Application of medicine for intervening mitochondrial pyruvic acid carrier in preparation of medicine for treating acute pancreatitis and medicine composition of medicine for intervening mitochondrial pyruvic acid carrier

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a medicine for intervening in a mitochondrial pyruvic acid carrier in preparation of a medicine for treating acute pancreatitis and a medicine composition of the medicine. The medicine for intervening in the mitochondrial pyruvic acid carrier can reduce the serum enzyme level, relieve pancreatic pathological injury, inhibit inflammatory response, inhibit calcium overload and protect the mitochondrial function, so that pancreatic acinus cell injury is relieved, the effect of treating acute pancreatitis is achieved, and the medicine has a wide application prospect. The invention provides a new choice for clinically treating acute pancreatitis, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of a drug for intervening in mitochondrial pyruvate carriers in the preparation of a drug for treating acute pancreatitis and its pharmaceutical composition. Background Art

[0002] Acute pancreatitis (AP) is an acute inflammatory disease caused by the damage of acinar cells in the pancreas responsible for exocrine function. It is one of the most common digestive system diseases that require emergency treatment, and its global incidence is on the rise. Approximately 20% of patients develop severe acute pancreatitis (SAP) mainly manifested by persistent organ failure, with a mortality rate as high as 30%-50%. Research shows that there is a "critical threshold" for the progression of AP to SAP, and its important indication is the "explosive cell death-inflammation cascade reaction", but the time window and specific molecular biological events of this trigger node are not yet clear. Large-scale clinical studies have confirmed that the critical threshold of SAP occurs in the early stage of the disease, suggesting the necessity of advancing the intervention time. At present, the lack of understanding of the core events and corresponding mechanisms in the critical threshold of SAP is the key factor restricting the research and development of targeted drugs and breakthroughs in clinical efficacy. Recent studies have shown that glucose metabolism (hereinafter referred to as "glucose metabolism") is one of the core events in the critical thresholds of many major diseases such as tumors, heart diseases, and sepsis, but its research in AP is very limited.

[0003] Pancreatic acinar cells are rich in mitochondria and have vigorous energy metabolism, with a classic stimulus-metabolism coupling phenomenon. Calcium signals and glycolysis and tricarboxylic acid (TCA) cycles in sugar metabolism play an important role in the production of ATP in acinar cells, ensuring normal protein synthesis and zymogen granule exocytosis of cells under physiological conditions. AP stimuli such as caerulein, bile acid and fatty acids all cause calcium overload and mitochondrial dysfunction in acinar cells. At this time, cell energy supply mainly depends on glycolysis, the total ATP of cytoplasm and mitochondria gradually decreases, lactic acid accumulates, cellular acidosis, and eventually cell necrosis. Pyruvate is the end product of glycolysis and an intermediate substance connecting glycolysis and TCA cycle: under aerobic conditions, pyruvate is actively transported into the mitochondrial matrix through the voltage-dependent anion channel (VDAC) located in the outer membrane of the mitochondria and the mitochondrial pyruvate carrier (MPC) in the inner membrane, further generating acetyl CoA to enter the TCA cycle, participate in oxidative phosphorylation, and produce ATP for energy supply; under hypoxic conditions, pyruvate can generate lactate under the catalysis of lactate dehydrogenase A (LDHA), and supply ATP through substrate-level phosphorylation. When AP occurs, mitochondrial membrane carriers such as the mitochondrial permeability transition pore (MPTP) become dysfunctional; targeted supplementation of pyruvate in the cell environment can reduce pancreatic necrosis, systemic inflammation and mortality in various AP animal models, indicating that the study of mitochondrial pyruvate metabolism-related carriers is of certain importance.

[0004] Currently, MPC is mainly a key target for controlling diabetes, non-alcoholic fatty liver disease or neurodegenerative diseases. Its function in AP is still unknown, and there is no literature reporting the preventive and therapeutic effects of MPC inhibitors in AP. Summary of the invention

[0005] In view of the problems of the prior art, the present invention provides the use of a drug for intervening in the mitochondrial pyruvate carrier in the preparation of a drug for treating acute pancreatitis and a pharmaceutical composition thereof.

[0006] The present invention provides the use of a drug that intervenes in mitochondrial pyruvate carriers in the preparation of a drug for treating acute pancreatitis, and the drug that intervenes in mitochondrial pyruvate carriers is selected from at least one of a mitochondrial pyruvate carrier inhibitor, a drug that inhibits the expression of mitochondrial pyruvate carriers, a drug that knocks out or knocks down mitochondrial pyruvate carriers, and a drug that degrades mitochondrial pyruvate carriers.

[0007] Preferably, the mitochondrial pyruvate carrier inhibitor is selected from at least one of small molecule compounds, polypeptides, and antibodies; and / or, the drug that inhibits the expression of mitochondrial pyruvate carriers is selected from at least one of circular RNAs, antisense nucleic acids, small interfering nucleic acids, nucleic acid aptamers, small activating nucleic acids, microRNAs, mRNA drugs, and ribozymes; and / or, the drug that knocks out or knocks down mitochondrial pyruvate carriers achieves gene knockout or knockdown through a gene editing tool; the gene editing tool is at least one of the CRISPR / Cas9 gene editing system and the Cre-loxP system; and / or, the drug that degrades mitochondrial pyruvate carriers is selected from proteolysis agents.

[0008] Preferably, the drug that intervenes in mitochondrial pyruvate carriers is selected from a mitochondrial pyruvate carrier inhibitor, a drug that knocks out or knocks down mitochondrial pyruvate carriers.

[0009] Preferably, the mitochondrial pyruvate carrier inhibitor is selected from small molecule compounds.

[0010] Preferably, the small molecule compound is 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-acrylic acid.

[0011] Preferably, the drug that knocks out or knocks down mitochondrial pyruvate carriers achieves gene knockout or knockdown through the Cre-loxP system.

[0012] Preferably, the drug can reduce the serum enzyme level; the serum enzyme is at least one of amylase, lipase, and lactate dehydrogenase.

[0013] Preferably, the drug can inhibit the inflammatory response.

[0014] Preferably, the drug can reduce pancreatic acinar cell damage.

[0015] The present invention also provides a pharmaceutical composition for treating acute pancreatitis, which is a preparation prepared with a drug that intervenes in mitochondrial pyruvate carriers as an active ingredient and pharmaceutically acceptable excipients.

[0016] The present invention first discovers the relationship between intervening in mitochondrial pyruvate carriers as targets and acute pancreatitis. Drugs that intervene in mitochondrial pyruvate carriers can reduce serum enzyme levels, alleviate pancreatic pathological damage, inhibit inflammatory responses, and at the same time inhibit calcium overload and protect mitochondrial function, thereby reducing pancreatic acinar cell damage and playing a role in treating acute pancreatitis. This provides a new option for drugs used to treat acute pancreatitis clinically and has good application prospects.

[0017] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0018] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0019] Figure 1 The protective effects of the MPC-specific inhibitor UK-5099 on CER-AP model mice, including reducing serum enzyme activity, alleviating pancreatic tissue damage and improving pathological scores. A. Effects of different treatment groups on the expression levels of amylase, lipase and lactate dehydrogenase in the sera of mice; B. HE staining results of CER-AP model mice in different treatment groups; C. Edema, inflammatory exudation, acinar cell necrosis and overall scoring of pancreatic tissues in different treatment groups.

[0020] Figure 2 The anti-inflammatory effects of the MPC-specific inhibitor UK-5099 on CER-AP model mice and the effects on MPC2 protein expression. A. mRNA expression levels of inflammation-related genes IL-6, TNF-α and IL-1β in different treatment groups; B. Expression levels of MPC2 protein in different treatment groups.

[0021] Figure 3 The effects of Pdx1-Cre-mediated Mpc2 gene knockout on MPC2 mRNA expression, serum enzyme activity, pancreatic tissue damage, pathological scores and inflammation-related gene expression in CER-AP model mice. A. Expression levels of MPC2 mRNA in different treatment groups; B. Expression levels of amylase and lipase in the sera of mice in different treatment groups; C. HE staining results of CER-AP model mice in different treatment groups; D. Edema, inflammatory exudation, acinar cell necrosis and overall scoring of pancreatic tissues in different treatment groups; E. mRNA expression levels of inflammation-related genes IL-6, TNF-α and IL-1β in different treatment groups.

[0022] Figure 4 To detect the changes in intracellular calcium ion concentration in cells of different treatment groups using the Fluo-4 AM fluorescent probe.

[0023] Figure 5 To investigate the effect of the MPC-specific inhibitor UK-5099 on the changes in intracellular calcium ion concentration in pancreatic acinar cells induced by CCK. A. The graph showing the change in intracellular calcium ion concentration over time in different treatment groups; B. The effect of different concentrations of UK5099 on the fold change in intracellular calcium ion concentration in pancreatic acinar cells induced by CCK.

[0024] Figure 6 To investigate the effect of the MPC-specific inhibitor UK-5099 on the mitochondrial membrane potential of pancreatic acinar cells. A. Detection of changes in mitochondrial membrane potential in different treatment groups using the JC-1 probe; B. The quantitative analysis result graph showing the effect of different treatment groups on the mitochondrial membrane potential of pancreatic acinar cells. Detailed implementation methods

[0025] In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available products.

[0026] Example 1 Mitochondrial pyruvate carrier inhibitor (MPC inhibitor) can be used to treat acute pancreatitis I. Experimental methods 1. Acute pancreatitis mouse model (CER-AP) Wild-type C57BL / 6J mice were selected, and an acute pancreatitis mouse model (CER-AP) was constructed by intraperitoneal injection of cerulein (dose: 50 μg / kg, injected once every 1 hour for 7 consecutive injections).

[0027] 2. Experimental grouping Control group (Ctrl group): C57BL / 6J mice received an intraperitoneal injection of an equal volume of PBS (n = 4).

[0028] Model group (CER group): Acute pancreatitis mouse model (n = 6).

[0029] CER + UK5099 group: Mice in the treatment group received an intraperitoneal injection of 10 mg / kg of the MPC-specific inhibitor 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-acrylic acid (UK5099, CAS number: 56396-35-1) 0.5 hours before the first injection of cerulein for constructing the acute pancreatitis mouse model (n = 6).

[0030] Twelve hours after the first injection of caerulein, mouse serum was collected to detect the serum enzyme levels. Subsequently, in vivo cardiac perfusion was performed on the mice to remove the blood in the circulation. The pancreatic tissues were quickly removed and fixed and dehydrated to prepare paraffin sections stained with HE for histopathological scoring (see Table 1). At the same time, a part of the pancreatic tissue was cryopreserved for subsequent experiments.

[0031] Table 1 3. PCR was used to detect the mRNA levels of inflammatory factors IL-6, TNF-α and IL-1β.

[0032] 4. WB was used to detect the expression level of Mpc2 protein.

[0033] II. Experimental Results As Figure 1 shown in A, pre-injection of the MPC specific inhibitor UK5099 significantly reduced the levels of amylase, lipase and lactate dehydrogenase in the serum of CER-AP model mice. The edema, inflammatory exudation and acinar cell necrosis of pancreatic tissues are important indicators for measuring the severity of CER-AP disease. As can be seen from Figure 1 B to 1C, in the CER-AP model, the pancreatic tissue showed obvious edema, increased inflammatory infiltration and increased acinar cell necrosis, while UK5099 could significantly reduce the edema, inflammatory cell infiltration, tissue necrosis and overall pathological damage of the pancreas in mice. Further detection by PCR found that ( Figure 2 A), UK5099 significantly reduced the levels of inflammatory factors IL-6, TNF-α and IL-1β in pancreatic tissues; at the same time, UK5099 decreased the expression of Mpc2 protein ( Figure 2 B). The above results indicate that MPC inhibitors can be used to treat acute pancreatitis.

[0034] Example 2 Pancreatic-specific knockout of MPC2 can be used to treat acute pancreatitis I. Experimental Methods By crossing Pdx1-Cre mice with Mpc2 gene conditional knockout mice, Pdx1-Cre Mpc2 mice were obtained. The experimental groups included littermate control mice and gene mice, and each group was set with a control group and a model group. The mice in the model group were intraperitoneally injected with cerulein (dose: 50 μg / kg, injected once every hour for 7 consecutive injections) to construct an acute pancreatitis model (CER-AP), while the control group mice received an equal volume of PBS intraperitoneally. At 12 hours after the first injection of cerulein, mouse serum was collected to detect the serum enzyme levels. Subsequently, in vivo cardiac perfusion was performed on the mice to remove the circulating blood, and the pancreatic tissues were quickly removed and fixed and dehydrated to make paraffin sections stained with HE for histopathological scoring. At the same time, a part of the pancreatic tissue was cryopreserved for subsequent experiments.

[0035] II. Experimental Results As Figure 3 shown in A, the expression of Mpc2 in the pancreatic tissues of Pdx1-Cre Mpc2 mice was significantly decreased, confirming that Mpc2 was successfully knocked out. Figure 3 As shown in B, compared with the control group, the levels of amylase and lipase in the serum of Pdx1-Cre Mpc2 mice decreased in the CER-AP model. Pancreatic edema, inflammatory infiltration, and acinar cell necrosis are the key indicators for evaluating the severity of CER-AP. Figure 3 As shown in C and 3D, pancreatic edema, inflammatory cell infiltration, tissue necrosis, and overall pathological damage in Pdx1-Cre Mpc2 mice were significantly alleviated in the CER-AP model. Detection by PCR found that Mpc2 gene knockout reduced the levels of inflammatory factors IL-6, TNF-α, and IL-1β in pancreatic tissues ( Figure 3 E). These results indicate that Pdx1-Cre-mediated Mpc2 gene knockout can effectively alleviate the severity of CER-AP, thereby treating acute pancreatitis.

[0036] Example 3 MPC inhibitor can inhibit pancreatic acinar cell damage I. Experimental Methods Pancreatic acinar cells were freshly isolated from the pancreas of healthy adult mice, and the cells were divided into 4 groups: CCK group, low-concentration UK5099 group (5 μM), medium-concentration UK5099 group (10 μM), and high-concentration UK5099 group (20 μM). The CCK group was only treated with cholecystokinin (CCK) at a final concentration of 10 -7 mol / L, while each concentration group of UK5099 was pre-incubated with the corresponding concentration of UK5099 for 30 minutes and then added with a final concentration of 10 -7Treatment with CCK at a concentration of mol / L. The change in intracellular calcium ion concentration was detected using the Fluo-4AM fluorescent probe. The higher the fluorescence intensity, the higher the intracellular calcium ion concentration. The change in mitochondrial membrane potential was detected using the JC-1 probe. The operation was carried out according to the kit instructions. Each experiment was repeated 3 times, and the average value was taken for statistical analysis.

[0037] II. Experimental Results As Figure 4 shown, after freshly isolated pancreatic acinar cells were pre-incubated with the MPC specific inhibitor UK5099 (5, 10, and 20 μM) for 30 minutes and then calcium overload was induced with cholecystokinin (CCK), the change in intracellular calcium ion concentration was detected using the Fluo-4 AM fluorescent probe. The higher the fluorescence intensity, the higher the intracellular calcium ion concentration. The results showed that cells treated only with 100 nM CCK showed obvious green fluorescence; compared with the CCK group, the green fluorescence intensity in the UK5099 incubation group was significantly weakened, indicating that UK-5099 could significantly inhibit the increase in intracellular calcium ion concentration induced by CCK. As Figure 5 shown in A, the peak value of calcium overload and the area under the curve in the UK5099 incubation group were significantly decreased compared with the CCK group alone, but no obvious dose-dependence was shown; Figure 5 B shows the results of the fold change in intracellular calcium ion concentration in pancreatic acinar cells induced by CCK with different concentrations of UK5099, which is consistent with the above results. In further experiments, 5 μM UK5099 significantly protected acinar cells and prevented CCK-induced mitochondrial membrane potential depolarization ( Figure 6 A), Figure 6 and B is the result graph of quantitative analysis of Figure 6 A by measuring the green / red fluorescence ratio. The results indicate that the MPC inhibitor UK5099 can reduce pancreatic acinar cell damage by inhibiting calcium overload and protecting mitochondrial function.

[0038] In summary, the drug of the present invention that intervenes in the mitochondrial pyruvate carrier can reduce serum enzyme levels, alleviate pancreatic pathological damage, inhibit inflammatory responses, and at the same time inhibit calcium overload and protect mitochondrial function, thereby reducing pancreatic acinar cell damage and playing a role in the treatment of acute pancreatitis, providing a new option for drugs for the clinical treatment of acute pancreatitis and having good application prospects.

Claims

1. Use of a drug that intervenes in the mitochondrial pyruvate carrier in the preparation of a drug for treating acute pancreatitis, characterized in that: The drugs that intervene in mitochondrial pyruvate carriers are selected from at least one of mitochondrial pyruvate carrier inhibitors, drugs that inhibit the expression of mitochondrial pyruvate carriers, drugs that knockout or knockdown mitochondrial pyruvate carriers, and drugs that degrade mitochondrial pyruvate carriers.

2. The use according to claim 1, characterized in that: The mitochondrial pyruvate carrier inhibitors are selected from at least one of small molecule compounds, polypeptides, and antibodies; and / or, the drugs that inhibit the expression of mitochondrial pyruvate carriers are selected from at least one of circular RNAs, antisense nucleic acids, small interfering nucleic acids, nucleic acid aptamers, small activating nucleic acids, microRNAs, mRNA drugs, and ribozymes; and / or, the drugs that knockout or knockdown mitochondrial pyruvate carriers achieve gene knockout or knockdown through gene editing tools; the gene editing tools are at least one of the CRISPR / Cas9 gene editing system and the Cre-loxP system; and / or, the drugs that degrade mitochondrial pyruvate carriers are selected from proteolysis agents.

3. The use according to claim 1 or 2, characterized in that: The drugs that intervene in mitochondrial pyruvate carriers are selected from mitochondrial pyruvate carrier inhibitors, drugs that knockout or knockdown mitochondrial pyruvate carriers.

4. The use according to claim 3, characterized in that: The mitochondrial pyruvate carrier inhibitors are selected from small molecule compounds.

5. The use according to claim 4, characterized in that: The small molecule compound is 2-cyano-3-(1-phenyl-1H-indol-3-yl)-2-acrylic acid.

6. The use according to claim 3, characterized in that: The drugs that knockout or knockdown mitochondrial pyruvate carriers achieve gene knockout or knockdown through the Cre-loxP system.

7. The use according to claim 1, characterized in that: The drugs can reduce the levels of serum enzymes; the serum enzymes are at least one of amylase, lipase, and lactate dehydrogenase.

8. The use according to claim 1, characterized in that: The drugs can inhibit inflammatory responses.

9. The use according to claim 1, characterized in that: The drugs can reduce pancreatic acinar cell damage.

10. A pharmaceutical composition for treating acute pancreatitis, characterized in that: It is a preparation prepared with a drug that intervenes in mitochondrial pyruvate carriers as the active ingredient and pharmaceutically acceptable excipients.

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