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

Abstract

The invention relates to application of 12-ketolithocholic acid in preparation of a medicine for preventing or treating acute ischemic intestinal injury. According to the application, 12-ketolithocholic acid (12-KLCA) is used for preventing or treating acute ischemic intestinal injury and is verified on a constructed classical intestinal ischemia reperfusion model, and a verification result shows that the 12-KLCA obviously improves intestinal histopathologic injury induced by intestinal ischemia reperfusion of a mouse, improves the survival rate of the mouse, and has a good application prospect in prevention or treatment of acute ischemic intestinal injury. The protein expression level of the apoptosis factor activated caspase-3 is reduced, the effect is obvious, safety and non-toxicity are achieved, and the side effect is small.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and particularly 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 tissue and organ injury in clinical practice, often occurring during diagnostic and treatment processes such as abdominal aortic aneurysm surgery, cardiopulmonary resuscitation, small intestine transplantation surgery, and surgical operations under extracorporeal circulation. Research shows that the occurrence of intestinal I / R injury has a wide coverage and a high mortality rate, seriously endangering the life and health safety of surgical patients. However, at present, people have not found a sensitive detection index that can warn or early diagnose intestinal I / R injury, and there is also a lack of effective treatment means. It still mainly focuses on actively correcting the primary disease and symptomatic supportive treatment, and drugs for improving intestinal injury caused by intestinal I / R are still very lacking. Therefore, the research and development of new drugs for the prevention and treatment of acute ischemic intestinal injury is of great significance for improving the postoperative rehabilitation of critically ill patients and surgical patients.

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

[0004] Formula (1)

[0005] 12-KLCA is generated by the oxidation of lithocholic acid in the intestine or liver through the intestinal flora enzyme system, participates in the regulation of body metabolism, inflammation and oxidative stress, and intestinal barrier protection, and has a broad application prospect. For example, research has confirmed that the abundance of 12-KLCA decreases in patients with ulcerative colitis, and at the same time, 12-KLCA can slow down the occurrence and development of colitis in mice by inhibiting the secretion of pro-inflammatory factor IL-17A by type 3 innate lymphoid cells; 12-KLCA can significantly counteract CCl4-induced liver injury by inhibiting the inflammatory pathways mediated by IL-17 and TNF. It can be seen that the current mechanism and pathological research on 12-KLCA mainly focus on its anti-inflammatory activity, and its effect on acute ischemic intestinal injury has not been reported. Summary of the Invention

[0006] Based on this, this 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 solution is as follows:

[0008] Application of 12-ketolithocholic acid in the preparation of a drug 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, application of 12-ketolithocholic acid in the preparation of a drug for preventing acute ischemic intestinal injury.

[0011] In one embodiment, preventing or treating refers to improving the pathological damage of acute ischemic intestinal tissue.

[0012] In one embodiment, preventing or treating refers to reducing the protein expression level of activated caspase-3 (Cleaved caspase-3).

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

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

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

[0016] In one embodiment, the dosage form of the drug includes injection, oral liquid, pill, powder, plaster, 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) for preventing or treating acute ischemic intestinal injury, and it has been verified on the constructed classical intestinal ischemia-reperfusion model. The verification results show that 12-KLCA significantly improves the intestinal tissue pathological damage induced by intestinal ischemia-reperfusion in mice, increases the survival rate of mice, reduces the protein expression level of the apoptotic factor activated caspase-3, and the effect is obvious, safe and non-toxic, with little side effect. Description of the Drawings

[0019] Figure 1 It is a result graph of 12-KLCA increasing the survival rate of mice with intestinal ischemia-reperfusion.

[0020] Figure 2 It is a pathological result graph of 12-KLCA improving the intestinal tissue injury induced by intestinal ischemia-reperfusion in mice. Among them, (A) is the HE staining graph of the morphological changes of intestinal tissues in each group, and (B) is the quantitative scoring result of the intestinal tissue injury in each group.

[0021] Figure 3 Figure showing the results of reducing the protein expression level of apoptotic factor caspase-3 in intestinal tissue of mice after intestinal ischemia-reperfusion by 12-KLCA. Among them, (A) is the Western Blotting band diagram of intestinal tissue in each group, and (B) is the quantitative scoring result of Western Blotting of intestinal tissue in each group. Detailed implementation manners

[0022] The following further elaborates on the application of 12-ketolithocholic acid of the present application in the preparation of drugs for preventing or treating acute ischemic intestinal injury in combination with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

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

[0024] In the present application, among the technically characterized features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0025] In the present application, the percentage content involved, unless otherwise specified, refers to the mass percentage for solid-liquid mixing and solid-solid mixing, and refers to the volume percentage for liquid-liquid mixing.

[0026] In the present application, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding this component.

[0027] In the present application, the temperature parameters, unless otherwise specifically limited, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0028] In the present application, the term "ischemia" refers to a condition that may occur in any organ or tissue lacking oxygen supply and / or metabolite 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 the formation of thrombus, the presence of stenotic atherosclerosis, restenosis, anemia, stroke, arterial coagulation, vasoconstriction, and / or endothelial dysfunction of the microvascular system (Takotsubo syndrome).

[0029] In the present application, the term "ischemia / reperfusion injury" refers to organ or tissue damage caused by insufficient blood supply to an organ or tissue during ischemia prior to the start of reperfusion (i.e., ischemic injury is the damage caused by ischemia during the time period between the onset of ischemia and the start of reperfusion). The typical and pathological manifestation of ischemic injury is that the ischemic area becomes pale. In contrast, during reperfusion, the non-necrotic ischemic tissue resumes its physiological color.

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

[0031] Ischemic injury may involve the following symptoms: chest discomfort, shortness of breath, discomfort in other areas of the upper body, nausea, and / or anxiety.

[0032] As used herein, the term "reperfusion" refers 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 give rise to a series of adverse reactions that paradoxically harm the tissue.

[0033] As used herein, the term "reperfusion injury" refers to organ or tissue damage that occurs when blood supply returns to an organ or tissue after the ischemic period. Thus, reperfusion injury is the damage caused during the time period between the start and end of reperfusion (usually, the major part of this damage will be caused within the first few minutes of reperfusion). The underlying mechanisms of reperfusion injury are complex and multifactorial. Without limitation, the lack of oxygen and nutrients in the blood during the ischemic period results in the restoration of circulation by inducing oxidative stress rather than restoring normal function. The oxidative stress associated with reperfusion can cause damage to the affected tissue or organ. The biochemical signature of reperfusion injury is oxygen depletion during the ischemic event, followed by reoxygenation during reperfusion and the concomitant production of reactive oxygen species. The injury that occurs with reperfusion is the result of the interaction between substances accumulated during ischemia and substances delivered during reperfusion. The basis for these events is oxidative stress, which is defined as an imbalance between oxygen free radicals and the endogenous scavenging system. The result is cell injury and death, which are initially local but, if not controlled, ultimately become systemic.

[0034] Reperfusion injury can be caused by, for example, mechanical events, or by one or more surgical procedures or other therapeutic interventions that restore blood flow to tissues or organs that have experienced a reduction in blood supply. Such surgical procedures include, for example, coronary artery bypass graft surgery, coronary angioplasty, and organ transplantation surgery. In a specific embodiment, reperfusion injury is caused by the treatment of an ischemic process that is due to atherosclerotic plaque rupture / erosion and superimposed thrombus, thromboembolism, lipid embolism, hemorrhage, stent, surgery, angioplasty, bypass termination during surgery, organ transplantation, total ischemia, vasoconstriction, or microvascular dysfunction or a combination thereof.

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

[0036] As used herein, the term "prevention" means reducing the risk of disease occurrence in an individual or population, or alleviating the severity of the disease, delaying the progression of the disease, by taking a series of proactive intervention measures. Specifically in this application, "prevention" means that before ischemia-reperfusion, administering the drug of this application can relieve the injury of intestinal tissue after ischemia-reperfusion. Preventing disease is an active concept of health management, avoiding or reducing the harm of disease through "upstream intervention", and its value far exceeds the treatment after the disease occurs.

[0037] As used herein, the term "treatment" means a series of medical behaviors targeted at an existing disease through medical means, techniques or methods, with the goal of eliminating the cause, relieving symptoms, controlling the development of the disease, promoting tissue repair, restoring function, or reducing pain. Specifically in this application, "treatment" means that after intestinal ischemia-reperfusion injury, administering the drug of this application can improve the pathological injury of intestinal tissue.

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

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

[0040] In some of these examples, the use of 12-ketolithocholic acid in the preparation of a drug for preventing acute ischemic intestinal injury.

[0041] In some of these examples, prevention or treatment means improving the pathological injury of acute ischemic intestinal tissue.

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

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

[0044] In some of these examples, the drug includes a pharmaceutically acceptable excipient.

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

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

[0047] For the experimental parameters not specified in the following specific examples, preferentially refer to the guidance given in this application document, and you can also refer to the experimental manuals in this field or other experimental methods known in this field, or refer to the experimental conditions recommended by the manufacturers.

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

[0049] Example 1:

[0050] This example provides an experimental study 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] 60 male C57BL / 6J mice aged 6 to 8 weeks and weighing 18 g to 22 g were selected for the experiment. They were purchased from the Animal Center of Southern Hospital, and the breeding place was the SPF-level Animal Experiment Department of Southern Medical University Southern Hospital. All operations involved in the animal breeding process were approved by the ethics committee and met the animal ethics requirements.

[0054] 1.2, Reagents and Instruments

[0055] 12-KLCA (Med Chem Express, USA); Isoflurane (Rewards Life Science Co., Ltd.); Microvascular artery clip (Chengdu North America Jiarui Biotechnology Co., Ltd.); Sterile silk thread (Ningbo Medical Sewing Needle Co., Ltd.); Normal saline (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.); Phosphate buffer saline (PBS) at pH 7.4 (Gibco).

[0056] 2. Experimental Methods and Results

[0057] 2.1. Animal Experiments

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

[0059] 1) Fast the mice for 12 h before surgery, allow free access to water, anesthetize the mice by inhaling isoflurane, and clamp the superior mesenteric artery with a non-invasive microvascular artery clip to block blood flow.

[0060] 2) After 60 min of intestinal ischemia, release the artery clip to restore blood supply and perform intestinal reperfusion. After checking that there is no bleeding in the abdominal cavity, suture the peritoneum, muscle, and skin layer by layer with sterile silk thread.

[0061] 3) Inject about 0.5 mL of warm normal saline at about 37 °C subcutaneously for fluid resuscitation during and after reperfusion, and observe and record the survival perfusion time of the mice.

[0062] (2) Experimental grouping:

[0063] Randomly divide 60 C57BL / 6 mice aged 6 to 8 weeks into a sham operation group (Sham), an intestinal I / R group (I / R), and an intestinal I / R + 12-KLCA group (I / R+12-KLCA).

[0064] 1) Sham operation group (Sham): After pretreatment with an intraperitoneal injection of PBS solution for 1 h, only perform laparotomy, isolate the superior mesenteric artery but do not clamp it;

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

[0066] 3) Intestinal I / R + 12-KLCA group (I / R+12-KLCA): After pretreatment with an intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h, establish an intestinal I / R model.

[0067] 2.2. Experimental Results

[0068] Please refer to the Figure 1 , Figure 1 result graph of 12-KLCA improving the survival rate of mice with intestinal ischemia-reperfusion; Figure 1 The meanings of the marked symbols in the figure are as follows: The data were analyzed using the Log-rank (Mantel-Cox) test, and * indicates a statistically significant difference compared with the I / R group (p < 0.05). Figure 1 The results showed that after treatment with 12-KLCA, the survival time of mice with 60 min of ischemia followed by reperfusion was significantly increased, and the survival rate of mice was improved.

[0069] Example 2:

[0070] This example provides an experimental study on 12-KLCA alleviating the intestinal tissue 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, 6 to 8 weeks old and weighing 18 g to 22 g, were purchased from the Animal Center of Nanfang Hospital and housed in the SPF-level Animal Experiment Department of Nanfang Hospital, Southern Medical University. All operations involved in the animal feeding 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 (Reword Biotechnology Co., Ltd.); microvascular artery clips (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk threads (Ningbo Medical Sewing Needle Co., Ltd.); normal saline (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.); phosphate buffer saline (PBS) with pH 7.4 (Gibco); hematoxylin-eosin staining (Beijing Regen Biotech Co., Ltd.); absolute ethanol (Guangdong Guanghua Sci-Tech Co., Ltd.); xylene (Guangdong Guanghua Sci-Tech Co., Ltd.); paraffin (Leica); 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.); neutral gum (Solarbio); automatic fluorescence microscope (Olympus).

[0076] 2. Experimental methods and results

[0077] 2.1 Animal experiment:

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

[0079] 1) Fast the mice for 12 h before surgery, allow free access to water, anesthetize the mice by inhalation of isoflurane, and occlude the superior mesenteric artery with a non-invasive microvascular arterial clamp to block blood flow.

[0080] 2) After 60 min of intestinal ischemia, release the arterial clamp to restore blood supply and perform intestinal reperfusion. After checking that there is no intraperitoneal bleeding, suture the peritoneum, muscle, and skin layer by layer with sterile silk thread.

[0081] 3) Perform fluid resuscitation by subcutaneous injection of about 0.5 mL of warm saline at about 37 °C during occlusion and reperfusion. After 2 h of perfusion, take the intestinal tissue of the mice for examination.

[0082] (2) Experimental grouping:

[0083] Randomly divide 24 C57BL / 6 mice aged 6 - 8 weeks into a sham operation group (Sham), an intestinal I / R group (I / R), and an intestinal I / R + 12-KLCA group (I / R+12-KLCA) evenly.

[0084] 1) Sham operation group (Sham): After pretreatment with intraperitoneal injection of PBS solution for 1 h, only perform laparotomy, isolate the superior mesenteric artery without occlusion;

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

[0086] 3) Intestinal I / R + 12-KLCA group (I / R+12-KLCA): After pretreatment with intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h, establish an intestinal I / R model.

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

[0088] Immerse the fresh intestinal tissue in 4% paraformaldehyde for fixation for 24 h, then perform dehydration, embedding, sectioning, followed by hematoxylin-eosin staining, and seal the sections with neutral gum. Observe the pathological morphological changes of the intestinal tissue under an automatic fluorescence microscope, and then grade and score the intestinal mucosal injury using the modified Chiu's method.

[0089] 2.3 Experimental results

[0090] The results are shown in Figure 2 , Figure 2 which is the pathological result diagram of 12-KLCA improving intestinal tissue injury induced by intestinal ischemia-reperfusion in mice. Among them: Figure 2 (A) is the HE staining diagram of the morphological changes of intestinal tissue in each group, Figure 2 (B) is the quantitative scoring result of intestinal tissue injury, and the picture scale is 100 µm; Figure 2The meanings of the marked symbols are as follows: The data were analyzed by one-way ANOVA. * indicates a statistically significant difference compared with the I / R group (p < 0.05), and ** indicates a statistically significant difference compared with the I / R group (p < 0.01). Figure 2 (A) and Figure 2 The results of HE staining and scoring of intestinal tissues in (B) showed that the intestinal villi at the top of the I / R model group were shed and the capillaries were dilated. After treatment with 12-KLCA, the morphological pathological changes of intestinal tissues induced by intestinal I / R in mice were significantly improved. The above data indicate that 12-KLCA can slow down the pathological morphological changes of intestinal tissues in mice with intestinal ischemia-reperfusion.

[0091] Example 3:

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

[0093] 1. Experimental materials

[0094] 1.1 Experimental animals

[0095] Twenty-four male C57BL / 6J mice aged 6 to 8 weeks with a body weight of 18 g to 22 g were selected. They were purchased from the Animal Center of Southern Hospital and housed in the SPF-level Animal Experiment Department of Southern Medical University Southern Hospital. All operations involved in the animal feeding 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 (Reword Life Science Co., Ltd.); Microvascular artery clips (Chengdu North America Jiarui Biotechnology Co., Ltd.); Sterile silk threads (Ningbo Medical Sewing Needle Co., Ltd.); Normal saline (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.); Phosphate buffer saline (PBS) with pH 7.4 (Gibco); BCA detection kit (Thermo Fisher Scientific, USA); Rabbit monoclonal primary antibody against caspase-3 (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); Blotting fluorescence chemiluminescence imaging instrument (Bio-Red, USA).

[0098] 2. Experimental Methods and Results

[0099] 2.1 Animal Experiments:

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

[0101] 1) Fast the mice for 12 h before surgery, allow free access to water, anesthetize the mice by inhaling isoflurane, and clamp the superior mesenteric artery with a non-invasive microvascular artery clamp to block blood flow.

[0102] 2) After 60 min of intestinal ischemia, release the artery clamp to restore blood supply and perform intestinal reperfusion. After checking that there is no bleeding in the abdominal cavity, suture the peritoneum, muscle, and skin layer by layer with sterile silk thread.

[0103] 3) Inject about 0.5 mL of warm normal saline at about 37 °C subcutaneously for fluid resuscitation during and after reperfusion. After 2 hours of perfusion, take the intestinal tissue of the mice for examination.

[0104] (2) Experimental grouping:

[0105] Randomly divide 24 C57BL / 6 mice aged 6 - 8 weeks into a sham operation group (Sham), an intestinal I / R group (I / R), and an intestinal I / R + 12-KLCA group (I / R + 12-KLCA) evenly.

[0106] 1) Sham operation group (Sham): After pretreatment with an intraperitoneal injection of PBS solution for 1 h, only perform laparotomy, isolate the superior mesenteric artery without clamping;

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

[0108] 3) Intestinal I / R + 12-KLCA group (I / R + 12-KLCA): After pretreatment with an intraperitoneal injection of 12-KLCA (10 mg / kg) for 1 h, establish the intestinal I / R model.

[0109] 2.2 Detection of the protein expression level of apoptotic factor activated caspase-3 in intestinal tissue

[0110] Quick-freeze the fresh intestinal tissue in liquid nitrogen and then mechanically grind it. Add lysis buffer to decompose the tissue, perform BCA protein quantitative analysis after centrifugation, and then prepare the gel, load the sample, perform electrophoresis, transfer the membrane, block, and incubate with antibodies. Then use a blot fluorescence chemiluminescence imager for imaging, analyze the gray value of each band with Image JV1.8.0, and finally perform quantitative scoring.

[0111] 2.3 Experimental Results

[0112] See the results in Figure 3 , Figure 3 Figure showing the protein expression level of apoptotic factor caspase-3 in intestinal tissues of mice after intestinal ischemia-reperfusion with 12-KLCA. Among them: Figure 3 (A)is the Western Blotting band diagram of intestinal tissues in each group, Figure 3 (B)is the quantitative scoring result of Western Blotting of intestinal tissues in each group; Figure 3 The meanings of the symbols marked are: The data were analyzed by one-way ANOVA test, and * indicates that there is a statistically significant difference compared with the I / R group, p < 0.05. Figure 3 (A)and Figure 3 The Western Blotting bands and quantitative scoring results of intestinal tissues in (B) show that the protein expression of apoptotic factor caspase-3 in intestinal tissues of the I / R model group is significantly the highest, and after treatment with 12-KLCA, it can significantly reduce the protein expression of apoptotic factor caspase-3 in intestinal tissues of mice after intestinal I / R. The above data indicate that 12-KLCA can reduce the apoptosis level of intestinal tissues in mice after intestinal ischemia-reperfusion.

[0113] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0114] The above-described embodiments only express several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope 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 based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. Use of 12-ketolithocholic acid in the preparation of a medicament for preventing or treating acute ischemic intestinal injury.

2. The application according to claim 1, wherein The acute ischemic intestinal injury includes intestinal ischemia-reperfusion injury.

3. The application according to claim 1 or 2, characterized in that, Use of 12-ketolithocholic acid in the preparation of a medicament for preventing acute ischemic intestinal injury.

4. The application according to claim 1 or 2, characterized in that, Preventing or treating refers to improving the pathological injury of acute ischemic intestinal tissue.

5. The application according to claim 1 or 2, characterized in that, Preventing or treating refers to reducing the protein expression level of activated caspase-3.

6. The application according to claim 1 or 2, characterized in that, The medicament includes an active ingredient, and the active ingredient includes the 12-ketolithocholic acid.

7. The application according to claim 6, wherein The active ingredient is the 12-ketolithocholic acid.

8. The application according to claim 1 or 2, characterized in that, The medicament includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The dosage form of the medicament includes injection, oral liquid, pill, powder, plaster, tablet, granule, powder or capsule.

10. The application according to claim 8, characterized in that, The administration route of the medicament includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

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