mouse bone marrow macrophages by Gly-β-MCA in Example 1.

[0022] Figure 2 This is the result of Gly-β-MCA inhibiting pyroptosis of human macrophages in Example 2.

[0023] Figure 3 These are the results of the effect of bile acid receptor S1PR2 regulation on macrophage pyroptosis in Example 3.

[0024] Figure 4 This is the result of Gly-β-MCA alleviating pyroptosis by inhibiting macrophage S1PR2 in Example 4.

[0025] Figure 5 These are the results of the effect of macrophage S1PR2 on macrophage pyroptosis-related pathways and gene expressions in Example 5.

[0026] Figure 6 These are the results of the effect of injection of S1PR2 inhibitor on liver ischemia-reperfusion injury in Example 6.

[0027] Figure 7 These are the results of the effect of inhibiting S1PR2 of bone marrow macrophages on liver reperfusion injury in mice in Example 7.

[0028] Figure 8 This is the first related result of Example 8 in which injection of Gly-β-MCA alleviated liver reperfusion injury in mice.

[0029] Figure 9 This is the second related result of Example 8 on alleviating liver reperfusion injury in mice after injection of Gly-β-MCA. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] In one embodiment, the specific mechanism of action of the bile acid derivative Gly-β-MCA in alleviating liver ischemia-reperfusion injury was explored at the molecular, cellular, and animal levels. It was proposed that the bile acid Gly-βMCA can be injected intravenously into the blood to reduce the release of liver injury markers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after reperfusion in mice, and reduce the area of ​​liver tissue necrosis. It was found that Gly-βMCA inhibits macrophage pyroptosis by inhibiting the bile acid receptor S1PR2 on proinflammatory macrophages, thereby reducing the release of inflammatory factors such as interleukin-1β and lactate dehydrogenase. This indicates that Gly-βMCA can be used as an injectable drug for the treatment of liver ischemia-reperfusion injury, providing a new molecular target and therapeutic means for the clinical prevention or treatment of acute liver inflammation.

[0032] Example 1 Gly-β-MCA inhibits pyroptosis of mouse macrophages

[0033] Experimental groups: Gly-β-MCA: 0, 50, 100, 150, 200 μM (the control group used the same amount of DMSO)

[0034] Mouse primary macrophages treated with macrophage colony stimulating factor were seeded in 12-well plates and treated with 0.1 μg / ml LPS and different concentrations of Gly-β-MCA for 3 hours, and then treated with 5 mM ATP for 30 minutes. The treated cells and cell supernatants were collected for detection. Western blot experiments were used to detect the protein levels of GSDMD / GSDMD-N, Pro-IL-1β, CASP1, CASP1-p20, S1PR2, p-AKT1, p-ERK1 / 2, and β-actin; ELISA kits were used to detect the level of IL-1β in the supernatant; and a cytotoxicity detection kit was used to detect the level of LDH in the supernatant. The results are shown in Figure 2. Figure 1 As shown, Figure 1 This indicates that Gly-β-MCA can inhibit pyroptosis and S1PR2 expression of mouse bone marrow macrophages in a concentration-dependent manner, and reduce the release of IL-1β and LDH. Figure 1 A shows that treatment with different concentrations of Gly-β-MCA can reduce the activation of GSDMD and CASP1 (GSDMD-N and CASP1-p20), and inhibit the protein levels of S1PR2 and its downstream molecules p-AKT1 and p-ERK1 / 2. Figure 1 B and Figure 1 C shows that treatment with different concentrations of Gly-β-MCA can reduce the release of IL-1β and LDH.

[0035] Example 2 Gly-β-MCA inhibits pyroptosis in human macrophages

[0036] Experimental groups: Gly-β-MCA: 0, 50, 100, 150, 200 μM (the control group used the same amount of DMSO)

[0037] The experiment was conducted using the human mononuclear macrophage cell line THP-1. First, 100 ng / mL PMA was used to induce differentiation for 36 hours, followed by 1 μg / mL LPS and different concentrations of Gly-β-MCA for 4 hours. The treated cells and cell supernatants were collected for detection. Western blot experiments were used to detect the protein levels of GSDMD / GSDMD-N, Pro-IL-1β, CASP1, CASP1-p20, S1PR2, and β-actin; ELISA kits were used to detect the level of IL-1β in the supernatant; and a cytotoxicity detection kit was used to detect the level of LDH in the supernatant. The results are shown in Figure 2. Figure 2 As shown, Figure 2 The results showed that Gly-β-MCA could inhibit pyroptosis and S1PR2 expression of human macrophage cell line THP-1 in a concentration-dependent manner, and reduce the release of IL-1β and LDH. Figure 2A shows that treatment with different concentrations of Gly-β-MCA can reduce the activation of GSDMD and CASP1 (GSDMD-N and CASP1-p20), and inhibit the protein level of S1PR2. Figure 2 B and 2C show that treatment with different concentrations of Gly-β-MCA can reduce the release of IL-1β and LDH.

[0038] Example 3 Bile acid receptor S1PR2 inhibitor JTE-013 alleviates macrophage pyroptosis

[0039] Experimental groups: DMSO, ATP, LPS, LPS+ATP, JTE-013+LPS+ATP, CYM5520 (S1PR2 agonist)+LPS+ATP

[0040] Primary mouse macrophages treated with macrophage colony-stimulating factor were seeded in 12-well plates, and 0.1 mg / mL LPS was added to the cells, followed by 5 μM JTE-013 or CYM-5520. The control group was added with 0.1 mg / mL LPS or 5 mM ATP or DMSO in an equal volume to the drug. After incubation in the incubator for 3 hours, 5 mM ATP was added, and the cell supernatant and cells were collected after 30 minutes of treatment. The protein levels of GSDMD / GSDMD-N, Pro-IL-1β, CASP1, CASP1-p20, and β-actin were detected by western blot experiments; the IL-1β level in the supernatant was detected by ELISA kit; and the LDH level in the supernatant was detected by cytotoxicity detection kit. The results are shown in Figure 2. Figure 3 As shown, Figure 3 This indicates that regulating S1PR2 can regulate pyroptosis of mouse bone marrow macrophages and reduce the release of IL-1β and LDH. Figure 3 A shows that the control group ATP or LPS or LPS+ATP treated macrophages, and LPS+ATP induced pyroptosis macrophages were given S1PR2 inhibitor JTE-013 or agonist CYM-5520, respectively. It shows that JTE-013 treatment inhibited the activation of GSDMD and CASP1 (GSDMD-N and CASP1-p20), while the agonist CYM-5520 treatment promoted the activation of GSDMD and CASP1 (GSDMD-N and CASP1-p20). Figure 3 B shows that the inhibitor JTE-013 inhibited the release of IL-1β and LDH, while the agonist CYM5520 promoted the activation of GSDMD and CASP1.

[0041] Example 4 Gly-β-MCA alleviates pyroptosis by inhibiting macrophage S1PR2

[0042] Experimental grouping: si-Control, si-Control+Gly-β-MCA (100μM, 150μM), si-S1pr2, si-S1pr2+Gly-β-MCA (100μM, 150μM)

[0043] BMDMs transfected with siControl and siS1pr2 were treated with Gly-β-MCA (100, 150 μM) combined with LPS (0.1 μg / mL) for 3 h, and then activated with ATP (5 mM, 30 min). The protein levels of GSDMD / GSDMD-N, Pro-IL-1β, CASP1, CASP1-p20, and β-actin were detected by western blot; the IL-1β level in the supernatant was detected by ELISA kit; and the LDH level in the supernatant was detected by cytotoxicity detection kit. The results are shown in Figure 2. Figure 4 As shown, Figure 4 This indicates that Gly-β-MCA relies on the inhibition of macrophage S1PR2 to exert its anti-pyroptosis effect. Figure 4 A shows siRNA transfection of mouse bone marrow macrophages. Green fluorescence indicates high transfection efficiency. Figure 4 BD shows that when S1PR2 was not knocked down by siRNA, the inhibitory effect on the levels of GSDMD-N, IL-1β and LDH in the supernatant gradually increased with the increase of the treatment concentration of Gly-β-MCA; however, after S1PR2 was knocked down by siS1pr2, the increase of the treatment concentration of Gly-β-MCA could not further inhibit the levels of GSDMD-N, IL-1β and LDH in the supernatant.

[0044] Example 5 Macrophage S1PR2 mediates upregulation of pyroptosis-related genes

[0045] Experimental groups: DMSO+LPS+ATP, JTE-013+LPS+ATP, CYM5520+LPS+ATP

[0046] LPS-pretreated macrophages were treated with DMSO (control group), 5μM JTE-013, and 5μM CYM-5520 for 3 hours, and then treated with 5mM ATP for 30 minutes. The RNA of the cells was collected for transcriptome sequencing. Differential gene expression was analyzed for JTE-013 vs DMSO and CYM-5520 vs DMSO, and the enrichment pathways of the differential genes were analyzed by IPA. Furthermore, the differential genes in the pyroptosis pathway were analyzed by IPA. The results are shown in Figure 2. Figure 5 As shown, Figure 5A shows that in the intersection of pathways that were downregulated after JTE-013 treatment and upregulated after CYM-5520 treatment, interleukin-1 family signaling and cell pyroptosis signaling were significantly enriched. Figure 5 B shows that the expression of genes related to pyroptosis was downregulated after JTE-013 treatment, while the expression of genes related to pyroptosis was upregulated after CYM-5520 treatment.

[0047] Example 6 Intraperitoneal injection of the S1PR2 inhibitor JTE-013 alleviates liver reperfusion injury in mice

[0048] Experimental groups: Pre-ischemic injection of drugs: Vehicle (control) + IR, 0.1 mg / kg + IR, 1 mg / kg + IR; Post-ischemic injection of drugs: IR + Vehicle, IR + 0.1 mg / kg, IR + 1 mg / kg

[0049] JTE-013 (0.1 mg / kg, 1 mg / kg) or a control solvent was injected intraperitoneally 1 hour before or after ischemia, and liver tissue and serum were collected. Serum ALT / AST levels were measured using a liver function test kit, and serum IL-1β levels were measured using an ELISA kit. Liver tissue sections were stained with H&E staining to assess necrosis. Figure 6 The results show that in vivo injection of S1PR2 inhibitor JTE-013 can alleviate liver ischemia-reperfusion injury. Figure 6 A is a schematic diagram of JTE-013 injection. Figure 6 BC showed that as the concentration of JTE-013 injection increased, the protective effect against liver injury was enhanced, and serum IL-1β and liver tissue necrosis area were further reduced.

[0050] Example 7 Inhibition of S1PR2 in bone marrow macrophages alleviates liver reperfusion injury in mice

[0051] Experimental groups: DT (Diphtheria toxin) + IR, DT + siControl BMDMs + IR, DT + siS1pr2 BMDMs + IR

[0052] The day before establishing the liver reperfusion injury model, CD11b-DTR mice were intraperitoneally injected with DT (25 μg / kg, -1 day) to deplete CD11b+ cells in vivo. The next day, 1 hour before ischemia, siControl- or siS1pr2-transfected BMDMs or PBS were transfused back into the mice. Liver reperfusion injury models were established, and liver tissue and serum were collected. Serum ALT / AST levels were measured using a liver function test kit; liver S1pr2 and inflammatory factor mRNA levels were measured by qRT-PCR; necrotic area was analyzed by H&E staining of liver tissue sections; GSDMD / GSDMD-NT and S1PR2 protein levels in liver tissue were measured by western blot; and serum IL-1β levels were measured by ELISA kit. Figure 7 The results indicate that inhibiting S1PR2 on bone marrow cells can alleviate liver ischemia-reperfusion injury. Figure 7 A is a diagram of the experimental model. After pretreatment with DT to eliminate bone marrow cells, the reperfusion injury model was established by reinfusing macrophages transfected with siControl or siS1pr2 before ischemia. Figure 7 B shows that the reinfusion of S1PR2 knockdown macrophages significantly alleviated liver function after reperfusion injury. Figure 7 C shows that the infusion of S1PR2-knockdown macrophages significantly reduced the mRNA level of S1pr2 in liver tissue. Figure 7 DG shows that the infusion of macrophages with knockdown of S1PR2 significantly reduced the necrosis area of ​​liver tissue, the mRNA levels of inflammatory factors, the protein levels of GSDMD-N and S1PR2, and the level of serum IL-1β.

[0053] Example 8 Gly-β-MCA alleviates liver ischemia-reperfusion injury by inhibiting macrophage S1PR2

[0054] Experimental groups: Models were established after injection of different concentrations of Gly-β-MCA: Sham, Vehicle (control) + IR, 2.5 mg / kg + IR, 5 mg / kg + IR, 10 mg / kg + IR

[0055] A liver ischemia-reperfusion injury model was established by injecting different concentrations of Gly-β-MCA (2.5, 5, 10 mg / kg) or a control vehicle (Vehicle) into the tail vein 1 hour before ischemia, and liver tissue and serum were collected. Serum ALT / AST levels were detected by liver function test kit; mRNA levels of inflammatory factors were detected by qRT-PCR; liver tissue necrosis was analyzed by H&E; Ly6C and S1PR2 levels were analyzed by immunohistochemistry; GSDMD / GSDMD-NT and S1PR2 protein levels were analyzed by western blot; and serum IL-1β levels were analyzed by ELISA kit. The results are shown in Figure 2. Figure 8-9 shown. Figure 8 A is the experimental model diagram, in which different concentrations of Gly-β-MCA were injected 1 hour before ischemia. Figure 8 B shows that as the concentration of injected Gly-β-MCA increases, the level of liver function ALT / AST gradually decreases. Figure 8 C shows that the mRNA level of inflammatory factors was inhibited as the concentration of injected Gly-β-MCA increased. Figure 9 A shows that as the concentration of injected Gly-β-MCA increases, the necrotic area of ​​liver tissue gradually decreases and the fluorescence intensity of S1PR2 observed by immunofluorescence is significantly reduced. Figure 9 B is the quantitative analysis result of necrotic area. Figure 9 C is the analysis result of the fluorescence intensity of S1PR2. Figure 9 D shows that as the concentration of injected Gly-β-MCA increases, the protein levels of GSDMD-N and S1PR2 in liver tissue decrease. Figure 9 E shows that as the concentration of injected Gly-β-MCA increases, the level of IL-1β in serum decreases.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of glycine-β-muricholic acid as an inhibitor of proinflammatory macrophage bile acid receptor S1PR2 in the preparation of a drug, characterized in that: The medicine is used for preventing and / or treating liver ischemia-reperfusion injury; the medicine is used for intravenous administration.

2. The use according to claim 1, characterized in that The drug has the function of inhibiting macrophage pyroptosis, inhibiting the release of the pro-inflammatory factor IL-1β, or reducing lactate dehydrogenase.

3. The use according to claim 1, characterized in that The drug is used to reduce the release of alanine aminotransferase and aspartate aminotransferase and reduce the area of ​​liver tissue necrosis.

4. The use according to claim 1, characterized in that The triggering factors of liver ischemia-reperfusion injury include liver cysts, liver transplantation, thrombolytic therapy, portal vein occlusion and hepatic coma.

5. The use according to claim 1, characterized in that The drug further includes a pharmaceutically acceptable carrier.

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