Application of MCU inhibitor in preparation of medicine for preventing or treating intestinal ischemia-reperfusion injury

Abstract

The invention discloses application of an MCU (microprogrammed control unit) inhibitor in preparation of a medicine for preventing or treating intestinal ischemia-reperfusion injury, and relates to the technical field of biological medicine, and the MCU inhibitor comprises one or two of a reagent for inhibiting MCU gene expression and a reagent for inhibiting MCU protein activity. The MCU inhibitor disclosed by the invention is beneficial to relieving intestinal mucosa injury and inflammatory change of intestinal tissues, improving completeness of a villus structure, reducing pathological injury degree score of the intestinal tissues and relieving cell apoptosis degree in the intestinal tissues, so that the MCU inhibitor is beneficial to relieving programmed death of intestinal mucosa cells; as a main component, the pharmaceutical composition is beneficial for preventing or treating intestinal ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of MCU inhibitors in the preparation of drugs for preventing or treating intestinal ischemia-reperfusion injury. Background Art

[0002] Intestinal ischemia-reperfusion (IIR) occurs widely in clinical critically ill patients, such as those undergoing intestinal obstruction, cardiac surgery, liver transplantation, and severe trauma. It can lead to a series of complications, including damaged intestinal mucosal barrier, bacterial translocation, and inflammatory cytokine storm. It is an important cause of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).

[0003] Research has shown that during intestinal ischemia-reperfusion, mitochondria, as key organelles for energy metabolism, calcium homeostasis, and cell death control, play a crucial role in intestinal ischemia-reperfusion injury. Mitochondrial DNA release triggered by mitochondrial damage induces a sterile inflammatory response, which exacerbates intestinal ischemia-reperfusion injury. The mitochondrial calcium uniporter (MCU) is the primary channel for calcium ion uptake on the inner mitochondrial membrane and is involved in regulating mitochondrial homeostasis. However, the role of MCU in intestinal ischemia-reperfusion injury is currently unclear, and no MCU-regulated components have been reported in drugs for the prevention or treatment of intestinal ischemia-reperfusion injury. Summary of the Invention

[0004] In view of the deficiencies of the above-mentioned related technologies, the present invention provides the use of MCU inhibitors in the preparation of drugs for preventing or treating intestinal ischemia-reperfusion injury.

[0005] On the one hand, the use of the MCU inhibitor provided by the present invention in the preparation of a drug for preventing or treating intestinal ischemia-reperfusion injury adopts the following technical solution: Use of MCU inhibitors in the preparation of drugs for preventing or treating intestinal ischemia-reperfusion injury.

[0006] Preferably, the MCU inhibitor comprises one or both of an agent that inhibits MCU gene expression and an agent that inhibits MCU protein activity.

[0007] Preferably, the reagent for inhibiting MCU gene expression includes RNA expressed by the MCU gene and / or its vector.

[0008] Preferably, the nucleotide sequence of the RNA that inhibits MCU gene expression is shown as SEQ ID NO: 1 and SEQ ID NO: 2.

[0009] Preferably, the agent for inhibiting the activity of MCU protein includes one or more of Ru360 inhibitor, Ru265 inhibitor, MCU-i4 and berberine.

[0010] Preferably, the agent that inhibits the activity of MCU protein is a Ru360 inhibitor.

[0011] Preferably, the prevention or treatment of intestinal ischemia-reperfusion injury includes improving the intestinal mucosal villus structure and reducing tissue edema, lowering the level of intestinal tissue apoptosis and lowering the intestinal tissue injury score.

[0012] Preferably, the MCU inhibitor is beneficial for inhibiting mitochondrial DNA release and inflammatory response triggered by mitochondrial damage.

[0013] Preferably, the MCU inhibitor is beneficial to maintaining mitochondrial function and intestinal mucosal barrier stability.

[0014] Preferably, the drug for preventing or treating intestinal ischemia-reperfusion injury is administered by intravenous injection and / or intraperitoneal injection.

[0015] On the other hand, the present invention provides a pharmaceutical composition used in the above application using the following technical solution: A pharmaceutical composition comprising an effective amount of the MCU inhibitor and pharmaceutically acceptable carriers and excipients.

[0016] In summary, the present invention includes at least one of the following beneficial technical effects: 1. MCU inhibitors can help alleviate intestinal mucosal damage and inflammatory changes, improve the integrity of villus structure, reduce the pathological damage score of intestinal tissue, and reduce the degree of cell apoptosis in intestinal tissue. This suggests that MCU inhibitors can alleviate the programmed cell death of intestinal mucosal cells and, as a major component, can help prevent or treat intestinal ischemia-reperfusion injury.

[0017] 2. MCU inhibitors are beneficial for lowering MDA and LDH levels while upregulating SOD content, thereby improving oxidative stress, and reducing mitochondrial calcium content, which is beneficial for regulating the normalization of mitochondrial calcium ion levels; MCU inhibitors are beneficial for increasing mitochondrial membrane potential; alleviating mitochondrial membrane structure damage, discontinuity, and broken or agglomerated distribution phenomena, enhancing membrane structure continuity, and alleviating mitochondrial structural damage; and restoring ATP levels, which is beneficial for improving mitochondrial energy metabolism function; reducing the release of mtDNA and alleviating mitochondrial damage.

[0018] 3. MCU inhibitors effectively inhibit MCU activity, thereby improving the survival rate of cells after hypoxia / reoxygenation treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a comparison of the mRNA expression levels of MCU in the intestinal ischemia-reperfusion model of Comparative Example 1 and the sham operation group; Figure 2 This is a comparison of the MCU protein expression levels in the intestinal ischemia-reperfusion model of Comparative Example 1 and the sham operation group; Figure 3 This is a comparison of the mRNA expression levels and contents of inflammatory factors IL-1β and IL-6 in mice in the intestinal ischemia-reperfusion model of Comparative Example 1 and the sham operation group; Figure 4 is a comparison of the mRNA expression levels of MCU in Caco-2 cells in the hypoxia / reoxygenation model of comparative example 2 and the control group; Figure 5 This is a comparison of the MCU protein expression levels of Caco-2 cells in the hypoxia / reoxygenation model of comparative example 2 and the control group; Figure 6 1. H&E staining of intestinal tissues and comparison of intestinal tissue injury scores of the intestinal ischemia-reperfusion model of Comparative Example 1 and the intestinal ischemia-reperfusion model of Example 1; Figure 7 1 is a comparative diagram of TUNEL staining of intestinal tissues and quantitative analysis results of apoptosis rates in the intestinal ischemia-reperfusion model of Comparative Example 1 and the intestinal ischemia-reperfusion model of Example 1; Figure 8 3. This is a comparison chart of the test results of the content of oxidative stress-related indicators in intestinal tissues of the intestinal ischemia-reperfusion model of Comparative Example 1 and the intestinal ischemia-reperfusion model of Example 1; Figure 9 is a comparison of the calcium ion content in the intestinal tissue of the intestinal ischemia-reperfusion model of Comparative Example 1 and the intestinal ischemia-reperfusion model of Example 1; Figure 10 3. It is a comparison diagram of the fluorescence images and quantitative analysis results of the changes in mitochondrial membrane potential in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 11 This is a comparison of cell viability in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 12 is a comparison of immunofluorescence assessment of mitochondrial membrane integrity in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 13 3. It is a comparison chart of ROS levels and quantitative analysis results in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 14 3. This is a comparison diagram of the mitochondrial calcium ion distribution and quantitative analysis results in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 15 This is a comparison of ATP content in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 16 3. It is a comparison diagram of the release of mtDNA in the cytoplasmic components in the hypoxia / reoxygenation model of Comparative Example 2 and the hypoxia / reoxygenation model of Example 2; Figure 17 This is a comparison chart of the hypoxia / reoxygenation model of Comparative Example 2, the hypoxia / reoxygenation model of Examples 3-5, and the cell viability of normal human colon adenocarcinoma cell line Caco-2 cells. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to Examples. The following examples are intended to illustrate the present invention only and are not to be construed as limiting the scope of the present invention. In the following examples, if no specific conditions are specified, the methods used are conventional methods well known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0021] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0022] Example 1 Example 1 of the present invention provides the use of an MCU inhibitor in preparing a medicament for preventing or treating intestinal ischemia-reperfusion injury, comprising the following steps: Male C57BL / 6J mice, 7–9 weeks old and weighing 22–26 g, were selected. After weighing, the mice were intraperitoneally injected with a Ru360 inhibitor (50 nmol / kg). One hour after the injection, anesthesia was achieved with an intraperitoneal injection of 2% sodium pentobarbital based on body weight. The mice were placed in the supine position and secured to a surgical board. The abdomen was depilated, and a longitudinal incision was made midline to access the abdominal cavity. The abdominal aorta and mesenteric regions were exposed, and the superior mesenteric artery (SMA) was identified and isolated, then clamped with an atraumatic vascular clip. Successful ischemia was considered established when the intestinal color changed from rosy to pale and vascular pulsation disappeared. After 45 minutes of ischemia, the abdominal cavity was re-entered through the original incision, and the vascular clamp was removed to restore intestinal blood supply. Successful reperfusion was determined by the restoration of pulsation and a return of intestinal mucosal color to rosy red, thus establishing an intestinal ischemia-reperfusion model.

[0023] Example 2 Example 2 of the present invention provides the use of an MCU inhibitor in preparing a medicament for preventing or treating intestinal ischemia-reperfusion injury, comprising the following steps: Human colon adenocarcinoma cell line Caco-2 cells (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) were cultured in MEM medium containing 10% fetal bovine serum and 1% double-antibody at 37°C in a 5% CO2 incubator. The medium was changed every 48 hours. When the cells reached 80%-90% confluence, they were digested with trypsin and seeded into 6-well or 96-well plates and cultured in a cell culture incubator. Adherent cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended to a cell density of 1×10 4 When the cell confluence reached about 80%, the cells were plated with Lipofectamine 5000 containing RNA that inhibited MCU gene expression. TM Plasmids were transiently transfected into cells using 3000 transfection reagent. Four hours after transfection, the culture medium in each well was replaced with medium containing 10% fetal bovine serum and cultured for an additional 48 hours. The cells were washed with PBS and then incubated in an incubator with 94% N₂, 5% CO₂, and 1% O₂ for 12 hours. The cells were then transferred to a normoxic incubator and cultured for 2 hours to establish a hypoxia / reoxygenation (H / R) model.

[0024] In this embodiment, the nucleotide sequence of the RNA that inhibits the expression of the MCU gene is shown in Table 1 below: Table 1:

[0025] Example 3 Example 3 of the present invention provides the use of an MCU inhibitor in preparing a medicament for preventing or treating intestinal ischemia-reperfusion injury, comprising the following steps: Caco-2 cells, a human colon adenocarcinoma cell line (purchased from Wuhan Punosai Life Science Co., Ltd.), were cultured in MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. The medium was changed every 48 hours. When the cell confluence reached 80%-90%, they were trypsinized and evenly seeded into 6-well or 96-well plates and cultured until stable attachment was achieved.

[0026] To establish the hypoxia / reoxygenation model, cells were washed with PBS buffer and then cultured in culture medium. Ru360 inhibitor was added to a final concentration of 10 μM and pretreated under normoxic conditions for 1 hour. Following pretreatment, cells were incubated in a hypoxic incubator with 94% N₂, 5% CO₂, and 1% O₂ for 12 hours. Subsequently, cells were transferred to normoxic conditions and reoxygenated for 2 hours to complete the hypoxia / reoxygenation model.

[0027] Example 4 Example 4 of the present invention provides the use of an MCU inhibitor in the preparation of a medicament for preventing or treating intestinal ischemia-reperfusion injury. Example 4 differs from Example 3 in that the final concentration of the Ru360 inhibitor added in Example 4 is 1 μM.

[0028] Example 5 Example 5 of the present invention provides the use of an MCU inhibitor in the preparation of a medicament for preventing or treating intestinal ischemia-reperfusion injury. Example 5 differs from Example 3 in that the final concentration of the Ru360 inhibitor added in Example 5 is 20 μM.

[0029] Comparative Example 1 Comparative Example 1 provides an intestinal ischemia-reperfusion model. The difference between Comparative Example 1 and Example 1 is that: Comparative Example 1 does not perform intraperitoneal injection of Ru360 inhibitor.

[0030] Comparative Example 2 Comparative Example 2 provides a hypoxia / reoxygenation model. The difference between Comparative Example 2 and Example 2 is that in Comparative Example 2, the Caco-2 cells are not subjected to the RNA transfection treatment operation for inhibiting the expression of the MCU gene.

[0031] Test and Inspection (1) The mRNA expression levels of MCU were detected in the intestinal ischemia-reperfusion model (labeled as IIR) of comparative example 1 and the mice in the sham operation group (labeled as Sham). The results are as follows: Figure 1 As shown; Western blot detection of MCU protein expression was performed, and the results were as follows Figure 2 As shown; the mRNA expression levels of inflammatory factors IL-1β and IL-6 were detected, and the results were as shown Figure 3 As shown in A; the levels of inflammatory factors IL-1β and IL-6 were detected respectively, and the results were as follows Figure 3 As shown in B.

[0032] (2) The mRNA expression levels of MCU were detected in the hypoxia / reoxygenation model of comparative example 2 (marked as HR) and the Caco-2 cells of the control group (marked as C). The results are as follows: Figure 4 As shown; Western blot detection of MCU protein expression was performed, and the results were as follows Figure 5 shown.

[0033] (3) H&E staining of intestinal tissues was performed on the intestinal ischemia-reperfusion model of comparative example 1 (labeled as IIR) and the intestinal ischemia-reperfusion model of example 1 (labeled as Ru360). The results are as follows: Figure 6 As shown in A, the intestinal tissues were pathologically evaluated and the Chiu's scoring method was used to grade the degree of intestinal mucosal damage. The results are shown in Figure 6As shown in B; TUNEL staining of intestinal tissues Figure 7 As shown in A, quantitative analysis of apoptosis rate was performed, and the results were as follows Figure 7 As shown in B; the levels of MDA, LDH and SOD, the indicators related to oxidative stress in intestinal tissue, were detected respectively. The results are shown in Figure 8 As shown; the calcium ion content in the intestinal tissue was tested, and the results were as follows Figure 9 shown.

[0034] (4) The fluorescence images of the changes in mitochondrial membrane potential were detected for the hypoxia / reoxygenation model of comparative example 2 (labeled as HR) and the hypoxia / reoxygenation model of example 2 (labeled as siMCU). Figure 10 A, and its quantitative analysis results are shown in Figure 10 As shown in B; Cell viability was tested respectively, and the results were as follows Figure 11 As shown; TOM20 staining was used for immunofluorescence assessment of mitochondrial membrane integrity, and the results were shown Figure 12 As shown; ROS fluorescent probes were used to detect the fluorescence microscopy images of intracellular ROS levels, and the results were as follows Figure 13 As shown in A, the results of ROS fluorescence quantitative analysis are as follows Figure 13 As shown in B; the distribution of mitochondrial calcium ions was detected by Rhod-2AM fluorescent probe. Figure 14 As shown in A, the quantitative analysis results of calcium ion concentration are as follows Figure 14 As shown in B; the cell ATP content was detected, and the results were as follows Figure 15 As shown; quantitative detection of the release of mtDNA (ND1, D-LOOP, ATP-6, CO2 mitochondrial encoding genes) in the cytoplasmic fraction was performed respectively, and the results were shown Figure 16 shown.

[0035] (5) Cell viability was tested on the hypoxia / reoxygenation model of Comparative Example 2, the hypoxia / reoxygenation model of Examples 3-5, and the normal human colon adenocarcinoma cell line Caco-2 cells. The results are as follows: Figure 17 shown.

[0036] Result Analysis The following is based on Figure 1-17 The present invention is described in detail based on the experimental results.

[0037] Reference Figure 1-3 The results showed that the mRNA expression level of MCU, the protein expression level of MCU, the mRNA expression level of inflammatory factors IL-1β and IL-6, and the content of inflammatory factors IL-1β and IL-6 in the intestinal tissue of the intestinal ischemia-reperfusion model of comparative example 1 without injection of Ru360 inhibitor were significantly increased compared with those of the sham operation group, and the differences were statistically significant. Figure 4-5The results showed that the mRNA expression level and protein expression level of MCU in comparative example 2, in which Caco-2 cells were not transfected with RNA to inhibit MCU gene expression, were significantly increased compared with those in the control group, and the difference was statistically significant.

[0038] Reference Figure 6 A. The results showed that the intestinal mucosal structure of the intestinal tissue of the comparative example 1 without the injection of Ru360 inhibitor was disordered, the villi were shed, and the edema was significant, accompanied by a large number of inflammatory cells infiltration. However, the intestinal mucosal damage of the intestinal tissue of the intestinal ischemia-reperfusion model of Example 1 with the injection of Ru360 inhibitor was significantly reduced, the villi structure was relatively intact, and the inflammatory changes were reduced. Figure 6 B, The results showed that after further quantification of the degree of intestinal tissue pathological damage, the score of the Ru360 group decreased compared with the score of the IIR group, which was statistically significant; Figure 7 The results showed that the apoptotic cells in the intestinal ischemia-reperfusion model of comparative example 1 without injection of Ru360 inhibitor increased significantly, while the degree of apoptosis in the intestinal tissue of the intestinal ischemia-reperfusion model of Example 1 injected with Ru360 inhibitor was significantly reduced, indicating that the injection of Ru360 inhibitor is beneficial to alleviate the programmed cell death of intestinal mucosal cells; Figure 8 The results showed that the MDA and LDH levels in the IIR group increased significantly, while the SOD level decreased significantly; the Ru360 group significantly reduced the MDA and LDH levels, while increasing the SOD content, indicating that the injection of Ru360 inhibitors is beneficial to improving the oxidative stress state. Figure 9 The results showed that the mitochondrial calcium load in the IIR group was significantly increased, while the mitochondrial calcium content in the Ru360 group was significantly decreased, indicating that the injection of Ru360 inhibitors is beneficial to the regulation of mitochondrial calcium ion levels to normalize, and the difference is statistically significant.

[0039] Reference Figure 10 The results showed that in the hypoxia / reoxygenation model of Comparative Example 2, in which the Caco-2 cells were not transfected with RNA that inhibited MCU gene expression, the mitochondrial membrane potential decreased significantly, while in the hypoxia / reoxygenation model of Example 2, in which the Caco-2 cells were transfected with RNA that inhibited MCU gene expression, the membrane potential increased significantly, and the difference was statistically significant. Figure 11 The results showed that the cell viability of the siMCU group was significantly higher than that of the HR group, indicating that RNA transfection treatment that inhibits MCU gene expression is beneficial to improving cell viability; Figure 12 The results showed that the mitochondrial membrane structure of the HR group was damaged, discontinuous, and distributed in a broken or agglomerated manner, while the mitochondrial membrane morphology of the siMCU group was restored and the continuity of the membrane structure was enhanced, indicating that RNA transfection treatment that inhibits MCU gene expression is beneficial to reducing mitochondrial structural damage. Figure 13 The results showed that the ROS fluorescence intensity of the siMCU group was significantly lower than that of the HR group, indicating that RNA transfection treatment that inhibits MCU gene expression is beneficial to reducing ROS levels and alleviating oxidative stress damage. Figure 14 The results showed that the calcium ion accumulation in the siMCU group was significantly lower than that in the HR group, indicating that RNA transfection that inhibits MCU gene expression is beneficial to alleviating mitochondrial calcium load. Figure 15 The results showed that the ATP level in the siMCU group was higher than that in the HR group, and the ATP level was restored, indicating that RNA transfection that inhibits MCU gene expression is beneficial to improving mitochondrial energy metabolism function. Figure 16 The results showed that the HR group significantly promoted the release of mtDNA from mitochondria to the cytosol, while the siMCU group reduced the release of mtDNA and alleviated mitochondrial damage.

[0040] Reference Figure 17 The results showed that normal human colon adenocarcinoma cell line Caco-2 cells had the highest cell viability. The cell viability of the hypoxia / reoxygenation models of Examples 3-5 all recovered to a certain extent compared to the hypoxia / reoxygenation model of Comparative Example 2 without the addition of Ru360 inhibitor. After treatment with different concentrations of Ru360 inhibitor, cell viability showed a dose-dependent change. The cell viability of the hypoxia / reoxygenation model of Example 3 was significantly increased compared to the cell viability of the hypoxia / reoxygenation models of Examples 4 and 5, indicating that under conditions of hypoxia / reoxygenation injury, the addition of 10 μM Ru360 inhibitor can more effectively inhibit MCU activity and improve the cell viability of human colon adenocarcinoma cell line Caco-2 cells.

[0041] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons of ordinary skill in the field to which the present invention belongs. The words "first", "second", "third" and similar terms used in the specification and claims of the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "one" or "a" and similar terms do not indicate a quantity limitation, but rather indicate the existence of at least one. Words such as "include" or "comprise" mean that the elements or objects appearing before "include" or "comprises" cover the elements or objects listed after "include" or "comprises" and their equivalents, and do not exclude other elements or objects. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0042] The above are all optional embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of MCU inhibitors in the preparation of drugs for preventing or treating intestinal ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: The MCU inhibitor includes one or both of an agent that inhibits MCU gene expression and an agent that inhibits MCU protein activity.

3. The use according to claim 2, characterized in that: The reagent for inhibiting MCU gene expression includes RNA expressed by the MCU gene and / or its vector.

4. The use according to claim 3, characterized in that: The nucleotide sequences of the RNA that inhibits MCU gene expression are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

5. The use according to claim 2, characterized in that: The reagent for inhibiting the activity of MCU protein includes one or more of Ru360 inhibitor, Ru265 inhibitor, MCU-i4 and berberine.

6. The use according to claim 1, characterized in that: The prevention or treatment of intestinal ischemia-reperfusion injury includes improving the intestinal mucosal villus structure and reducing tissue edema, reducing the level of intestinal tissue apoptosis and reducing the intestinal tissue injury score.

7. The use according to claim 1, characterized in that: The MCU inhibitor is beneficial for inhibiting mitochondrial DNA release and inflammatory response caused by mitochondrial damage.

8. The use according to claim 1, characterized in that: The MCU inhibitor is beneficial for maintaining mitochondrial function and intestinal mucosal barrier stability.

9. The use according to claim 1, characterized in that: The drug for preventing or treating intestinal ischemia-reperfusion injury is administered by intravenous injection and / or intraperitoneal injection.

10. A pharmaceutical composition for use according to any one of claims 1 to 9, characterized in that: The invention comprises an effective amount of the MCU inhibitor and pharmaceutically acceptable carriers and excipients.

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