Use of milodil in the preparation of a medicament for the prevention of liver damage

Abstract

The application discloses application of minoxidil in preparation of a medicine for preventing liver injury and belongs to the technical field of medicines. The application pre-administers 0.5 mg / kg of minoxidil to a mouse once a day for 30 min, so as to reduce liver ischemia-reperfusion injury, reduce serum ALT and AST levels of a liver ischemia-reperfusion model mouse by 70%, reduce serum inflammatory cytokine levels, and reduce liver tissue pathological injury, which indicates that minoxidil has a function of preventing liver ischemia-reperfusion injury. The application provides application of minoxidil in prevention of liver ischemia-reperfusion injury, and provides a new prevention idea for liver ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of milodil in the preparation of drugs for the prevention of liver damage. Background Technology

[0002] Hepatic ischemia-reperfusion injury (IRI) is a serious complication that can occur during liver transplantation, resection surgery, trauma, or hemorrhagic shock. IRI involves a complex series of events following numerous pathophysiological processes. Specifically, the release of pro-inflammatory cytokines, chemokines, and reactive oxygen species (ROS) during the acute inflammatory response to ischemia and subsequent reperfusion of the organ or tissue can lead to significant cellular damage and organ dysfunction. Hepatic IRI can be categorized into cold ischemia and warm ischemia based on the clinical scenario. Cold ischemia occurs during the in vitro preservation phase of the transplanted liver, where the leading damaged cells are primarily sinusoidal endothelial cells, and reperfusion injury following cold ischemia is often accompanied by warm ischemia-reperfusion injury. Warm ischemia mainly occurs during liver transplantation, partial hepatectomy, and shock, where the leading damaged cells are hepatocytes, exhibiting similar pathophysiological processes. IRI comprises an ischemic injury phase and a reperfusion injury phase. During the ischemic injury period, due to hypoxia, glucose depletion, and a sharp decrease in the production of adenosine triphosphate (ATP), local hepatocyte edema, degeneration, and even necrosis occur. During the reperfusion injury period, the liver blood supply is restored, and secondary metabolic disorders and activation of inflammatory signaling pathways lead to the infiltration of various immune cells in the liver, further aggravating liver damage.

[0003] Although extensive research has deepened our understanding of the mechanisms of hepatic ischemia-reperfusion injury (IRI), the underlying mechanisms remain unclear, and no approved pharmacological interventions have yet been developed to prevent organ damage caused by hepatic ischemia-reperfusion (I / R). Furthermore, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive indicators of liver damage and are currently the most commonly used liver function tests. When hepatocytes are damaged, ALT and AST are released into the bloodstream, leading to elevated levels. ALT is mainly distributed in the hepatocyte cytoplasm, and its elevation reflects damage to the hepatocyte membrane; AST is mainly distributed in the hepatocyte cytoplasm and hepatocyte mitochondria, and its elevation indicates that hepatocyte damage has reached the organelle level. Minoxidil was first introduced to my country in the 1970s, initially as a clinical antihypertensive drug, usually used in combination with diuretics and beta-blockers. As a potassium channel opener, minoxidil can directly relax vascular smooth muscle, dilate arterioles, and lower blood pressure. Subsequent clinical studies have found that systemic use of minoxidil in most adults leads to hirsutism, while topical application of minoxidil can promote hair growth by affecting hair follicle cells; therefore, it is also used to treat androgenetic alopecia. In recent years, some studies have found that minoxidil also has anti-tumor effects; for example, minoxidil can inhibit the growth of hepatocellular carcinoma in vitro, and the combination of paclitaxel and minoxidil can inhibit tumor growth. Furthermore, minoxidil can reduce the neurotoxicity caused by paclitaxel. However, whether minoxidil exacerbates or alleviates liver ischemia-reperfusion injury has not yet been reported globally.

[0004] Based on this, the present invention accidentally discovered that minoxidil has a preventive effect on the prevention of liver ischemia-reperfusion injury, opening up a new application scenario for the clinical treatment of minoxidil. Summary of the Invention

[0005] Based on the above analysis, this invention specifically explores the pharmacological use of minoxidil, a potassium channel opener, in the prevention of hepatic ischemia-reperfusion injury. An ischemia-reperfusion model was established in mice with 70% liver clamping and reperfusion. This model was pre-treated with minoxidil by gavage for 30 minutes. The results clearly showed that a single dose of minoxidil significantly reduced hepatic ischemia-reperfusion injury, thus demonstrating that minoxidil has a preventive effect on hepatic ischemia-reperfusion injury. This invention is achieved through the following technical means:

[0006] This invention discloses the use of milodil in the preparation of drugs for the prevention of liver damage.

[0007] Furthermore, the liver injury is liver ischemia-reperfusion injury.

[0008] Furthermore, the animal model of liver ischemia-reperfusion injury is a mouse.

[0009] Furthermore, the route of administration of minoxidil is by gavage.

[0010] Furthermore, the gavage is administered only once.

[0011] Furthermore, the gavage time is 30 minutes for a pre-existing 70% liver ischemia-reperfusion injury model.

[0012] Furthermore, the oral gavage dose is 0.5 mg / kg.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention, through experiments, found that a single oral administration of minoxidil 30 minutes prior to liver ischemia-reperfusion injury in mice can alleviate this injury, reducing serum ALT and AST levels in a 70% reduction in mice with a liver ischemia-reperfusion injury model, decreasing serum inflammatory cytokine levels, and alleviating pathological damage to liver tissue. This demonstrates that minoxidil has a preventive effect against liver ischemia-reperfusion injury. This invention provides the application of minoxidil in the prevention of liver ischemia-reperfusion injury, offering a new approach to its prevention. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the technical solutions of the present invention and form part of the specification. They are used together with the specific embodiments of this application to explain the technical solutions of the present invention and do not constitute a limitation on the technical solutions of the present invention.

[0016] Figure 1 Effects of minoxidil pretreatment on serum ALT in mice with 70% liver ischemia-reperfusion injury;

[0017] Figure 2 Effects of minoxidil pretreatment on serum AST in mice with 70% liver ischemia-reperfusion injury;

[0018] Figure 3 Effects of minoxidil pretreatment on serum tumor necrosis factor-α (TNF-α) in mice with 70% liver ischemia-reperfusion injury;

[0019] Figure 4 Effects of minoxidil pretreatment on serum interferon-γ (IFN-γ) in mice with 70% liver ischemia-reperfusion injury;

[0020] Figure 5 Liver histopathology in mice with 70% liver ischemia-reperfusion injury pretreated with minoxidil. Detailed Implementation

[0021] To better understand the content of this invention, the following description, in conjunction with specific implementation methods, further illustrates the invention. However, before describing the specific embodiments of this invention, it should be understood that the scope of protection of this invention is not limited to the following examples.

[0022] The drug reagents and consumables used in the present invention are all ordinary commercially available products and can be purchased on the market.

[0023] Through experiments, the present invention found that single intragastric administration of minoxidil 30 minutes in advance alleviates liver ischemia-reperfusion injury, reduces the serum ALT and AST levels in 70% of the mice with liver ischemia-reperfusion model, reduces the serum inflammatory cytokine levels, and alleviates the pathological damage of liver tissue, indicating that minoxidil has the effect of preventing liver ischemia-reperfusion injury.

[0024] Example 1

[0025] Application of miloridine in the preparation of drugs for preventing liver injury

[0026] Single intragastric administration of minoxidil 30 minutes in advance in the present invention alleviates liver ischemia-reperfusion injury, reduces the serum ALT and AST levels in 70% of the mice with liver ischemia-reperfusion model, reduces the serum inflammatory cytokine levels, and alleviates the pathological damage of liver tissue, indicating that minoxidil has the effect of preventing liver ischemia-reperfusion injury. The specific scheme is as follows:

[0027] (1) Materials and reagents:

[0028] Animals: Male C57BL / 6J mice, SPF level, 6 - 8 weeks old, weighing 20 ± 2 g, purchased from the Experimental Animal Center of Chongqing Medical University, and adaptively fed in a SPF-level laboratory for one week. Experimental animal production license number: SCXK(YU)2022 - 0010; Experimental animal use license number: SYXK(YU)2022 - 0016.

[0029] Reagents: Minoxidil, MCE Company; ALT and AST test kits, Nanjing Jiancheng Bioengineering Institute; TNF-α ELISA kit, Shenzhen Dakewei Biotechnology Co., Ltd.; IFN-γ ELISA kit, Shenzhen Dakewei Biotechnology Co., Ltd.; 4% paraformaldehyde, Biosharp; hematoxylin, eosin staining solution, phosphate buffer (powder), neutral gum, Shanghai Sangon.

[0030] Instrument consumables: Electronic analytical balance, Shanghai Jingtian Electronic Instrument Company; low-temperature high-speed centrifuge, Eppendorf, Germany; microplate reader, Molecular Devices, USA; vortex oscillator, Scientific Industries, USA; 0.22um filter, Millipore; disposable sterile syringe (1ml), Shandong Weigao Group Co., Ltd.

[0031] Minoxidil solution preparation: Weigh minoxidil powder in a clean bench, dissolve in sterile PBS to prepare a working solution with a concentration of 0.05 mg / ml, vortex to mix, filter with a 0.22 μm filter and use immediately.

[0032] (2) Experiment on the protection of liver ischemia-reperfusion injury by minoxidil:

[0033] Twenty male C57BL / 6J mice, SPF grade, 6–8 weeks old, weighing 20±2g, were randomly divided into four groups: PBS group, minoxidil (M) group, PBS+IRI group, and M+IRI group, with five mice in each group. After 3 days of acclimatization, mice in the PBS and PBS+IRI groups were administered an equal volume of PBS by gavage; mice in the minoxidil (M) and M+IRI groups were administered an equal volume of minoxidil (0.5mg / kg) by gavage. Thirty minutes after gavage, sham surgery was performed in the PBS and M groups, while 70% hepatic vascular clipping was performed in the PBS+IRI and M+IRI groups. One hour later, the vascular clips were released, and the abdominal cavity of the mice was sutured closed.

[0034] (3) Detection of serum ALT and AST levels: Serum ALT and AST levels were detected 6 hours after modeling. After anesthetizing the mice, blood was expelled from the eyeballs, allowed to stand at room temperature for 30 min, centrifuged at 3000 rpm for 10 min at 4℃, the supernatant was collected, diluted appropriately, and then detected using ALT and AST kits.

[0035] (4) Detection of serum inflammatory factors TNF-α and IFN-γ levels: Serum TNF-α and IFN-γ levels were detected 6 hours after modeling. After anesthetizing mice, blood was expelled from the eyeballs, allowed to stand at room temperature for 30 min, centrifuged at 4℃ and 4000 rpm for 10 min, and the supernatant was collected, diluted appropriately, and detected using an ELISA kit.

[0036] (5) Hematoxylin-eosin (H&E) staining of liver tissue: Six hours after mouse modeling, the right middle lobe of liver tissue was taken from anesthetized mice and fixed in 4% paraformaldehyde for 48 hours. After dehydration by alcohol gradient in a glass jar, the tissue was embedded in paraffin and sectioned. Hematoxylin-eosin staining was performed, and the sections were mounted with neutral resin and observed under an optical microscope for pathological changes in liver tissue.

[0037] result:

[0038] (1) Effects of minoxidil on serum ALT and AST in mice with liver injury induced by hepatic ischemia-reperfusion and the results

[0039] Mice were randomly divided into four groups: PBS group, minoxidil (M) group, PBS+IRI group, and M+IRI group, with N=5. The PBS group was administered the same volume of PBS by gavage, the M group was administered M by gavage, the PBS+IRI group was administered PBS by gavage beforehand, and the M+IRI group was administered M by gavage beforehand. The ischemia-reperfusion surgery was performed 30 minutes later.

[0040] Six hours after modeling, serum ALT and AST levels were detected: after anesthetizing mice, blood was expelled from the eyeballs, allowed to stand at room temperature for 30 minutes, centrifuged at 4000 rpm for 10 minutes at 4°C, the supernatant was collected, diluted appropriately, and then detected using ALT and AST kits.

[0041] The results are as follows Figure 1 , Figure 2 As shown, ALT and AST levels in mice in the PBS and M groups were within normal ranges. ALT and AST levels were significantly elevated in the PBS+IRI model group, but significantly decreased in the model group after minoxidil intervention (P < 0.05). These results indicate that a single oral administration of minoxidil 30 minutes prior to liver ischemia-reperfusion injury can reduce serum ALT and AST levels in mice, demonstrating a protective effect against liver injury induced by ischemia-reperfusion.

[0042] (2) Effects of minoxidil on serum TNF-α and IFN-γ in mice with liver injury induced by hepatic ischemia-reperfusion and the results

[0043] Mice were randomly divided into four groups: PBS group, minoxidil (M) group, PBS+IRI group, and M+IRI group, with N=5. The PBS group was administered the same volume of PBS by gavage, the M group was administered M by gavage, the PBS+IRI group was administered PBS by gavage beforehand, and the M+IRI group was administered M by gavage beforehand. The ischemia-reperfusion surgery was performed 30 minutes later.

[0044] Six hours after modeling, blood was collected from the eyeballs of mice, allowed to stand at room temperature for 30 minutes, centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was collected, diluted appropriately, and detected using TNF-α and IFN-γ kits.

[0045] The results are as follows Figure 3 , Figure 4 As shown, TNF-α and IFN-γ levels in the PBS+IRI model group were significantly higher than those in the M+IRI model group (P < 0.05). The experimental results indicate that a single oral administration of minoxidil 30 min beforehand can reduce serum TNF-α and IFN-γ levels in mice with hepatic ischemia-reperfusion injury, demonstrating a protective effect against hepatic ischemia-reperfusion-induced liver injury.

[0046] (3) Effects and results of minoxidil on liver histopathology in mice with liver ischemia-reperfusion-induced liver injury.

[0047] Mice were randomly divided into four groups: PBS group, minoxidil (M) group, PBS+IRI group, and M+IRI group, with N=5. The PBS group was administered the same volume of PBS by gavage, the M group was administered M by gavage, the PBS+IRI group was administered PBS by gavage beforehand, and the M+IRI group was administered M by gavage beforehand. The ischemia-reperfusion surgery was performed 30 minutes later.

[0048] Six hours after modeling, mouse livers were harvested for H&E staining, and the pathological changes in liver tissue were observed under an optical microscope.

[0049] The results are as follows Figure 5 As shown, hepatocytes in the PBS and M drug groups had clear outlines and normal morphology. In the PBS+IRI group, liver vessels and sinusoids showed significant congestion and hemorrhage, with inflammatory cell infiltration in necrotic areas. In contrast, the liver structure in the M+IRI group returned to normal, and hepatic hemorrhage and inflammatory infiltration were significantly reduced. These results suggest that a single pre-administration of minoxidil can significantly alleviate the pathological manifestations of liver injury induced by ischemia-reperfusion in mice.

[0050] In summary, this study, by detecting serum ALT and AST levels, serum TNF-α and IFN-γ levels, and observing liver tissue pathological manifestations in mice with liver ischemia-reperfusion-induced liver injury, found that a single pre-administration of minoxidil can reduce serum ALT and AST levels, decrease serum TNF-α and IFN-γ levels, and improve liver tissue pathological manifestations in mice with liver ischemia-reperfusion injury, thus clarifying that minoxidil can protect against liver ischemia-reperfusion-induced liver injury.

[0051] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. In addition, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed by the present invention.

Claims

1. Application of milodil as the sole active ingredient in the preparation of drugs for the prevention of liver damage.

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