Application of diphenyl iodized chloride in preparation of medicine for preventing liver ischemia reperfusion injury

Abstract

The invention discloses application of diphenyl iodized salt in preparation of a medicine for preventing hepatic ischemia-reperfusion injury, which is characterized in that 1 mg / kg of diphenyl iodized salt is injected into the abdominal cavity of a hepatic ischemia-reperfusion injury model animal, and the time of intraperitoneal injection is 30 minutes before 70% of the hepatic ischemia-reperfusion injury model. Experiments show that by intraperitoneal injection of diphenyl iodized salt for 30 min in advance, mouse ischemia-reperfusion injury can be relieved, the levels of ALT and AST in mouse serum of a 70% liver ischemia-reperfusion injury model can be reduced, the liver oxidative stress level can be relieved, and pathological injury of liver tissue can be relieved; it is indicated that the diphenyl iodized salt has the effect of preventing the liver ischemia-reperfusion injury. The invention provides the application of the diphenyl iodized salt in prevention of the hepatic ischemia reperfusion injury, and a new prevention thought is provided for the hepatic ischemia reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of diphenyl iodonium chloride in the preparation of a medicine for preventing liver ischemia-reperfusion injury. Background Art

[0002] Hepatic ischemia-reperfusion injury (IRI) is a serious complication that can occur during liver transplantation, resection surgery, trauma, or hemorrhagic shock. Hepatic ischemia-reperfusion injury involves a complex series of events that follow many pathophysiological processes. Specifically, during the acute inflammatory response to ischemia and the subsequent reperfusion of the organ or tissue, the release of proinflammatory cytokines, chemokines, and reactive oxygen species (ROS) may lead to significant cellular damage and organ dysfunction. In addition, histopathological changes such as cellular swelling and neutrophil infiltration are observed during liver I / R injury. Since current treatment strategies only involve supportive care, an effective approach to manage HIRI is needed.

[0003] In recent years, the number of patients with end-stage liver disease has increased year by year, and liver transplantation is the best treatment for end-stage liver disease. Liver IRI can be divided into cold ischemia and warm ischemia according to the clinical scenario type. Cold ischemia exists in the stage of in vitro preservation of transplanted livers, and its leading damaged cells are mainly hepatic sinusoidal endothelial cells. Reperfusion injury after cold ischemia is often accompanied by warm ischemia-reperfusion injury; warm ischemia mainly occurs in liver transplantation, partial liver resection and shock and other diseases, and its leading injury is hepatocytes, which have similar pathological and physiological processes. IRI includes ischemic injury period and reperfusion injury period. In the ischemic injury period, due to hypoxia, glucose depletion and sharp decrease in adenosine triphosphate (ATP) production and many other reasons, local hepatocytes are edematous, degenerated and even necrotic; in the reperfusion injury period, liver blood supply is restored, secondary metabolic disorders and activation of inflammatory signaling pathways promote the infiltration of various immune cells in the liver, further aggravating liver damage. Although a large number of studies have deepened our understanding of the mechanism of liver IRI, the mechanism of liver IRI is still not clear enough, and no approved drug intervention has been developed to avoid organ damage caused by liver ischemia-reperfusion (I / R). In addition, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive indicators of liver damage and are currently the most commonly used liver function test indicators. When liver cells are damaged, ALT and AST are released into the blood, and the levels of ALT and AST in the blood increase. ALT is mainly distributed in the cytoplasm of liver cells, and its increase reflects the damage of liver cell membranes; AST is mainly distributed in the cytoplasm of liver cells and mitochondria of liver cells, and its increase indicates that liver cell damage has reached the level of organelles.

[0004] Diphenyleneiodonium chloride (DPI) is a known NADPH oxidase inhibitor that is widely used to study the generation and function of reactive oxygen species (ROS). DPI reduces the generation of ROS by inhibiting members of the NADPH oxidase family, especially NOX2. This compound has important applications in pharmacological research, especially in the study of oxidative stress-related diseases such as inflammation and tumors. DPI can block the reaction catalyzed by NADPH oxidase, reduce the generation of superoxide anions, and thus affect downstream signaling and cell function. Due to its effective inhibitory effect on ROS generation, DPI plays a key role in studying immune cell function, inflammatory response, and cell apoptosis. In experiments, DPI is often used to simulate low oxidative stress conditions to explore its effects on cell behavior. Studies have provided examples of the application of DPI in inhibiting ROS generation and regulating neutrophil function. In recent years, the application of DPI in stem cell therapy and molecular imaging has also made progress. The latest study in August this year further confirmed the role of DPI in inhibiting cell death caused by specific bacterial toxins. These studies highlight the importance of DPIs as research tools and shed light on their potential therapeutic applications.

[0005] Diphenyl iodonium chloride (DPI) has been studied for the treatment of cardiovascular diseases such as atherosclerosis and myocardial infarction because of its ability to inhibit NADPH oxidase activity and reduce ROS production. However, there are no reports on whether diphenyl iodonium chloride (DPI) can aggravate or alleviate liver ischemia-reperfusion injury. Summary of the invention

[0006] The invention discloses the application of diphenyl iodine chloride salt in the prevention of liver ischemia-reperfusion injury, and specifically studies the drug use of DPI diphenyl iodine chloride salt in the prevention of liver ischemia-reperfusion injury. An ischemia-reperfusion model is established by clamping and reperfusing 70% of the liver of mice, and diphenyl iodine chloride salt is injected intraperitoneally 30 minutes in advance to intervene in the model, and it is clarified that a single administration of diphenyl iodine chloride salt can significantly reduce liver ischemia-reperfusion injury, that is, it is proved that diphenyl iodine chloride salt has a preventive effect on liver ischemia-reperfusion injury.

[0007] The present invention is achieved by the following technical means:

[0008] The invention discloses application of diphenyl iodonium chloride salt in preparing medicine for preventing liver ischemia-reperfusion injury.

[0009] Furthermore, the drug is any acceptable drug in pharmacy containing diphenyl iodonium chloride salt.

[0010] Furthermore, the medicament is a tablet, capsule, pill, freeze-dried powder or injection containing diphenyl iodonium chloride.

[0011] Furthermore, the medicament is an injection containing diphenyl iodonium chloride salt.

[0012] Furthermore, the administration route is intraperitoneal injection; the intraperitoneal injection time is 30 minutes before the 70% liver ischemia-reperfusion injury model.

[0013] Furthermore, the intraperitoneal injection is performed once.

[0014] Furthermore, the liver ischemia-reperfusion injury model animal is a mouse.

[0015] Furthermore, the intraperitoneal injection dose is 1 mg / kg.

[0016] The beneficial effects of the present invention are:

[0017] The present invention has found through experiments that intraperitoneal injection of diphenyl iodine chloride 30 minutes in advance can reduce liver ischemia-reperfusion injury in mice, reduce serum ALT and AST levels of 70% of mice with liver ischemia-reperfusion injury model, reduce liver oxidative stress levels, and reduce liver tissue pathological damage, indicating that diphenyl iodine chloride has the effect of preventing liver ischemia-reperfusion injury. The present invention provides the use of diphenyl iodine chloride in preventing liver ischemia-reperfusion injury, and provides a new prevention idea for liver ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the specific implementation methods of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0019] Figure 1 :The effect of pretreatment with diphenyl iodonium chloride on serum ALT in mice with 70% liver ischemia-reperfusion liver injury;

[0020] Figure 2 :The effect of pretreatment with diphenyl iodonium chloride on serum AST in mice with 70% liver ischemia-reperfusion liver injury;

[0021] Figure 3 :The effect of pretreatment with diphenyl iodonium chloride on GSH in liver tissue of mice with 70% liver ischemia-reperfusion liver injury;

[0022] Figure 4 :The effect of pretreatment with diphenyl iodonium chloride on MDA in liver tissue of mice with 70% liver ischemia-reperfusion liver injury;

[0023] Figure 5: Histopathological diagram of liver tissue of mice with 70% liver ischemia-reperfusion injury pretreated with diphenyliodonium chloride (×200). Detailed implementation manners

[0024] Through experiments, the present invention found that intraperitoneal injection of diphenyliodonium chloride 30 minutes in advance can alleviate liver ischemia-reperfusion injury, reduce the levels of serum ALT and AST in mice with 70% liver ischemia-reperfusion model, alleviate the liver oxidative stress level, and reduce the pathological injury of liver tissue, indicating that diphenyliodonium chloride has the effect of preventing liver ischemia-reperfusion injury.

[0025] The reagents and consumables used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0026] To better understand the content of the present invention, the content of the present invention will be further described below in combination with specific implementation methods. However, before describing the specific implementation manners of the present invention, it should be understood that the content protected by the present invention is not limited to the following examples.

[0027] The present invention intraperitoneally injects diphenyliodonium chloride 30 minutes in advance to alleviate liver ischemia-reperfusion injury, reduce the levels of serum ALT and AST in mice with 70% liver ischemia-reperfusion model, alleviate the liver oxidative stress level, and reduce the pathological injury of liver tissue, indicating that diphenyliodonium chloride has the effect of preventing liver ischemia-reperfusion injury.

[0028] The specific scheme is as follows:

[0029] (1) Materials and reagents:

[0030] 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.

[0031] Reagents: Diphenyliodonium chloride, Selleck; ALT and AST test kits, GSH kit, MDA kit, Nanjing Jiancheng Bioengineering Institute; 4% paraformaldehyde, Biosharp; BCA protein content detection kit, Nanjing KeyGen Biotech; Hematoxylin, eosin staining solution, phosphate buffer (powder), neutral gum, Shanghai Sangon.

[0032] Instrument consumables: electronic analytical balance, Shanghai Jingtian Electronic Instrument Co., Ltd.; low-temperature high-speed centrifuge, Eppendorf, Germany; microplate reader, Molecular Devices, USA; vortex oscillator, Scientific Industries, USA; 0.22um bacteria filter, Millipore; disposable sterile syringe (1 ml), Shandong Weigao Group Co., Ltd.

[0033] Preparation of related solutions: Preparation of diphenyl iodonium chloride salt solution: weigh diphenyl iodonium chloride salt powder on a clean bench, dissolve it in sterile deionized water, prepare a working solution with a concentration of 0.088 mg / ml, shake on a vortex machine to mix, filter with a 0.22 um filter and use directly. Prepare and use immediately.

[0034] (1) Experiment on the protection of diphenyliodonium chloride against liver ischemia-reperfusion injury: 20 male C57BL / 6J mice, SPF level, 6-8 weeks old, weighing 20±2g, were randomly divided into PBS group, diphenyliodonium chloride (D) group, PBS+IRI group, and D+IRI group, with 5 mice in each group. After 3 days of adaptive feeding, mice in the PBS group and PBS+IRI group were intraperitoneally injected with an equal volume of PBS; mice in the diphenyliodonium chloride (D) group and D+IRI group were intraperitoneally injected with an equal volume of diphenyliodonium chloride (1 mg / kg). 30 minutes after intraperitoneal injection, mice in the PBS group and D group underwent sham surgery, and mice in the PBS+IRI group and D+IRI group underwent 70% liver vascular clamping. The clamped vascular clamps were released 1 hour later, and the abdominal cavity of the mice was sutured and closed.

[0035] (2) Detection of serum ALT and AST levels: 6 hours after modeling, serum ALT and AST levels were detected. After anesthetizing the mice, the eyeballs were bled, and the mixture was allowed to stand at room temperature for 30 minutes. The mixture was centrifuged at 4°C, 3000 rpm, and the supernatant was taken and diluted to a reasonable multiple before detection using ALT and AST kits.

[0036] (3) Detection of GSH and MDA content in liver tissue: 6 hours after modeling, the levels of GSH and MDA in liver tissue were detected. Accurately weigh the liver tissue (about 0.05 g), add physiological saline to make a tissue homogenate at a ratio of weight (g): volume (mL) = 1:9, centrifuge at 2500 rpm for 10 minutes, and take the homogenate supernatant (part of the homogenate supernatant is used to determine the protein concentration) and detect it with GSH and MDA kits.

[0037] (4) Hematoxylin-eosin (H&E) staining of liver tissue: 6 hours after the mouse model was established, the right middle lobe of the liver tissue was removed from the anesthetized mice and fixed in 4% paraformaldehyde for 48 hours. The tissue was dehydrated in a glass jar with an alcohol gradient, embedded in paraffin and sliced. The tissue was stained with hematoxylin-eosin and sealed with neutral resin, and the pathological changes of liver tissue were observed under an optical microscope.

[0038] Example 1

[0039] Effects of diphenyl iodonium chloride on serum ALT and AST in mice with liver injury induced by hepatic ischemia-reperfusion

[0040] The mice were randomly divided into PBS group, diphenyl iodonium chloride (D) group, PBS+IRI group, and D+IRI group, N = 5. The PBS group was intraperitoneally injected with the same volume of PBS, the D group was intraperitoneally injected with M, the PBS+IRI group was pre-injected with PBS, and the D+IRI group was pre-injected with DPI, and then the ischemia-reperfusion surgery was performed 30 minutes later.

[0041] 6 hours after modeling, serum ALT and AST levels were detected: after anesthetizing the mice, the eyeballs were bled and the mixture was allowed to stand at room temperature for 30 minutes. Then, the mixture was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was taken and diluted to an appropriate multiple before detection using ALT and AST kits.

[0042] The results are as follows Figure 1 , Figure 2 As shown: ALT and AST values ​​of mice in PBS group and D group were at normal levels, while ALT and AST values ​​of mice in PBS+IRI model group were significantly increased. However, after intervention with diphenyl iodine chloride, ALT and AST levels of mice in model group were significantly decreased (P<0.05). The experimental results showed that intraperitoneal injection of diphenyl iodine chloride 30 minutes beforehand could reduce serum ALT and AST levels in mice with liver ischemia-reperfusion, and had a preventive and protective effect on liver injury induced by liver ischemia-reperfusion.

[0043] Example 2

[0044] Effects of diphenyl iodonium chloride on GSH and MDA in liver tissue of mice with liver injury induced by hepatic ischemia-reperfusion

[0045] The animals were grouped and dosed according to Example 1. The liver tissue of the mice was taken 6 h after modeling, and the liver tissue weight (about 0.05 g) was accurately weighed. Physiological saline was added to prepare a tissue homogenate at a ratio of weight (g): volume (mL) = 1:9, and centrifuged at 2500 rpm for 10 minutes. The homogenate supernatant was taken (part of the homogenate supernatant was retained for determining the protein concentration) and detected with GSH and MDA kits.

[0046] The results are as follows Figure 3 , Figure 4As shown: the GSH content in the liver tissue of mice in the PBS+IRI model group was significantly higher than that in the D+IRI model group (P<0.05), and the MDA content in the liver tissue of mice in the PBS+IRI model group was significantly lower than that in the D+IRI model group. The experimental results show that intraperitoneal injection of diphenyl iodonium chloride 30 minutes beforehand can adjust the GSH content in the liver tissue of mice with liver ischemia-reperfusion, reduce the MDA content in the liver tissue of mice with liver ischemia-reperfusion, and have a preventive and protective effect on liver injury induced by liver ischemia-reperfusion.

[0047] Example 3

[0048] Effects of diphenyl iodonium chloride on liver histopathology in mice with liver injury induced by hepatic ischemia-reperfusion

[0049] The animals were grouped and dosed according to Example 1. The livers of mice were taken out 6 hours after modeling for H&E staining, and the pathological changes of liver tissue were observed under an optical microscope.

[0050] The results are as follows Figure 5 As shown: The hepatocytes in the PBS group and the DPI group had clear outlines and normal morphological structures. The hepatic blood vessels and hepatic sinusoids of the mice in the PBS+IRI group were obviously congested and hemorrhagic, and the necrotic area was infiltrated with inflammatory cells, while the liver structure of the mice in the D+IRI group returned to normal, and the hepatic hemorrhage and inflammatory infiltration phenomena were significantly alleviated. The results suggest that pre-intraperitoneal injection of diphenyl iodonium chloride can significantly alleviate the liver pathological manifestations of mice with liver injury induced by liver ischemia-reperfusion.

[0051] This study detected the serum ALT and AST levels of mice with liver damage induced by liver ischemia-reperfusion, detected the GSH and MDA levels of mice liver tissue, and observed the pathological manifestations of liver tissue. It was found that pre-intraperitoneal injection of diphenyl iodonium chloride salt can reduce the serum ALT and AST of mice with liver ischemia-reperfusion model, adjust the GSH content in liver tissue, reduce the MDA content in liver tissue, and improve the pathological manifestations of liver tissue. It is clear that diphenyl iodonium chloride salt can protect liver damage induced by liver ischemia-reperfusion.

[0052] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of equivalent transformations, and these equivalent transformations all belong to the protection scope of the present invention. It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further explain various possible combinations. In addition, the various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Application of diphenyl iodonium chloride in the preparation of drugs for preventing liver ischemia-reperfusion injury.

2. The use according to claim 1, wherein: The drug is any acceptable drug in pharmacy containing diphenyl iodonium chloride salt.

3. The use according to claim 2, wherein: The medicament is a tablet, capsule, pill, freeze-dried powder injection or injection containing diphenyl iodonium chloride salt.

4. The use according to claim 3, wherein: The medicament is an injection containing diphenyl iodonium chloride salt.

5. The use according to claim 4, wherein: The administration route is intraperitoneal injection; The intraperitoneal injection time was 30 minutes before the 70% liver ischemia-reperfusion injury model was established.

6. The use according to claim 5, wherein: The intraperitoneal injection was performed once.

7. The use according to claim 5, wherein: The liver ischemia-reperfusion injury model animal is a mouse.

8. The use according to claim 5, wherein: The intraperitoneal injection dose is 1 mg / kg.

Images