Compound for preventing and treating organ ischemia-reperfusion injury, and use thereof

Abstract

The use of 7-ketolithocholic acid or a pharmaceutically acceptable salt, prodrug or metabolite thereof in the preparation of a drug for preventing, treating or assisting in the treatment of ischemia-reperfusion injury, and the use of 12-ketodeoxycholic acid in the preparation of a drug for preventing, ameliorating, treating or assisting in the treatment of ischemia-reperfusion injuries of the heart, intestine and kidney. The aforementioned uses provide new therapeutic methods and strategies for the clinical prevention and alleviation of ischemia-reperfusion injury.

Description

A class of compounds for the prevention and treatment of organ ischemia-reperfusion injury and their applications

[0001] This application claims priority to an earlier application filed on March 20, 2025, with the China National Intellectual Property Administration, patent application number 2025103354519, entitled "Application of 7-Ketolithocholic Acid in the Prevention and Treatment of Organ Ischemia-Reperfusion Injury". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicine, specifically relating to a class of compounds for the prevention and treatment of organ ischemia-reperfusion injury and their applications. Background Technology

[0003] Ischemia-reperfusion injury (IRI) is a condition in which tissue damage worsens or even becomes irreversible after blood flow is restored following ischemic injury. This process involves both ischemic and reperfusion damage. Common ISI injuries include cardiac, cerebral, pulmonary, and other organ ischemia-reperfusion injuries. ISI is a common complication of various organ ischemic diseases, severely impacting patient survival and prognosis. However, currently, there are still relatively few safe and effective drugs for the prevention and treatment of ISI.

[0004] 7-Keto lithocholic acid is an endogenous metabolite produced in the bile acid metabolic pathway. It is generated by the dehydrogenation of the hydroxyl group of the primary bile acid chenodeoxycholic acid by 7α-hydroxysteroid dehydrogenase (7α-HSDH). 12-Ketodeoxycholic acid is a secondary bile acid. Currently, there are no reports or studies on the relationship between 7-keto lithocholic acid, 12-ketodeoxycholic acid, and the prevention and treatment of organ ischemia and reperfusion injury. Summary of the Invention

[0005] In view of the deficiencies in the prior art and / or the needs in the art, one of the objects of the present invention is to provide 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof for use in the preparation of a drug for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury.

[0006] The 7-keto lithocholic acid, CAS number 4651-67-6, has the molecular formula C7. 24 H 38 O4, with a molecular weight of 390.56, has the following structural formula:

[0007] According to an embodiment of the present invention, the ischemia-reperfusion injury is selected from one or more ischemic conditions formed by myocardial ischemia, cerebral ischemia, renal ischemia, hepatic ischemia, ischemia-reperfusion cardiomyopathy, skin ischemia, intestinal ischemia, gastric ischemia, pulmonary ischemia, pancreatic ischemia, skeletal muscle ischemia, abdominal muscle ischemia, limb ischemia, ischemia-reperfusion colitis, mesenteric ischemia, and asymptomatic ischemia. Preferably, the ischemia-reperfusion injury is caused by ischemia resulting from ischemic diseases, organ-related surgery, trauma, or organ transplantation.

[0008] According to an embodiment of the present invention, the ischemia-reperfusion injury is selected from ischemia-reperfusion injuries of organs such as the heart, brain, kidney, liver, intestine and lung.

[0009] According to embodiments of the present invention, pharmaceutically acceptable salts of 7-ketolithocholic acid include salts that may be present in the acidic group of 7-ketolithocholic acid. Examples include sodium, calcium, and potassium salts.

[0010] According to an embodiment of the present invention, in the above-described application, the drug can be administered before, during, or after ischemia-reperfusion injury occurs, for example, as a pretreatment for reperfusion surgery.

[0011] According to an embodiment of the present invention, in the above-described application, the drug can improve the survival rate after ischemia-reperfusion injury; further, the drug can improve one or more of the following after cardiac ischemia-reperfusion injury: survival rate, physical activity capacity, myocardial infarction area, cardiac function, and cardiac troponin-I level.

[0012] According to an embodiment of the present invention, in the above-described application, the drug can improve the survival rate after intestinal ischemia-reperfusion injury; further, the drug can improve the overall survival rate, the pathological degree of intestinal injury, and one or more intestinal injury markers after intestinal ischemia-reperfusion injury.

[0013] According to an embodiment of the present invention, in the above-described application, the drug can improve the survival rate after cerebral ischemia-reperfusion injury; furthermore, the drug can improve the overall survival rate after cerebral ischemia-reperfusion injury, reduce the infarct area, and reduce the degree of neurological function impairment, among one or more of the following:

[0014] According to embodiments of the present invention, in the above applications, the drug further includes a pharmaceutically acceptable carrier and / or excipients (such as diluents), suitable for preparation into formulations for intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, etc.

[0015] According to embodiments of the present invention, in the above applications, the drug may be an oral preparation, an injectable preparation, or an enema liquid preparation. Oral preparations include tablets, capsules, pills, powders, granules, suspensions, drops, etc., and injectable preparations include injection solutions, powder injections, etc.; more preferably, the tablets include ordinary tablets, orally disintegrating tablets, dispersible tablets, or sustained-release tablets.

[0016] According to embodiments of the present invention, in the above applications, the drug may be administered alone or in combination with one or more other therapeutic agents.

[0017] The present invention also provides the use of a pharmaceutical composition containing 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof in the preparation of a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury.

[0018] The present invention also provides a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury, comprising 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0019] The present invention also provides a method for preventing, treating or adjunctive treating ischemia-reperfusion injury, the method comprising administering to a subject in need a therapeutically effective amount of 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0020] A second objective of this invention is to provide a 7-ketolithocholic acid analogue for use in the prevention, improvement, treatment, or adjunctive treatment of ischemia-reperfusion injury of the heart, intestine, and kidney. The 7-ketolithocholic acid analogue is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof, or a prodrug or metabolite thereof. The 12-ketodeoxycholic acid has the CAS number 5130-29-0 and the molecular formula C7. 24 H 38 O4 has a molecular weight of 390.56.

[0021] According to embodiments of the present invention, pharmaceutically acceptable salts of 12-ketodeoxycholic acid include salts that may be present in the acidic group of 12-ketodeoxycholic acid. Examples include sodium, calcium, and potassium salts.

[0022] The present invention also provides a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury, comprising a 7-ketolithocholic acid analogue, and optionally a pharmaceutically acceptable carrier and / or excipient, wherein the 7-ketolithocholic acid analogue is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0023] The present invention also provides a method for preventing, treating or adjunctive treating ischemia-reperfusion injury, the method comprising administering a therapeutically effective amount of a 7-ketolithocholic acid analogue to a subject in need, said 7-ketolithocholic acid analogue being 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0024] The present invention also provides the use of a pharmaceutical composition containing a 7-ketolithocholic acid analog in the preparation of a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury, wherein the 7-ketolithocholic acid analog is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0025] The present invention also provides a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury, comprising a 7-ketolithocholic acid analogue, and optionally a pharmaceutically acceptable carrier and / or excipient, wherein the 7-ketolithocholic acid analogue is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0026] The present invention also provides a method for preventing, treating or adjunctive treating ischemia-reperfusion injury, the method comprising administering a therapeutically effective amount of a 7-ketolithocholic acid analogue to a subject in need, said 7-ketolithocholic acid analogue being 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

[0027] According to embodiments of the present invention, the effective dose for mice of the 7-ketolithocholic acid or its pharmaceutically acceptable salt or its prodrug or metabolite, or an analogue of the 7-ketolithocholic acid, is 10–60 mg / kg, preferably 30–60 mg / kg, more preferably 40 mg / kg; and the effective dose for humans is 2.45–4.88 mg / kg.

[0028] According to an embodiment of the invention, the subject in need may be a mammal, such as a human. Beneficial effects

[0029] This invention verifies the effects of 7-ketolithocholic acid or its pharmaceutically acceptable salts, prodrugs, or metabolites on ischemia-reperfusion injury, demonstrating its ability to improve the survival rate of cardiac, cerebral, renal, hepatic, intestinal, and pulmonary cells after ischemia-reperfusion injury, increase overall survival after ischemia-reperfusion, and improve physical activity, myocardial infarction area, cardiac function, and cardiac troponin-I levels. Furthermore, the invention also verifies the effects of 7-ketolithocholic acid analogues on ischemia-reperfusion injury in the heart, intestine, and kidney. The overall protective effect of 7-ketolithocholic acid is significantly superior to other 7-ketolithocholic acid analogues. These findings also indicate that 7-ketolithocholic acid has high specificity and significant advantages in the prevention, treatment, or adjuvant therapy of organ ischemia-reperfusion injury. In addition, experimental results show that 12-ketodeoxycholic acid can improve the survival rate of cardiac, renal, and intestinal cells after ischemia-reperfusion injury, indicating its therapeutic or adjuvant therapeutic efficacy for ischemia-reperfusion injury in the heart, intestine, and kidney, providing a new treatment method and strategy for the clinical prevention and mitigation of ischemia-reperfusion injury. Attached Figure Description

[0030] Figure 1 shows the time-varying results of reperfusion extent in distal ischemic pretreatment.

[0031] Figure 2 shows the increase in endogenous 7-ketolithocholic acid content in mice after distal ischemia pretreatment.

[0032] Figure 3 shows the results of 7-ketolithocholic acid in improving ischemia / reperfusion injury of cardiac cells (where ** indicates P<0.01; *** indicates P<0.001).

[0033] Figure 4 shows the results of 7-ketolithocholic acid in improving ischemia / reperfusion injury in liver, lung, kidney, brain and intestinal cells (where * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001).

[0034] Figure 5 shows the results of 7-ketolithocholic acid improving the overall survival rate of mice after myocardial ischemia-reperfusion (where * indicates P<0.05).

[0035] Figure 6 shows the results of 7-ketolithocholic acid improving the activity of the mouse heart after ischemia-reperfusion (where *** indicates P<0.001).

[0036] Figure 7 shows the results of 7-ketolithocholic acid improving the infarct area in mice (where *** indicates P<0.001).

[0037] Figure 8 shows the results of 7-ketolithocholic acid improving cardiac function EF and FS in mice (where **** indicates P<0.0001).

[0038] Figure 9 shows the results of 7-ketolithocholic acid reducing the level of cardiac troponin-I in mice (where ** indicates P<0.01).

[0039] Figure 10 shows the results of 7-ketolithocholic acid improving the overall survival rate of mice after intestinal ischemia-reperfusion (where * indicates P<0.05).

[0040] Figure 11 shows the results of 7-ketolithocholic acid improving intestinal pathological scores and intestinal fatty acid-binding protein (I-FABP) in mice after intestinal ischemia-reperfusion (where * indicates P<0.05, *** indicates P<0.001).

[0041] Figure 12 shows the results of 7-ketolithocholic acid improving the overall survival rate of mice after cerebral ischemia-reperfusion (where * indicates P<0.05).

[0042] Figure 13 shows the results of 7-ketolithocholic acid improving the cerebral infarction area and neurological function score in mice after cerebral ischemia-reperfusion (*** indicates P<0.001, **** indicates P<0.0001).

[0043] Figure 14 compares the effects of intraperitoneal injection of the same concentration of 7-ketolithocholic acid, cholic acid, and ursodeoxycholic acid on the activity level and infarct area of ​​mice after myocardial ischemia-reperfusion injury (where * indicates P<0.05; ** indicates P<0.01; **** indicates P<0.0001).

[0044] Figure 15 shows the survival rate of 7-ketolithocholic acid structural analogues in the treatment of ischemia-reperfusion injury of heart, brain, kidney, lung, liver and intestinal cells (where * indicates P<0.05; ** indicates P<0.01; **** indicates P<0.0001).

[0045] Terminology Definition

[0046] The term "7-ketolithocholic acid prodrug" in this document refers to the conversion of a compound into 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug compounds of this invention can be esters; in existing inventions, esters that can serve as prodrugs include phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, one compound in this invention contains a hydroxyl group, meaning it can be acylated to yield the prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a hydroxyl group on the parent compound.

[0047] The term "7-ketolithocholic acid metabolite" in this document refers to the product obtained in vivo through the metabolism of 7-ketolithocholic acid or its pharmaceutically acceptable salt. A metabolite of a compound can be identified using techniques known in the art, and its activity can be characterized by experimental methods as described in this invention. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this invention includes metabolites of compounds, including metabolites produced by sufficient contact of the compounds of this invention with mammals for a period of time.

[0048] The term "12-ketodeoxycholic acid prodrug" in this article refers to the conversion of a compound into 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or by enzymatic conversion of the prodrug into its parent structure in the blood or tissues.

[0049] The term "12-ketodeoxycholic acid metabolite" in this article refers to the product obtained in vivo through the metabolism of 12-ketodeoxycholic acid or its pharmaceutically acceptable salt.

[0050] The term "ischemia-reperfusion injury" in this article refers to the rapid increase in tissue damage after an organ or tissue that has suffered ischemia for a certain period of time has resumed blood flow and reperfusion. This includes ischemia-reperfusion injuries in organs such as the heart, brain, kidneys, liver, intestines, and lungs.

[0051] The term "remote ischemic preconditioning (RIPC)" in this article refers to the protective effect that a brief, reversible ischemic stimulus to distal organs (such as the upper or lower limbs) on other organs (such as the heart, brain, liver, and kidneys) against subsequent severe ischemia.

[0052] The term "adjunctive therapy" in this article refers to additional treatment, usually given after surgery.

[0053] The term "subject" in this article refers to an animal, such as a mammal. Mammals include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cattle, and humans.

[0054] The term "therapeutic effective amount" or "effective amount" herein refers to the amount of a compound disclosed and / or described herein that, when administered to a patient requiring this treatment, is sufficient to achieve the treatment as defined herein. A therapeutically effective amount of a compound may be an amount sufficient to treat ischemia-reperfusion injury. Therapeuticly effective amounts will vary depending on factors such as the subject being treated and their disease condition, the subject's weight and age, the severity of the disease condition, the specific compound, the dosing regimen to be followed, the timing of administration, and the method of administration, all of which can be readily determined by one of ordinary skill in the art.

[0055] The term “treatment” in this article includes one or more of the following: suppressing a disease or condition; slowing or halting the development of clinical symptoms of a disease or condition; and / or alleviating a disease or condition (i.e., causing relief or resolution of clinical symptoms), and both complete or partial reduction of clinical symptoms of a disease or condition.

[0056] The term "therapeutic effect" in this article refers to the effect resulting from treatment, which at the cellular level manifests as an inhibition rate of cell growth or a cell death rate, and at the animal level manifests as an alteration, usually a reduction or improvement of the symptoms of a disease or disease condition, or a cure of a disease or disease condition.

[0057] The term "pharmaceutically acceptable salt" in this article refers to a salt that may be present in the acidic group of 7-ketolithocholic acid or in the acidic group of 12-ketodeoxycholic acid. Examples include sodium salts, calcium salts, and potassium salts. Detailed Implementation

[0058] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0059] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0060] Laboratory Animals: All experiments involving mice were approved by the Standing Animal Committee of the Second Affiliated Hospital of Chongqing Medical University (Program No.: IACUC-SAHCQMU-2023-0009). All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The number of mice used in this study was minimized as much as possible. The C57BL / 6J male mice (8-10 weeks old, weighing 22-30g) used in this study were purchased from Hunan Silek Jingda Laboratory Animal Co., Ltd., China. Five mice were housed per cage, with free access to food and water. The housing environment was 12 hours of light / 12 hours of darkness. All mice were housed for one week before the formal experiments began.

[0061] Cells: H9C2 rat cardiomyocytes, AC16 human cardiomyocytes, HT-22 mouse hippocampal neurons, 293T human kidney epithelial cells, BRL-3A rat liver cells, BEAS-2B human bronchial epithelial cells, and IEC-6 rat small intestinal crypt epithelial cells were all purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0062] Materials and reagents: 7-ketolithocholic acid (HY-W018512) was purchased from MedChemExpress; DMEM medium (11-966-025), PBS buffer (C14190500CP), penicillin-streptomycin (15140122), and trypsin-EDTA (25200056) were purchased from Gibco; Cell Counting Kit-8 (CK04-01) was purchased from Dojindo; and fetal bovine serum (BC-SE-FBS01) was purchased from Nanjing Shenghang Biotechnology Co., Ltd.

[0063] Example 1: Determination of reperfusion degree in mice after distal ischemic preconditioning (RIPC) at different time points.

[0064] 1. Experimental animals: C57BL / 6J mice were used as the research subjects. They were male and 8-10 weeks old. The environment was 12 hours of light / 12 hours of darkness. Water and food were freely available. The formal experiment began after 7-10 days of rearing. The mice were divided into two groups (ZT0 and ZT8 according to the lighting time of the rearing environment), with 4 or more mice in each group.

[0065] 2. Mouse model of distal ischemic preconditioning (RIPC): After anesthetizing mice, the right hind limb was shaved and disinfected, and the mice were placed in a supine position. The skin was carefully cut at the quadriceps femoris muscle, and the muscle was dissected to expose the femoral artery in the right hind limb. The femoral artery was clamped with a mouse arterial clamp for 5 minutes, and then reperfused for 5 minutes after releasing the arterial clamp, for a total of 4 cycles.

[0066] 3. Detection of reperfusion extent during RIPC in mice: Based on the mouse RIPC model constructed in step 2, the blood perfusion extent of the hind limbs of mice was recorded using a laser speckle blood flow imaging system (Reward, China). The results are shown in Figure 1. In Figure 1, ZT0 represents the mouse housing environment when the lights were first turned on, and ZT8 represents 8 hours after the lights were turned on. R1, R2, R3, and R4 represent the four reperfusion periods during RIPC, respectively.

[0067] According to Figure 1, during R1, the skin blood perfusion at ZT0 and ZT8 were 0.52±0.04 PU and 1.19±0.15 PU, respectively (P=0.0017), showing a significant time difference. During R2, the skin blood perfusion at ZT0 and ZT8 were 0.57±0.08 PU and 1.36±0.19 PU, respectively (P=0.0032), showing a significant time difference. During R3, the skin blood perfusion at ZT0 and ZT8 were 0.70±0.08 PU and 1.36±0.21 PU, respectively (P=0.0153), showing a significant time difference. During R4, the skin blood perfusion at ZT0 and ZT8 were 0.70±0.10 PU and 1.32±0.22 PU, respectively (P=0.0419), showing a significant time difference. This indicates that ZT8 is the dominant time point for RIPC.

[0068] Example 2: Determination of endogenous 7-ketolithocholic acid content in mice after distal ischemic preconditioning (RIPC).

[0069] 1. Experimental animals: C57BL / 6J mice were used as the research subjects. They were male and 8-10 weeks old. The environment was 12 hours of light / 12 hours of darkness. Water and food were freely available. After 7-10 days of rearing, the formal experiment began. The mice were divided into two groups of 22 each.

[0070] 2. Mouse model of distal ischemic preconditioning (RIPC): After anesthetizing mice, the right hind limb was shaved and disinfected, and the mice were placed in a supine position. The skin was carefully cut at the quadriceps femoris muscle, and the muscle was dissected to expose the femoral artery in the right hind limb. The femoral artery was clamped with a femoral clamp for 5 minutes, followed by reperfusion for 5 minutes, for a total of 4 cycles. The same procedure was performed on the control mice to expose the femoral artery, but no femoral artery clamping / reperfusion treatment was performed.

[0071] 3. Detection of endogenous 7-ketolithocholic acid in mice: Based on the mouse RIPC model constructed in step 2, myocardial, quadriceps femoris muscle, and plasma samples were collected from mice for the detection of 7-ketolithocholic acid-targeting metabolites. The results are shown in Figure 2, where RIPC represents the experimental mouse model and Control represents the control mouse model.

[0072] According to Figure 2, the abundance of 7-ketolithocholic acid metabolites in the control group at ZT0 in plasma was (6.7 × 10⁻⁶). 6 ±9.0×10 5 ), RIPC group is (9.9×10 6 ±2.0×10 6 The p-value was 0.2250, indicating no statistically significant difference; at ZT8, the control group (7.2 × 10⁻⁶) showed no significant difference. 6 ±2.0×10 6 ), RIPC group is (2.2×107 ±4.0×10 6 The difference was statistically significant (P=0.0031); and 7-ketolithocholic acid levels were significantly higher after ZT8 RIPC than after ZT0 RIPC (P=0.0119).

[0073] The abundance of 7-ketolithocholic acid metabolites in the heart at ZT0 in the control group was (1.2 × 10⁻⁶). 6 ±6.4×10 4 ), RIPC group is (1.3×10 6 ±1.0×10 5 The p-value was 0.2830, indicating no statistically significant difference; at ZT8, the control group (1.4 × 10⁻⁶) showed no significant difference. 6 ±8.6×10 4 ), RIPC group is (2.2×10 6 ±2.9×10 5 The difference was statistically significant (P=0.0119); and 7-ketolithocholic acid levels were significantly higher after ZT8 RIPC than after ZT0 RIPC (P=0.0105).

[0074] The abundance of 7-ketolithocholic acid metabolites in the quadriceps femoris muscle at ZT0 in the control group was (1.9 × 10⁻⁶). 6 ±1.2×10 5 ), RIPC group is (1.8×10 6 ±7.6×10 4 The p-value was 0.8668, indicating no statistically significant difference; at ZT8, the control group (1.7 × 10⁻⁶) showed no statistically significant difference. 6 ±6.6×10 4 ), RIPC group is (3.4×10 6 ±5.6×10 5 The difference was statistically significant (P=0.0067); and 7-ketolithocholic acid levels were significantly higher after ZT8 RIPC than after ZT0 RIPC (P=0.0107).

[0075] Example 3: Determination of the survival rate of cardiac cells affected by ischemia-reperfusion injury with 7-ketolithocholic acid.

[0076] A: Survival rate determination of H9C2 rat cardiomyocytes after ischemia-reperfusion injury:

[0077] 1. Construction of a cell ischemia-reperfusion injury model: H9C2 rat cardiomyocytes were used as the research subject, and were evenly seeded into 96-well plates (cell count: 10,000). After 4 hours of hypoxia treatment in a cell culture incubator (the oxygen content in the incubator was adjusted to 1% by adjusting the nitrogen ratio), the cells were returned to a cell culture incubator with 21% oxygen content for 24 hours of reoxygenation treatment to induce the construction of a cell ischemia-reperfusion injury model. The experimental group (denoted as 7-KLCA) cells were cultured with 1 μM 7-ketolithocholic acid, the blank control group (denoted as 0) cells were cultured in normal medium, the solvent control group (denoted as 1% DMSO) cells were cultured with 1% dimethyl sulfoxide, and the normoxic control group cells (denoted as control) were placed in a cell culture incubator with 21% oxygen content.

[0078] 2. Cell viability determination: The cell ischemia-reperfusion injury model constructed according to step 1 of Example 3(A) was used as the research object. Cells were removed, and 10 μL of Cell Counting Kit-8 solution was added to each well. The cells were placed in a cell culture incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0079] According to Figure 3, the cell survival rate of H9C2 cells in the normoxic control group was standardized to 100%. The survival rate of H9C2 cells after ischemia-reperfusion injury (blank control group) (16.93±3.27%) was significantly lower than that in the normoxic control group. The survival rate of H9C2 cells in the 1% DMSO group (13.60±2.23%) did not show significant improvement, while the survival rate of H9C2 cells in the 7-ketolithocholic acid group (i.e., the experimental group) (53.08±6.85%) was significantly higher than that in the ischemia-reperfusion injury group.

[0080] B: Determination of cardiomyocyte survival rate after ischemia-reperfusion injury in AC16 patients:

[0081] 1. Construction of a cellular ischemia-reperfusion injury model: AC16 human cardiomyocytes were used as the research subject, and were evenly seeded into 96-well plates (cell count: 10,000). After 4 hours of hypoxia treatment in a cell culture incubator (the oxygen content in the incubator was adjusted to 1% by adjusting the nitrogen ratio), the cells were returned to a cell culture incubator with 21% oxygen content for 24 hours of reoxygenation treatment to induce the construction of a cellular ischemia-reperfusion injury model. The experimental group (denoted as 7-KLCA) cells were cultured with 1 μM 7-ketolithocholic acid, the blank control group (denoted as 0) cells were cultured in normal medium, the solvent control group (denoted as 1% DMSO) cells were cultured with 1% dimethyl sulfoxide, and the normoxic control group cells (denoted as control) were placed in a cell culture incubator with 21% oxygen content throughout.

[0082] 2. Cell viability determination: The cell ischemia-reperfusion injury model constructed according to step 1 of Example 3(B) was used as the research object. Cells were removed, and 10 μL of Cell Counting Kit-8 solution was added to each well. The cells were placed in a cell culture incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0083] According to Figure 3, the cell survival rate of AC16 cells in the normoxic control group was standardized to 100%. The survival rate of AC16 cells after ischemia-reperfusion injury (blank control group) (48.20±1.69%) was significantly lower than that in the normoxic control group. The survival rate of AC16 cells in the 1% DMSO group (51.57±2.57%) did not show significant improvement, while the survival rate of AC16 cells in the 7-ketolithocholic acid group (i.e., the experimental group) (57.29±1.41%) was significantly higher than that in the ischemia-reperfusion injury group.

[0084] Example 4: Survival determination of brain, kidney, liver, intestine and lung cells after ischemia-reperfusion injury.

[0085] 1. Construction of a cellular ischemia-reperfusion injury model: HT-22 mouse hippocampal neurons, 293T human kidney epithelial cells, BRL-3A rat liver cells, BEAS-2B human bronchial epithelial cells, and IEC-6 rat small intestinal crypt epithelial cells were used as research subjects. Each type of cell was uniformly seeded into 96-well plates (cell number: 10,000). After 4 hours of hypoxia treatment in a cell culture incubator (the oxygen content in the incubator was adjusted to 1% by adjusting the nitrogen ratio), the cells were returned to a cell culture incubator with 20% oxygen content and reoxygenated for 24 hours to induce a cellular ischemia-reperfusion injury model. The experimental group (denoted as 7-KLCA) cell line was cultured with 1 μM 7-ketolithocholic acid. The blank control group (denoted as 0) cells for ischemia-reperfusion injury were cultured in normal medium. The solvent control group (denoted as 1% DMSO) cells for ischemia-reperfusion injury were cultured with 1% dimethyl sulfoxide. The normoxic control group cells (denoted as control) were always placed in a cell culture incubator with 20% oxygen content.

[0086] 2. Cell viability determination: The cell ischemia-reperfusion injury model constructed according to step 1 of Example 4 was used as the research object. Cells were removed, and 10 μL of Cell Counting Kit-8 solution was added to each well. The cells were placed in a cell culture incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0087] According to Figure 4, the cell survival rates of the normoxic control group for each cell line were standardized to 100%. After ischemia-reperfusion injury (i.e., blank control group), the survival rates of HT-22 (60.92±5.64%), 293T (18.10±2.49%), BRL-3A (34.31±1.66%), BEAS-2B (65.63±2.09%), and IEC-6 (66.95±2.29%) cells were significantly lower than those of the normoxic control group, by 1%. In the DMSO group, the survival rates of HT-22 (67.72±6.47%), 293T (17.72±3.86%), BRL-3A (40.06±4.90%), BEAS-2B (47.47±1.09%), and IEC-6 (75.55±3.61%) cells showed no significant improvement. However, in the 7-ketolithocholic acid group (experimental group), the survival rates of HT-22 (80.00±2.88%), 293T (37.84±2.83%), BRL-3A (42.54±1.20%), BEAS-2B (75.24±2.08%), and IEC-6 (80.90±2.93%) cells were significantly higher than those in the ischemia-reperfusion injury group.

[0088] Example 5: Constructing a mouse model of myocardial ischemia for 30 minutes followed by reperfusion for 24 hours.

[0089] 1. Experimental animals: C57BL / 6J mice were used as the research subjects. They were male and 8-10 weeks old. The environment was 12 hours of light / 12 hours of darkness. Water and food were freely available. The formal experiment began after 7-10 days of rearing. They were divided into 2 groups with 22 mice in each group.

[0090] 2. Preparation of 7-ketolithocholic acid administration solution: 7-ketolithocholic acid was dissolved in an aqueous solution containing 10% dimethyl sulfoxide and physiological saline, and the final concentration of 7-ketolithocholic acid was 51.21 mM.

[0091] 3. Establishment of a mouse model of cardiac ischemia-reperfusion: After anesthetizing the mice, the precordial region was shaved and disinfected, and the mice were placed in a supine position. A mouse laryngoscope was used for endotracheal intubation, and the mice were connected to a small animal ventilator (respiratory rate 110 bp, tidal volume 0.35 mL). The experimental group (denoted as the 7K+MIR group) received an intraperitoneal injection of the 7-ketolithocholic acid solution (40 mg / kg) prepared in step 2, while the control group (denoted as the MIR group) received an intraperitoneal injection of the same volume of a solution containing 10% dimethyl sulfoxide and physiological saline. The skin of the precordial region was then cut open, the muscle tissue was separated, and the thoracic cavity was accessed at the fourth intercostal space along the left sternal border. The pericardium was torn open to expose the heart, and a slipknot was applied to the left anterior descending branch of the heart using 7-0 silk suture approximately 1.5 mm below the tip of the left atrial auricle. The thoracic cavity was immediately closed layer by layer. After 30 minutes of ischemia, the slipknot was opened, and cardiac reperfusion was restored. The Sham group represents the sham-operated group, where the mouse heart was only sutured without ligation for ischemia.

[0092] Example 6: Determination of overall survival rate of mice after myocardial ischemia-reperfusion

[0093] The mouse model of myocardial ischemia for 30 minutes and reperfusion for 24 hours was constructed according to Example 5. The number of mice surviving myocardial ischemia-reperfusion was recorded within 48 hours. The survival rate of the model mice was calculated based on the total number of mice with myocardial ischemia-reperfusion. The results are shown in Figure 5. In Figure 5, MIR represents the control group mouse model and 7K+MIR represents the 7-ketolithocholic acid group mouse model.

[0094] Survival rate = (number of survivors within 48 hours / total number) * 100.

[0095] According to Figure 5, the survival rate of mice in the 7-ketolithocholic acid group (77.27%) was significantly higher than that of mice in the control group (48.00%).

[0096] Example 7: Determination of activity level in mice after myocardial ischemia-reperfusion

[0097] Based on Example 5, a mouse model of myocardial ischemia for 30 minutes followed by reperfusion for 24 hours was constructed as the research subject. The mice that had been reperfused for 24 hours were removed from the animal room and placed in a behavioral room for 1 hour to adapt. Subsequently, each mouse was subjected to an open field experiment. The total distance and average speed of the mice's free movement within 5 minutes were recorded using Smart behavioral software. The results are shown in Figure 6, where the Sham group represents the sham-operated group of mice, the MIR group represents the control group of mice, and the 7K+MIR group represents the 7-ketolithocholic acid group of mice.

[0098] According to Figure 6, the total distance traveled (333.96±72.35 cm) and average speed (1.11±0.24 cm / s) of the control group mice were significantly lower than those of the sham-operated group mice (1402.68±92.34 cm and average speed (4.68±0.31 cm / s). The total distance traveled (1000.20±134.37 cm) and average speed (3.46±0.49 cm / s) of the 7-ketolithocholic acid group mice were significantly better than those of the control group.

[0099] Example 8: Measurement of myocardial infarction area in mice

[0100] A mouse model of myocardial ischemia for 30 minutes followed by reperfusion for 24 hours was constructed according to Example 5. The experimental group received intraperitoneal injection of 7-ketolithocholic acid (40 mg / kg), while the control group received an equal volume of control solvent. After 24 hours of reperfusion, mouse hearts were harvested for Evans-Blue / TTC double staining. The thoracic cavity was opened to expose the heart, and the left anterior descending branch of the heart was religated in the original ligation site. 1% Evans-Blue was injected into the aorta. Hearts were removed and flash-frozen at -20°C for approximately 1 hour. The heart tissue was then uniformly transversely sectioned into 1 mm thick slices. The slices were stained in 1% triphenyltetrazolium chloride (TTC) solution at 37°C for 15 min in the dark, followed by fixation in 4% paraformaldehyde for 1-2 hours. Finally, the heart slices were photographed under a microscope. ImageJ software was used to quantify the area of ​​necrotic and viable myocardium. Infarcted areas were shown in white, danger zones in red, and non-ischemic areas in blue. The infarct area was expressed as a percentage of the infarcted area to the danger zone. The results are shown in Figure 7, where MIR represents the control group mouse model and 7K+MIR represents the 7-ketolithocholic acid group mouse model.

[0101] According to Figure 7, compared with the control group (43.45±2.17%), 7-ketolithocholic acid significantly reduced the infarct area (29.21±2.73%).

[0102] Example 9: Measurement of cardiac function in mice

[0103] Using the mouse model of myocardial ischemia for 30 minutes and reperfusion for 24 hours constructed in Example 5 as the research subject, the left ventricular ejection fraction (EF) and fractional shortening (FS) of the mice were detected by small animal echocardiography 24 hours after reperfusion and expressed as percentages. The results are shown in Figure 8. In the figure, Sham group represents sham-operated mice, MIR represents control mouse model, and 7K+MIR represents 7-ketolithocholic acid mouse model.

[0104] According to Figure 8, the EF (44.45±2.28%) and FS (21.11±1.33%) of the control group mice were significantly lower than those of the sham-operated group mice (76.94±1.59%) and FS (43.91±1.65%). The EF (67.41±2.22%) and FS (34.70±1.95%) of the 7-ketolithocholic acid group mice were significantly improved compared to the EF and FS of the model mice.

[0105] Example 10: Measurement of mouse troponin-1

[0106] Using a mouse model of myocardial ischemia for 30 minutes followed by reperfusion for 24 hours constructed in Example 5 as the research subject, blood was collected from the eyeballs and whole blood was collected from mice in EDTA anticoagulant tubes. The blood was centrifuged at 2000 rpm for 20 minutes at 4°C. The supernatant plasma was then collected, and the level of troponin-I in each plasma sample was detected using a mouse troponin-I enzyme-linked immunosorbent assay (ELISA) kit. The results are shown in Figure 9, where MIR represents the control mouse model and 7K+MIR represents the 7-ketolithocholic acid group mouse model.

[0107] According to Figure 9, compared with the control group (21.00±0.74ng / mL), the 7-ketolithocholic acid group significantly reduced the level of troponin-I (16.02±1.56ng / mL) in mice with myocardial ischemia-reperfusion injury.

[0108] Example 11: Constructing a mouse model of intestinal ischemia for 50 minutes followed by reperfusion for 2 hours

[0109] 1. Experimental Animals: C57BL / 6J mice were used as the research subjects, and the housing environment was the same as in Example 5. The experiment was divided into 3 groups: intestinal ischemia-reperfusion control group (denoted as IIR), 7-ketolithocholic acid 40 mg / kg administration group (denoted as 7K+IIR 40 mg / kg), and 7-ketolithocholic acid 80 mg / kg administration group (denoted as 7K+IIR 80 mg / kg).

[0110] 2. Preparation of 7-ketolithocholic acid administration solution: Same as in Example 5.

[0111] 3. Before modeling, the experimental group mice were injected intraperitoneally with the corresponding dose of 7-ketolithocholic acid solution, while the control group mice were injected with the same volume of solvent.

[0112] 4. Establishment of a mouse intestinal ischemia-reperfusion model: After anesthetizing the mice, the abdomen was shaved and disinfected before placing them in a supine position. The skin and muscles of the mouse abdomen were cut open to expose the superior mesenteric artery, which was then clamped using a microvascular clamp to induce intestinal ischemia. After 50 minutes of ischemia, the arterial clamp was released to restore intestinal blood flow and reperfusion.

[0113] Example 12: Determination of overall survival rate of mice after intestinal ischemia-reperfusion

[0114] A mouse model of intestinal ischemia for 50 minutes followed by reperfusion for 2 hours was constructed according to Example 11. The number of mice surviving the intestinal ischemia-reperfusion injury was recorded within 48 hours. Based on the total number of mice subjected to intestinal ischemia-reperfusion, the survival rate of the model mice was calculated, and the results are shown in Figure 10. The survival rate was calculated using the same method as in Example 6.

[0115] According to Figure 10, compared with the control group (0%), both doses of 7-ketolithocholic acid significantly improved the survival rate of mice with intestinal ischemia-reperfusion injury (40 mg / kg: 20%; 80 mg / kg: 31.25%).

[0116] Example 13: Determination of intestinal pathological score and intestinal fatty acid-binding protein (I-FABP) level after intestinal ischemia-reperfusion in mice

[0117] A mouse model of intestinal ischemia for 50 minutes followed by reperfusion for 2 hours was constructed according to Example 11. The experimental group received intraperitoneal injection of 7-ketolithocholic acid (40 mg / kg or 80 mg / kg), while the control group received an intraperitoneal injection of the same volume of control solvent. After 2 hours of reperfusion, mouse intestinal tissue was harvested for H&E staining. The damage to the intestinal mucosal structure was observed under a microscope, and pathological scoring was performed. Simultaneously, the level of I-FABP in each sample was detected using a mouse intestinal fatty acid-binding protein (I-FABP) enzyme-linked immunosorbent assay (ELISA) kit. The results are shown in Figure 11.

[0118] According to Figure 11, regarding intestinal histopathological scores, compared to the control group (6.14±0.69), both doses of 7-ketolithocholic acid significantly reduced the intestinal histopathological scores in mice with intestinal ischemia-reperfusion injury (40 mg / kg: 4.50±1.69; 80 mg / kg: 4.29±1.51). Regarding injury markers, compared to the control group (100±8.57%), both doses of 7-ketolithocholic acid significantly reduced the intestinal histopathological scores in mice with intestinal ischemia-reperfusion injury (40 mg / kg: 88±7.45%; 80 mg / kg: 84±7.36%).

[0119] Example 14: Constructing a mouse model of 90 minutes of cerebral ischemia followed by 24 hours of reperfusion (MCAO).

[0120] 1. Experimental animals: C57BL / 6J mice were used as the research subjects, and the breeding environment was the same as in Example 5. The experiment was divided into two groups: the cerebral ischemia-reperfusion control group (denoted as MCAO) and the 7-ketolithocholic acid 40mg / kg administration group (denoted as 7K+MCAO).

[0121] 2. Preparation of 7-ketolithocholic acid administration solution: Same as described in Example 5.

[0122] 3. Before modeling, the experimental group mice were injected intraperitoneally with the corresponding dose of 7-ketolithocholic acid solution, while the control group mice were injected with the same volume of solvent.

[0123] 4. Establishment of a mouse model of cerebral ischemia-reperfusion: Mice were anesthetized with 1.5% isoflurane by inhalation. After hair removal and disinfection of the neck, the mice were placed in a supine position. Using the classic suture ligation method, the mice were fixed to a heated pad to maintain body temperature after hair removal and disinfection of the neck. The skin was cut along the midline of the neck, and the neck glands and muscle tissue were bluntly dissected to clearly expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Surgical sutures were used to ligate the proximal end of the CCA and the distal end of the ECA, and the ICA was temporarily clamped with micro-arterial clamps. Subsequently, a small incision was made in the main trunk of the ECA, and a specially made nylon suture with silicone-coated ends was slowly inserted into the ICA through the ECA incision, advancing approximately 9 to 11 mm towards the head until slight resistance was felt, indicating that the suture had successfully reached and blocked the origin of the middle cerebral artery (MCA). The suture was gently ligated and fixed to the blood vessel with surgical sutures to prevent slippage. After 90 minutes of ischemia, the suture embolus was carefully removed to restore blood flow to the middle cerebral artery. The ECA stump was ligated, and the neck skin was sutured layer by layer. After modeling, the mice were placed in a warm environment until they regained consciousness, and then returned to their original cages for continued feeding and observation.

[0124] Example 15: Determination of overall survival rate in mice after cerebral ischemia-reperfusion

[0125] Using the mouse cerebral ischemia-reperfusion model constructed in Example 14 as the research subject, the survival status of the mice was recorded and the survival rate was calculated within 10 days after model establishment. The survival rate calculation method was the same as in Example 6.

[0126] According to Figure 12, the survival rate of mice in the 7-ketolithocholic acid group (48.57%) was significantly higher than that of mice in the control group (30.76%).

[0127] Example 16: Determination of cerebral infarction area and neurological function score after cerebral ischemia-reperfusion in mice

[0128] Using a mouse model of 90 minutes of cerebral ischemia followed by 24 hours of reperfusion, constructed according to Example 14, the experimental group received intraperitoneal injection of 7-ketolithocholic acid (40 mg / kg), while the control group received the same volume of control solvent. After model establishment, blinded observers used the National Institutes of Health Scale (NIHSS) to assess the degree of neurological deficit in the mice after reperfusion. Following the assessment, brain tissue was harvested and sectioned for TTC staining to accurately assess the size of the cerebral infarction area. The brain was removed and flash-frozen at -20°C for about 1 hour. The brain tissue was then uniformly transversely sectioned into 1 mm thick slices. The slices were stained in 1% triphenyltetrazolium chloride (TTC) solution at 37°C for 15 min in the dark, and then fixed in 4% paraformaldehyde for 1-2 hours. Finally, the heart slices were photographed under a microscope. The area of ​​necrotic and viable myocardium was quantified using ImageJ software. Infarcted areas were white and non-infarcted areas were black. The infarcted area of ​​the brain was expressed as a percentage of infarcted area / non-infarcted area. The results are shown in Figure 13.

[0129] According to Figure 13, TTC staining images and quantitative analysis results showed that, compared with the control group (53.78±7.27), administration of 7-ketolithocholic acid significantly reduced the cerebral infarction area of ​​mice after cerebral ischemia-reperfusion (16.22±2.68); in terms of neurological behavioral assessment, compared with the control group (6.72±2.68), 7-ketolithocholic acid significantly reduced the neurological function score of mice after cerebral ischemia-reperfusion (2.94±1.92).

[0130] Example 17: Comparison of the protective effects of previously common bile acids (cholic acid and ursodeoxycholic acid) with 7-ketolithocholic acid on organ damage.

[0131] A cardiac ischemia-reperfusion injury model was established according to Example 5. Three groups of mice were intraperitoneally injected with the same concentration of 7-ketolithocholic acid (7K), cholic acid (CA), and ursodeoxycholic acid (UDCA) at the same time. At the same time, the activity capacity of mice after cardiac ischemia-reperfusion injury (a specific indicator of the overall improvement of mouse survival function by 7-ketolithocholic acid) and myocardial infarction area (a commonly used indicator for judging ischemia-reperfusion injury) were detected and compared according to Examples 7 and 8.

[0132] According to Figure 14A, compared with the control group (47.11±4.72%), 7-ketolithocholic acid significantly reduced the infarct area (28.33±7.92%), cholic acid significantly reduced the infarct area (31.77±1.85%), and ursodeoxycholic acid significantly reduced the infarct area (36.28±9.43%). All three compounds reduced the infarct area, but 7-ketolithocholic acid showed a better improvement than cholic acid and ursodeoxycholic acid.

[0133] According to Figure 14B, the total distance traveled (330.13±238.35 cm) and the average speed (1.30±0.91 cm / s) of the control group mice were significantly lower than those of the sham-operated group mice (1323.65±249.86 cm) and the average speed (4.41±0.81 cm / s). The total distance traveled (1061.35±319.71 cm) and the average speed (3.64±1.50 cm / s) of the 7-ketolithocholic acid group mice were significantly better than those of the control group, the cholic acid group, and the ursodeoxycholic acid group. The total distance traveled (439.88±250.77 cm) and average speed (1.46±0.84 cm / s) of mice in the cholic acid group showed no significant improvement compared to the control group. Similarly, the total distance traveled (463.69±127.43 cm) and average speed (1.55±0.42 cm / s) of mice in the ursodeoxycholic acid group also showed no significant improvement compared to the control group. This indicates that 7-ketolithocholic acid can significantly improve the overall activity level of mice after myocardial ischemia-reperfusion injury, while cholic acid and ursodeoxycholic acid did not have this effect.

[0134] Example 18: Survival determination of 7-ketolithocholic acid structural analogues in ischemia-reperfusion injury of heart, brain, kidney, lung, liver and intestinal cells.

[0135] 1. Experimental Subjects: The ischemia-reperfusion injury models of heart, brain, kidney, lung, liver, and intestinal cell lines constructed according to Examples 3 and 4 were used as the research subjects. In addition to the normoxic control group (designated as Control group), the ischemia-reperfusion blank control group (designated as Group 0), and the 1% DMSO solvent control group (designated as 1% DMSO group), in order to further verify the structural specificity of 7-ketolithocholic acid in the prevention and treatment of ischemia-reperfusion injury, this example selected a variety of analogs with similar structures for parallel control experiments.

[0136] 2. 7-Ketolithocholic acid structural analogs: The selected structural analogs included lithocholic acid (LCA), 12-ketodeoxycholic acid (12-keto-DCA), 12-ketochenodeoxycholic acid (12-keto-CDCA), and 7-keto-Deoxycholic acid (7-keto-DCA). These analogs were added to the cell culture medium of the corresponding groups at final concentrations of 1 μM and 10 μM, respectively, for intervention.

[0137] Lithocholic acid, with CAS number 434-13-9, has the molecular formula C2. 24H 40 O3 is characterized by having one less 7-keto group than 7-ketolithocholic acid.

[0138] The 12-ketodeoxycholic acid, CAS number 5130-29-0, has the molecular formula C1. 24 H 38 O4.

[0139] The 12-ketochenodeoxycholic acid, CAS number 2458-08-4, has the molecular formula C1. 24 H 38 O5 is characterized by having one more 7-hydroxyl group than 12-ketodeoxycholic acid.

[0140] The 7-keto-deoxycholic acid, CAS number 911-40-0, has the molecular formula C7. 24 H 38 O5 is characterized by having one more hydroxyl group at position 12 than 7-ketolithocholic acid.

[0141] 3. Cell viability determination: The cell ischemia-reperfusion injury model constructed according to step 1 of Example 3 was used as the research object. Cells were removed, and 10 μL of Cell Counting Kit-8 solution was added to each well. The cells were placed in a cell culture incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0142] According to Figure 15, the cell viability of the normoxic control group was standardized to 100%. After ischemia-reperfusion injury (blank control group), the viability of cardiomyocytes H9C2 (29.45±1.36%), brain-derived neurons HT-22 (46.73±4.47%), kidney-derived 293T (53.78±0.99%), liver-derived BRL-3A (38.72±2.75%), lung-derived BEAS-2B (57.02±3.28%), and intestinal-derived IEC-6 (71.92±3.99%) were all significantly lower than those of the normoxic control group (1%). In the DMSO group, the survival rates of H9C2 (31.45±1.29%), HT-22 (46.50±2.17%), 293T (59.30±5.44%), BRL-3A (41.00±2.02%), BEAS-2B (55.74±3.12%), and IEC-6 (80.93±7.80%) cells showed no significant improvement. However, the survival rates of H9C2 cells (1μM group: 37.31±2.41%), 293T cells (1μM group: 74.18±3.35%; 10μM group: 71.30±2.69%), and IEC cells (10μM group: 95.57±7.05%) were significantly higher in the DMSO group than in the ischemia-reperfusion injury group. According to the foregoing embodiments, 7-ketolithocholic acid exhibits broad and efficient protective effects against ischemia-reperfusion injury, improving the survival rate of cardiac, cerebral, renal, hepatic, intestinal, and pulmonary cells after ischemia-reperfusion injury, increasing overall survival after ischemia-reperfusion, and improving physical activity, myocardial infarction area, cardiac function, and cardiac troponin-I levels. The overall protective effect of 7-ketolithocholic acid is significantly superior to other 7-ketolithocholic acid analogs, demonstrating that the 7-ketolithocholic acid structure has high specificity and significant advantages in the prevention, treatment, or adjuvant therapy of organ ischemia-reperfusion injury. Furthermore, 12-ketodeoxycholic acid can improve the survival rate of cardiac, renal, and intestinal cells after ischemia-reperfusion injury, indicating its therapeutic or adjuvant therapeutic efficacy against ischemia-reperfusion injury of the heart, intestine, and kidney.

[0143] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

The use of 1,7-ketolithocholic acid or a pharmaceutically acceptable salt thereof, or a prodrug or metabolite thereof, in the preparation of medicaments for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury. Preferably, the pharmaceutically acceptable salts of 7-ketolithocholic acid include salts that may be present in the acidic groups of 7-ketolithocholic acid, such as sodium salts, calcium salts, and potassium salts. Preferably, the ischemia-reperfusion injury is selected from one or more ischemic conditions grouped into myocardial ischemia, cerebral ischemia, renal ischemia, hepatic ischemia, ischemia-reperfusion cardiomyopathy, skin ischemia, intestinal ischemia, gastric ischemia, pulmonary ischemia, pancreatic ischemia, skeletal muscle ischemia, abdominal muscle ischemia, limb ischemia, ischemia-reperfusion colitis, mesenteric ischemia, and asymptomatic ischemia. More preferably, the ischemia-reperfusion injury is selected from ischemia-reperfusion injuries of organs such as the heart, brain, kidneys, liver, intestines, and lungs. Preferably, the ischemia-reperfusion injury is an injury caused by ischemia, including ischemic diseases, organ-related surgery, trauma, or organ transplantation.

2. According to claim 1, the drug can be administered before, during, or after ischemia-reperfusion injury occurs, for example, as a pretreatment for reperfusion surgery.

3. The application according to claim 1, wherein the drug can improve survival rate after ischemia-reperfusion injury; for example, the drug can improve one or more of the following after cardiac ischemia-reperfusion injury: survival rate, physical activity capacity, myocardial infarction area, cardiac function, and cardiac troponin-I level; for example, the drug can improve survival rate after intestinal ischemia-reperfusion injury, such as the drug can improve overall survival rate after intestinal ischemia-reperfusion injury, pathological degree of intestinal injury, and one or more of intestinal injury markers; for example, the drug can improve survival rate after cerebral ischemia-reperfusion injury, such as the drug can improve overall survival rate after cerebral ischemia-reperfusion injury, reduce cerebral infarction area, and reduce the degree of neurological function impairment.

4. The use of a pharmaceutical composition containing 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof in the preparation of a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury, preferably, the ischemia-reperfusion injury being selected from ischemia-reperfusion injuries of organs such as the heart, brain, kidney, liver, intestine and lung. 5.7-Ketolithocholic acid analogues are used in the preparation of drugs for the prevention, improvement, treatment or adjunctive treatment of ischemia-reperfusion injury of the heart, intestine and kidney, wherein the 7-ketolithocholic acid analogue is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

6. The use of a pharmaceutical composition containing a 7-ketolithocholic acid analog in the preparation of a medicament for the prevention, treatment or adjunctive treatment of ischemia-reperfusion injury of the heart, intestine and kidney, wherein the 7-ketolithocholic acid analog is 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

7. The application according to any one of claims 1-6, wherein the medicament further comprises a pharmaceutically acceptable carrier and / or excipients (such as diluents); Alternatively, the drug is suitable for preparation into formulations for intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, or oral administration. Preferably, the drug is an oral formulation, an injectable formulation, or an enema liquid formulation. Oral formulations include tablets, capsules, pills, powders, granules, suspensions, and drops. Injectable formulations include injection solutions and powder injections. More preferably, the tablets include ordinary tablets, orally disintegrating tablets, dispersible tablets, or sustained-release tablets.

8. A medicament for the prevention, treatment, or adjunctive treatment of ischemia-reperfusion injury, said medicament containing 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof, and optionally a pharmaceutically acceptable carrier and / or excipient, preferably, the ischemia-reperfusion injury being selected from ischemia-reperfusion injuries of organs such as the heart, brain, kidney, liver, intestine, and lung.

9. A medicament for the prevention, treatment, or adjunctive treatment of ischemia-reperfusion injury of the heart, intestine, and kidney, said medicament comprising a 7-ketolithocholic acid analogue, and optionally a pharmaceutically acceptable carrier and / or excipient, said 7-ketolithocholic acid analogue being 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

10. A method for preventing, treating, or adjunctive treating ischemia-reperfusion injury, the method comprising administering to a subject in need a therapeutically effective amount of 7-ketolithocholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof, or the method comprising administering to a subject in need a therapeutically effective amount of a 7-ketolithocholic acid analogue, said 7-ketolithocholic acid analogue being 12-ketodeoxycholic acid or a pharmaceutically acceptable salt thereof or a prodrug or metabolite thereof.

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