Application of 12-ketolithocholic acid in the preparation of medicine for preventing or treating acute ischemic intestinal injury

Abstract

This application relates to the use of 12-ketolithocholic acid in the preparation of a drug for preventing or treating acute ischemic intestinal injury. This application attempts to use 12-ketolithocholic acid (12-KLCA) for the prevention or treatment of acute ischemic intestinal injury, and the application has been validated in a constructed classic intestinal ischemia-reperfusion model. The validation results show that 12-KLCA significantly improves intestinal histopathological damage induced by intestinal ischemia-reperfusion in mice, increases the survival rate of mice, and reduces the protein expression level of the apoptotic factor activating caspase-3. The effect is significant, safe, non-toxic, and has minimal side effects.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical technology, and in particular to the use of 12-ketolithocholic acid in the preparation of a drug for preventing or treating acute ischemic intestinal injury. Background Art

[0002] Intestinal ischemia-reperfusion (I / R) injury is a common type of tissue and organ injury in clinical practice, often occurring during treatments such as abdominal aortic aneurysm surgery, cardiopulmonary resuscitation, small bowel transplantation, and extracorporeal circulation surgery. Studies have shown that intestinal I / R injury occurs widely and has a high mortality rate, seriously endangering the life, health, and safety of surgical patients. However, a sensitive detection indicator that can provide early warning or early diagnosis of intestinal I / R injury has not yet been found, and there is a lack of effective treatments. Active correction of the primary disease and symptomatic supportive treatment are still the main treatments, and drugs to improve intestinal injury caused by intestinal I / R are still very scarce. Therefore, the development of new drugs for the prevention and treatment of acute ischemic intestinal injury is of great significance for improving the postoperative recovery of critically ill patients and surgical patients.

[0003] 12-Ketolithocholic Acids (12-KLCA) is a secondary bile acid with the molecular formula C 24 H 38 O4, with a molecular weight of 390.56, has a chemical structure as shown in the following formula (1).

[0004] Formula (1)

[0005] 12-KLCA is produced by oxidation of lithocholic acid in the intestine or liver through the intestinal microbial enzyme system. It participates in metabolic regulation, inflammation and oxidative stress, and intestinal barrier protection, and has broad application prospects. For example, studies have demonstrated that 12-KLCA abundance is reduced in patients with ulcerative colitis. Furthermore, 12-KLCA can mitigate the development and progression of colitis in mice by inhibiting the secretion of the proinflammatory cytokine IL-17A by type 3 innate lymphocytes. Furthermore, by inhibiting inflammatory pathways mediated by IL-17 and TNF, 12-KLCA can significantly protect against CCl4-induced liver damage. Therefore, current research on the mechanisms and pathologies of 12-KLCA primarily focuses on its anti-inflammatory activity, and its effects on acute ischemic intestinal injury have not been reported. Summary of the Invention

[0006] Based on this, the present application provides the use of 12-ketolithocholic acid in the preparation of a drug for preventing or treating acute ischemic intestinal injury.

[0007] The specific technical solutions are as follows:

[0008] Application of 12-ketolithocholic acid in the preparation of medicines for preventing or treating acute ischemic intestinal injury.

[0009] In one embodiment, the acute ischemic intestinal injury includes intestinal ischemia-reperfusion injury.

[0010] In one embodiment, 12-ketolithocholic acid is used in the preparation of a drug for preventing acute ischemic intestinal injury.

[0011] In one embodiment, the prevention or treatment refers to ameliorating pathological damage in acute ischemic intestinal tissue.

[0012] In one embodiment, the prevention or treatment refers to reducing the expression level of activated caspase-3 protein.

[0013] In one embodiment, the drug comprises an active ingredient, and the active ingredient comprises the 12-ketolithocholic acid.

[0014] In one embodiment, the active ingredient is 12-ketolithocholic acid.

[0015] In one embodiment, the drug includes a pharmaceutically acceptable excipient.

[0016] In one embodiment, the dosage form of the drug includes injection, oral solution, pill, powder, ointment, tablet, granule, powder or capsule.

[0017] In one embodiment, the administration route of the drug includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0018] This application attempts to use 12-ketolithocholic acid (12-KLCA) to prevent or treat acute ischemic intestinal injury, and the application has been verified in a constructed classic intestinal ischemia-reperfusion model. The verification results show that 12-KLCA significantly improves intestinal tissue pathological damage induced by intestinal ischemia-reperfusion in mice, increases the survival rate of mice, and reduces the protein expression level of apoptosis factor-activated caspase-3. The effect is obvious, safe and non-toxic, and has few side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the results of 12-KLCA improving the survival rate of mice with intestinal ischemia-reperfusion.

[0020] Figure 2 The following are the pathological results of 12-KLCA improving intestinal tissue damage induced by intestinal ischemia-reperfusion in mice. (A) is the HE staining image of the morphological changes of intestinal tissue in each group, and (B) is the quantitative scoring result of intestinal tissue damage in each group.

[0021] Figure 3 The results show that 12-KLCA reduces the protein expression level of apoptotic factor activated caspase-3 in intestinal tissue of mice after intestinal ischemia-reperfusion. (A) is the Western Blotting band diagram of intestinal tissue of each group, and (B) is the quantitative scoring result of Western Blotting of intestinal tissue of each group. DETAILED DESCRIPTION

[0022] The following, in conjunction with specific examples, further details the use of the 12-ketolithocholic acid of the present application in the preparation of a medicament for preventing or treating acute ischemic intestinal injury. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0024] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0025] Unless otherwise specified, the percentage contents mentioned in this application refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0026] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0027] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0028] As used herein, the term "ischemia" refers to a condition that may occur in any organ or tissue due to a lack of oxygen and / or metabolic product supply. Ischemia occurs when there is an imbalance between oxygen supply and demand due to insufficient perfusion (i.e., blood supply). Insufficient oxygen supply may be caused by thrombosis, the presence of atherosclerotic narrowing, restenosis, anemia, stroke, arterial coagulation, vasoconstriction, and / or endothelial dysfunction of the microvasculature (Tacu-Subo syndrome).

[0029] As used herein, the term "ischemia / reperfusion injury" refers to damage to an organ or tissue caused by insufficient blood supply to the organ or tissue during the ischemic period before reperfusion begins (i.e., ischemic injury is damage caused by ischemia during the period between the onset of ischemia and the start of reperfusion). A typical and pathological manifestation of ischemic injury is paleness of the ischemic area. In contrast, during reperfusion, non-necrotic ischemic tissue regains its physiological color.

[0030] The ischemic injury can be caused, for example, by atherosclerosis, thrombosis, thromboembolism, lipid embolism, hemorrhage, stents, surgery, angioplasty, intraoperative bypass grafting, organ transplantation, global ischemia, myocardial infarction, vasoconstriction, microvascular dysfunction, and / or a combination of two or more thereof.

[0031] Ischemic injury may involve symptoms such as chest discomfort, shortness of breath, discomfort in other areas of the upper body, feeling nauseous, and / or anxious.

[0032] As used herein, the term "reperfusion" relates to the restoration of blood flow to ischemic tissue. Although there are clear benefits to reperfusing blood to ischemic tissue, it is well known that reperfusion itself can induce a series of adverse reactions that paradoxically harm the tissue.

[0033] As used herein, the term "reperfusion injury" refers to the organ or tissue damage caused when the blood supply returns to the organ or tissue after the ischemic period. Therefore, reperfusion injury is the damage caused during the time between the start of reperfusion and the end of reperfusion (usually, the major part of this damage will be caused within the first few minutes of reperfusion). The potential mechanism of reperfusion injury is complex and multifactorial. Without limitation, the lack of oxygen and nutrients in the blood during the ischemic period has caused the recovery of circulation therein by inducing oxidative stress rather than restoring normal function. The oxidative stress associated with reperfusion may cause damage to the affected tissue or organ. The biochemical characteristics of reperfusion injury are oxygen depletion during the ischemic event, followed by reoxygenation during the reperfusion period and the generation of reactive oxygen species. The damage that occurs with reperfusion is the result of interaction between the substances accumulated during the ischemic period and the substances transmitted during reperfusion. The basis of these events is oxidative stress, which is defined as the imbalance between oxygen free radicals and the endogenous scavenging system. The result is cell damage and death, which is initially local but eventually becomes systemic if not controlled.

[0034] Reperfusion injury can be caused by, for example, a mechanical event, or by one or more surgical procedures or other therapeutic interventions to restore blood flow to a tissue or organ that has experienced reduced blood flow. Such surgical procedures include, for example, coronary artery bypass graft surgery, coronary angioplasty, and organ transplant surgery. In specific embodiments, reperfusion injury is caused by the treatment of an ischemic process due to rupture / erosion of an atherosclerotic plaque and superimposition of a thrombus, thromboembolism, lipid embolism, hemorrhage, stents, surgery, angioplasty, termination of a bypass during surgery, organ transplantation, total ischemia, vasoconstriction, or microvascular dysfunction, or a combination thereof.

[0035] Reperfusion injury may involve symptoms of palpitations, acute respiratory distress, fatigue, and / or edema.

[0036] As used herein, the term "prevention" means reducing the risk of disease in individuals or groups, or reducing the severity of the disease and delaying the progression of the disease by taking a series of proactive intervention measures. Specifically in this application, "prevention" means administering the drug of this application before ischemia-reperfusion, which can alleviate the damage to intestinal tissue after ischemia-reperfusion. Disease prevention is a proactive health management concept. Avoiding or reducing the harm of disease through "upstream intervention" is far more valuable than treatment after the disease occurs.

[0037] As used herein, the term "treatment" means a series of medical actions that intervene in an existing disease through medical means, techniques, or methods, with the goal of eliminating the cause, alleviating symptoms, controlling the progression of the disease, promoting tissue repair, restoring function, or alleviating pain. Specifically, in this application, "treatment" refers to the administration of the drug of this application after intestinal ischemia-reperfusion injury to improve pathological damage to intestinal tissue.

[0038] Some examples of the present application provide the use of 12-ketolithocholic acid in the preparation of a medicament for preventing or treating acute ischemic intestinal injury.

[0039] In some examples, the acute ischemic intestinal injury includes intestinal ischemia / reperfusion injury.

[0040] Among some of these examples is the use of 12-ketolithocholic acid in the preparation of a medicament for preventing acute ischemic intestinal injury.

[0041] In some of these examples, prevention or treatment refers to ameliorating pathological damage to acute ischemic intestinal tissue.

[0042] In some examples, prevention or treatment refers to reducing the protein expression level of activated caspase-3. It is understood that activated caspase-3 is the main apoptotic factor in intestinal tissue after intestinal ischemia-reperfusion injury. Reducing its protein expression level can reflect a preventive or therapeutic effect on intestinal ischemia-reperfusion injury.

[0043] In some examples, the drug includes an active ingredient, and the active ingredient includes the 12-ketolithocholic acid. It is understandable that the active ingredient of the drug can be only 12-ketolithocholic acid, or 12-ketolithocholic acid can be used in combination with other active ingredients. Furthermore, the active ingredient is the 12-ketolithocholic acid, that is, effective prevention or treatment of intestinal ischemia-reperfusion injury can be achieved by administering only 12-ketolithocholic acid.

[0044] In some examples, the medicament includes a pharmaceutically acceptable excipient.

[0045] In some examples, the dosage form of the drug includes injection, oral solution, pill, powder, ointment, tablet, granule, powder or capsule.

[0046] In some examples, the administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0047] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0048] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0049] Example 1:

[0050] This example provides experimental research showing that 12-KLCA can improve the survival rate of mice with intestinal ischemia-reperfusion injury.

[0051] 1. Experimental Materials

[0052] 1.1 Experimental Animals

[0053] Sixty male C57BL / 6J mice aged 6 to 8 weeks, weighing 18 g to 22 g, were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital and raised at the SPF-level Animal Experimental Department of Nanfang Hospital, Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and complied with animal ethics requirements.

[0054] 1.2 Reagents and Instruments

[0055] 12-KLCA (Med Chem Express, USA); isoflurane (Reward Life Science Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk suture (Ningbo Medical Suture Co., Ltd.); normal saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffer saline (PBS), pH 7.4 (Gibco).

[0056] 2. Experimental methods and results

[0057] 2.1 Animal Experiments

[0058] (1) Establishment of the superior mesenteric artery I / R model in mice (the intestinal ischemia-reperfusion animal model is a classic perioperative intestinal injury model constructed by superior mesenteric artery clamping):

[0059] 1) Before surgery, mice were fasted for 12 hours with free access to water. They were anesthetized by isoflurane inhalation and the superior mesenteric artery was clamped with a noninvasive microvascular artery clamp to block blood flow.

[0060] 2) After intestinal ischemia lasts for 60 minutes, the arterial clamp is released to restore blood supply and intestinal reperfusion is performed. After checking that there is no bleeding in the abdominal cavity, the peritoneum, muscles and skin are sutured layer by layer with sterile silk sutures.

[0061] 3) After occlusion and during reperfusion, 0.5 mL of warm saline at approximately 37°C was subcutaneously injected for fluid resuscitation. The survival and perfusion time of the mice were observed and recorded.

[0062] (2) Experimental groups:

[0063] Sixty C57BL / 6 mice aged 6 to 8 weeks were randomly divided into sham operation group (Sham), intestinal I / R group (I / R), and intestinal I / R + 12-KLCA group (I / R+12-KLCA).

[0064] 1) Sham operation group (Sham): After 1-hour pretreatment with intraperitoneal injection of PBS solution, the patient underwent laparotomy and the superior mesenteric artery was isolated but not clamped;

[0065] 2) Intestinal I / R group (I / R): After 1 h of pretreatment with intraperitoneal injection of PBS solution, the intestinal I / R model was established;

[0066] 3) Intestinal I / R + 12-KLCA group (I / R + 12-KLCA): The intestinal I / R model was established after intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h pretreatment.

[0067] 2.2 Experimental Results

[0068] Please refer to the experimental results Figure 1 , Figure 1 This figure shows the results of 12-KLCA improving the survival rate of mice with intestinal ischemia-reperfusion; Figure 1 The symbols in the figure mean: data were analyzed using the Log-rank (Mantel-Cox) test, and * indicates that the difference compared with the I / R group was statistically significant at p < 0.05. Figure 1 The results showed that 12-KLCA treatment could significantly prolong the survival time of mice after 60 minutes of ischemia and reperfusion, and improve the survival rate of mice.

[0069] Example 2:

[0070] This example provides an experimental study on the effect of 12-KLCA on alleviating intestinal pathological morphological damage induced by intestinal ischemia-reperfusion in mice.

[0071] 1. Experimental Materials

[0072] 1.1 Experimental Animals

[0073] Twenty-four male C57BL / 6J mice aged 6 to 8 weeks, weighing 18 g to 22 g, were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital and raised at the SPF-level Animal Experimental Department of Nanfang Hospital, Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and complied with animal ethics requirements.

[0074] 1.2 Reagents and Instruments

[0075] 12-KLCA (Med Chem Express, USA); isoflurane (Reward Life Science Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk suture (Ningbo Medical Suture Co., Ltd.); normal saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffered saline (PBS, pH 7.4) (Gibco); hematoxylin-eosin staining (Beijing Regen Biological Company); anhydrous ethanol (Guangdong Guanghua Science and Technology Co., Ltd.); xylene (Guangdong Guanghua Science and Technology Co., Ltd.); paraffin (Leica); 4% paraformaldehyde (Beijing Solarbio Science and Technology Co., Ltd.); neutral gum (Solarbio); and an automated fluorescence microscope (Olympus).

[0076] 2. Experimental methods and results

[0077] 2.1 Animal Experimentation

[0078] (1) Establishment of the superior mesenteric artery I / R model in mice (the intestinal ischemia-reperfusion animal model is a classic perioperative intestinal injury model constructed by superior mesenteric artery clamping):

[0079] 1) Before surgery, mice were fasted for 12 hours with free access to water. They were anesthetized by isoflurane inhalation and the superior mesenteric artery was clamped with a noninvasive microvascular artery clamp to block blood flow.

[0080] 2) After intestinal ischemia lasts for 60 minutes, the arterial clamp is released to restore blood supply and intestinal reperfusion is performed. After checking that there is no bleeding in the abdominal cavity, the peritoneum, muscles and skin are sutured layer by layer with sterile silk sutures.

[0081] 3) After occlusion and during reperfusion, 0.5 mL of warm saline at approximately 37°C was subcutaneously injected for fluid resuscitation. After 2 hours of perfusion, the intestinal tissue of the mice was collected for examination.

[0082] (2) Experimental groups:

[0083] Twenty-four C57BL / 6 mice aged 6 to 8 weeks were randomly divided into sham group (Sham), intestinal I / R group (I / R), and intestinal I / R + 12-KLCA group (I / R+12-KLCA).

[0084] 1) Sham operation group (Sham): After 1-hour pretreatment with intraperitoneal injection of PBS solution, the patient underwent laparotomy and the superior mesenteric artery was isolated but not clamped;

[0085] 2) Intestinal I / R group (I / R): After 1 h of pretreatment with intraperitoneal injection of PBS solution, the intestinal I / R model was established;

[0086] 3) Intestinal I / R + 12-KLCA group (I / R + 12-KLCA): The intestinal I / R model was established after intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h pretreatment.

[0087] 2.2 Detection of pathological morphological changes in intestinal tissue

[0088] Fresh intestinal tissue was immersed in 4% paraformaldehyde and fixed for 24 hours, then dehydrated, embedded, and sectioned. Hematoxylin-eosin staining was performed and the sections were sealed with neutral gum. The pathomorphological changes of intestinal tissue were observed under an automatic fluorescence microscope, and the modified Chiu method was used to grade and score intestinal mucosal damage.

[0089] 2.3 Experimental Results

[0090] See the results Figure 2 , Figure 2 The figure shows the pathological results of 12-KLCA improving intestinal tissue damage induced by intestinal ischemia-reperfusion in mice, where: Figure 2 (A) HE staining of intestinal tissue morphological changes in each group. Figure 2 (B) Quantitative scoring results of intestinal tissue damage in each group, the image scale is 100µm; Figure 2The symbols in the figure mean: the data were analyzed by one-way ANOVA test, * indicates that the difference compared with the I / R group was statistically significant at p < 0.05, and ** indicates that the difference compared with the I / R group was statistically significant at p < 0.01. Figure 2 (A) and Figure 2 (B) HE staining and scoring of intestinal tissue showed that the I / R model group showed loss of villi and dilation of capillaries in the apical region. Treatment with 12-KLCA significantly ameliorated these morphological changes in the intestinal tissue of mice induced by intestinal I / R. These data suggest that 12-KLCA can mitigate the pathological changes in the intestinal tissue of mice induced by intestinal ischemia-reperfusion.

[0091] Example 3:

[0092] This example provides experimental research showing that 12-KLCA can reduce the protein expression level of caspase-3, apoptotic factor activated in intestinal tissues of mice after intestinal ischemia-reperfusion.

[0093] 1. Experimental Materials

[0094] 1.1 Experimental Animals

[0095] Twenty-four male C57BL / 6J mice aged 6 to 8 weeks, weighing 18 g to 22 g, were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital and raised at the SPF-level Animal Experimental Department of Nanfang Hospital, Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and complied with animal ethics requirements.

[0096] 1.2 Reagents and Instruments

[0097] 12-KLCA (Med Chem Express, USA); isoflurane (Reward Life Science Technology Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk suture (Ningbo Medical Suture Co., Ltd.); normal saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffered saline (PBS, pH 7.4) (Gibco); BCA assay kit (Thermo Fisher Scientific, USA); activated caspase-3 rabbit monoclonal primary antibody (Cell Signaling Technology, USA); Alexa Fluor® 594 donkey anti-rabbit IgG secondary antibody (Life technologies, USA); glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology, USA); PVDF membrane (Millipore, USA); and blot fluorescence chemiluminescence imager (Bio-Red, USA).

[0098] 2. Experimental methods and results

[0099] 2.1 Animal Experimentation

[0100] (1) Establishment of the superior mesenteric artery I / R model in mice (the intestinal ischemia-reperfusion animal model is a classic perioperative intestinal injury model constructed by superior mesenteric artery clamping):

[0101] 1) Before surgery, mice were fasted for 12 hours with free access to water. They were anesthetized by isoflurane inhalation and the superior mesenteric artery was clamped with a noninvasive microvascular artery clamp to block blood flow.

[0102] 2) After intestinal ischemia lasts for 60 minutes, the arterial clamp is released to restore blood supply and intestinal reperfusion is performed. After checking that there is no bleeding in the abdominal cavity, the peritoneum, muscles and skin are sutured layer by layer with sterile silk sutures.

[0103] 3) After occlusion and during reperfusion, 0.5 mL of warm saline at approximately 37°C was subcutaneously injected for fluid resuscitation. After 2 hours of perfusion, the intestinal tissue of the mice was obtained for examination.

[0104] (2) Experimental groups:

[0105] Twenty-four C57BL / 6 mice aged 6 to 8 weeks were randomly divided into sham operation group (Sham), intestinal I / R group (I / R), and intestinal I / R+12-KLCA group (I / R+12-KLCA).

[0106] 1) Sham operation group (Sham): After 1-hour pretreatment with intraperitoneal injection of PBS solution, the patient underwent laparotomy and the superior mesenteric artery was isolated but not clamped;

[0107] 2) Intestinal I / R group (I / R): After 1 h of pretreatment with intraperitoneal injection of PBS solution, the intestinal I / R model was established;

[0108] 3) Intestinal I / R + 12-KLCA group (I / R + 12-KLCA): The intestinal I / R model was established after intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h pretreatment.

[0109] 2.2. Detection of protein expression level of intestinal apoptosis factor-activated caspase-3

[0110] Fresh intestinal tissue was quickly frozen in liquid nitrogen and then mechanically ground. Lysis buffer was added to decompose the tissue and centrifuged for quantitative analysis using BCA protein. The tissue was then prepared with gel, loaded with samples, electrophoresed, transferred to a membrane, blocked, and incubated with antibodies. The blot was then developed using a fluorescent chemiluminescence imager. The grayscale value of each band was analyzed using Image JV1.8.0, and quantitative scoring was performed.

[0111] 2.3 Experimental Results

[0112] See the results Figure 3 , Figure 3 The results show that 12-KLCA reduces the protein expression level of apoptosis factor activated caspase-3 in intestinal tissue of mice after intestinal ischemia-reperfusion, where: Figure 3 (A) is the Western Blotting band diagram of intestinal tissues in each group. Figure 3 (B) Quantitative scoring results of Western Blotting of intestinal tissues in each group; Figure 3 The symbols in the figure mean: the data were analyzed by one-way ANOVA test, and * indicates that the difference compared with the I / R group was statistically significant (p<0.05). Figure 3 (A) and Figure 3 Western blotting and quantitative scoring of intestinal tissue (B) showed that the expression of caspase-3, a caspase-3-activated protein, was significantly higher in the I / R model group. Treatment with 12-KLCA significantly reduced caspase-3 protein expression in the intestinal tissue of mice after intestinal I / R. These data suggest that 12-KLCA can reduce the level of apoptosis in the intestinal tissue of mice after intestinal ischemia-reperfusion.

[0113] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0114] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. Use of 12-ketolithocholic acid in the preparation of a drug for preventing or treating acute ischemic intestinal injury, wherein the acute ischemic intestinal injury is intestinal ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that The invention discloses an application of 12-ketolithocholic acid in the preparation of a medicine for preventing acute ischemic intestinal injury, wherein the acute ischemic intestinal injury is intestinal ischemia-reperfusion injury.

3. The use according to claim 1, characterized in that The drug can improve pathological damage of acute ischemic intestinal tissue.

4. The use according to claim 1, characterized in that The drug can reduce the protein expression level of activated caspase-3.

5. The use according to claim 1, characterized in that The 12-ketolithocholic acid serves as the sole active ingredient.

6. The use according to claim 1, characterized in that The drug includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The dosage form of the drug includes injection, oral solution, pill, powder, ointment, tablet, granule, powder or capsule.

8. The use according to claim 6, characterized in that The administration routes of the drug include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

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