Application of mannose in preparation of drug for preventing drug-induced liver injury induced by acetaminophen

Abstract

The invention discloses an application of mannose in preparation of a medicine for preventing acetaminophen-induced drug-induced liver injury. The mannose with the mass concentration of 20% is used for intervening acetaminophen-induced (acetaminophen intraperitoneal injection dosage of 300mg / kg) drug-induced liver injury model animals as mice for four weeks in a free water drinking manner. Experiments show that after the mannose is fed for four weeks in advance, the serum ALT and AST levels of APAP-induced drug-induced liver injury model mice can be reduced, the serum inflammatory cytokine level can be reduced, and the pathological injury of liver tissues can be relieved, which indicates that the mannose can protect the APAP-induced drug-induced liver injury. The invention provides the application of mannose in the APAP-induced drug-induced liver injury, and a new prevention thought is provided for the APAP-induced drug-induced liver injury.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and specifically discloses an application of mannose in preparing a medicine for preventing drug-induced liver injury induced by acetaminophen. Background Art

[0002] Drug-induced liver injury (DILI) is a disease in which the body is damaged by drugs or other exogenous substances, and it is very common in clinical practice. Globally, most causes of acute liver injury are drug poisoning. Clinically, DILI is a common cause of unexplained liver disease, ranging from asymptomatic liver enzyme elevation to fulminant liver failure and even death. Acetaminophen (APAP) is the most commonly used drug for the treatment of pain and fever worldwide, and is also a common cause of acute liver failure. APAP is safe for adults at a therapeutic dose of less than 4g / d, and will not cause liver damage even in susceptible individuals. However, once APAP is overused, it is likely to cause severe liver damage and even acute liver failure.

[0003] Mannose is a naturally occurring monosaccharide and an isomer of glucose. It is often extracted from palm kernels, coffee grounds, konjac flour, and acai seeds. As a biologically active monosaccharide, mannose has shown its potential application value in many fields. A large number of clinical studies have shown that mannose can be used to prevent and treat urinary tract infections. It also has certain therapeutic potential in anti-inflammatory, blood sugar regulation, and tumor treatment. In terms of protection against liver damage, relevant studies have shown that mannose can reduce autoimmune hepatitis, reduce thioacetamide-induced rat liver fibrosis, improve alcoholic liver steatosis, and reduce chemotherapy-induced tissue toxicity.

[0004] Currently, no studies have shown that mannose has a protective effect on APAP-induced acute liver injury. Based on this, the present invention explores the use of mannose in the preparation of a drug for preventing acetaminophen-induced drug-induced liver injury. Summary of the invention

[0005] Based on the above analysis, the present invention discloses the application of the natural product mannose in the prevention of acute liver injury, and specifically studies the protective effect of mannose in drug-induced liver injury induced by acetaminophen. An acute liver injury model was established with 300 mg / kg APAP, and 20% mannose was added to the drinking water 4 weeks in advance. It was clarified that mannose feeding can reduce liver injury and improve the survival rate of mice with acute liver injury, which proves that mannose has a preventive effect in drug-induced liver injury induced by acetaminophen.

[0006] The present invention is achieved by the following technical means:

[0007] The invention firstly discloses the application of mannose in preparing a medicine for preventing drug-induced liver injury induced by acetaminophen, wherein the mass concentration of the mannose is 20%.

[0008] Furthermore, the liver damage drug is used for:

[0009] Reduce liver inflammation; and / or,

[0010] Reduce the area of ​​liver necrosis.

[0011] Furthermore, the liver damage drug has the following effects:

[0012] Reduce the levels of liver function transaminases ALT and AST;

[0013] Reduce serum TNF-α, IFN-γ, IL-6 and IL-18 levels.

[0014] Furthermore, the mannose is administered by free intake of drinking water.

[0015] Furthermore, the mannose intervention time is 4 weeks.

[0016] Furthermore, the intraperitoneal injection dose of acetaminophen is 300 mg / kg.

[0017] Furthermore, the medicine also includes:

[0018] Pharmaceutically acceptable adjuvants.

[0019] The beneficial effects of the present invention are:

[0020] The present invention has found through experiments that feeding mannose for 4 weeks in advance can reduce the serum ALT and AST levels of APAP-induced drug-induced liver injury model mice, reduce the serum inflammatory cytokine levels, and alleviate liver tissue pathological damage, indicating that mannose can protect APAP-induced drug-induced liver injury. The present invention provides the application of mannose in APAP-induced drug-induced liver injury, and provides a new prevention idea for APAP-induced drug-induced liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following 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.

[0022] Figure 1 To study the effect of mannose pre-feeding on serum ALT in mice with APAP-induced drug-induced liver injury.

[0023] Figure 2 To study the effect of mannose pre-feeding on serum AST in mice with APAP-induced drug-induced liver injury.

[0024] Figure 3 To study the effect of mannose pre-feeding on serum tumor necrosis factor α (TNF-α) in APAP-induced drug-induced liver injury.

[0025] Figure 4 To study the effect of pre-feeding with mannose on serum interferon-γ (IFN-γ) in mice with APAP-induced drug-induced liver injury.

[0026] Figure 5 To study the effect of mannose pre-feeding on serum IL-6 in mice with APAP-induced drug-induced liver injury.

[0027] Figure 6 To study the effect of mannose pre-feeding on serum IL-18 in mice with APAP-induced drug-induced liver injury.

[0028] Figure 7 The liver tissue pathology of mice with APAP-induced drug-induced liver injury after pre-feeding with mannose, where A is the liver tissue pathology of mice in the APAP group, and B is the liver tissue pathology of mice after mannose intervention. DETAILED DESCRIPTION

[0029] The present invention found through experiments that feeding mannose in drinking water for 4 weeks in advance can reduce drug-induced liver injury, reduce the serum ALT and AST levels of mice in the drug-induced liver injury model induced by acetaminophen, reduce the serum inflammatory cytokine levels, and reduce liver tissue pathological damage, indicating that mannose has the effect of preventing drug-induced liver injury.

[0030] The reagents and consumables used in the present invention are all common commercial products and can be purchased in the market.

[0031] In order to better understand the content of the present invention, the content of the present invention is further described below in conjunction with specific implementation methods. However, before describing the specific implementation methods of the present invention, it should be understood that the content protected by the present invention is not limited to the following examples.

[0032] The present invention reduces drug-induced liver injury by feeding mannose in drinking water for 4 weeks in advance, reduces serum ALT and AST levels in mice with drug-induced liver injury model induced by acetaminophen, reduces serum inflammatory cytokine levels, and reduces liver tissue pathological damage, indicating that mannose has the effect of preventing drug-induced liver injury. The specific scheme is as follows:

[0033] 1. Materials and reagents:

[0034] Animals: Male C57BL / 6J mice, SPF grade, 7 weeks old, weighing 20±2 g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and adaptively fed in an SPF-grade laboratory for one week. License number for experimental animal production: SCXK(Yu)2023-0005; License number for experimental animal use: SYXK(Yu)2021-0002.

[0035] Reagents: Mannose, Macklin; APAP: Sigma, USA; 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 Shengong.

[0036] Instruments and 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; 0.22 μm filter, Millipore; Disposable sterile syringe (1 ml), Shandong Weigao Group Co., Ltd.

[0037] Preparation of related solutions: Preparation of APAP: Weigh APAP powder, dissolve it in PBS to prepare a working solution with a concentration of 25 mg / ml, mix well with a vortex mixer, dissolve it in a 60°C water bath, and use it immediately after preparation.

[0038] 2. Experimental methods

[0039] (1) Mannose protection against drug-induced liver injury experiment: Male C57BL / 6J mice, SPF grade, 7 weeks old, weighing 20±2 g, a total of 20 mice, were randomly divided into a blank (C) group, a mannose (Man) group, an APAP group, and a Man+APAP group, with 5 mice in each group. After 7 days of adaptive feeding, the mice in the Man group and the Man+APAP group were given 20% mannose to drink; the mice in the C group and the APAP group drank normal water. After 4 weeks of feeding, the mice in the C group and the Man group were intraperitoneally injected with normal saline, and the mice in the APAP group and the Man+APAP group were intraperitoneally injected with 300 mg / kg APAP. The mice were sacrificed 24 hours later, and mouse serum and liver tissue were collected.

[0040] (2) Detection of serum ALT and AST levels: At 24 h after modeling, the serum ALT and AST levels were detected. After anesthetizing the mice, blood was collected by orbital bleeding, left to stand at room temperature for 30 min, centrifuged at 4°C and 3000 rpm for 10 min, the supernatant was taken, and after dilution by a reasonable multiple, it was detected using the ALT and AST kits.

[0041] (3) Detection of serum inflammatory factors TNF-α, IFN-γ, IL-6, and IL-18 levels: 24 h after modeling, serum TNF-α and IFNγ levels were detected. After anesthesia, the mice were bled from the eyeballs, left to stand at room temperature for 30 min, centrifuged at 4000 rpm for 10 min at 4°C, and the supernatant was taken and diluted to a reasonable multiple before detection using an ELISA kit.

[0042] (4) Hematoxylin-eosin (H&E) staining of liver tissue: 24 hours after modeling, the right middle lobe of liver tissue was removed from 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, sealed with neutral resin, and the pathological changes of liver tissue were observed under an optical microscope.

[0043] Embodiment 1:

[0044] Effects of mannose on serum ALT and AST in mice with drug-induced liver injury induced by acetaminophen and its results

[0045] The mice were randomly divided into APAP group and Man+APAP group, with 5 mice in each group. The APAP group had normal drinking water, while the Man+APAP group was fed with mannose freely with drinking water. Four weeks later, the APAP group and the Man+APAP group were intraperitoneally injected with 300 mg / kg APAP.

[0046] 24 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, centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was taken and diluted to an appropriate multiple before detection using ALT and AST kits.

[0047] The results are as follows Figure 1 , Figure 2 As shown: ALT and AST levels of mice in the APAP group and Man+APAP group were significantly increased, but after mannose intervention, ALT and AST levels of mice in the model group were significantly reduced (P < 0.05). The experimental results show that feeding mannose for 4 weeks in advance can reduce the serum ALT and AST levels of mice with APAP-induced drug-induced liver injury, and has a preventive and protective effect on APAP-induced drug-induced liver injury.

[0048] Embodiment 2:

[0049] Effects of mannose on serum TNF-α, IFN-γ, IL-6 and IL-18 in mice with drug-induced liver injury induced by acetaminophen and its results

[0050] The animals were grouped and dosed according to Example 1. Blood was collected from the mouse eyeballs 24 h after modeling, and the mixture was allowed to stand at room temperature for 30 min. The mixture was centrifuged at 4°C, 4000 rpm, and the supernatant was taken and diluted to an appropriate multiple before detection using TNF-α, IFN-γ, IL-6, and IL-18 kits.

[0051] The results are as follows Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown: TNF-α, IFN-γ, IL-6 and IL-18 in the APAP model group were significantly higher than those in the APAP model group (P < 0.05). The experimental results showed that feeding mannose for 4 weeks in advance can reduce the serum TNF-α, IFN-γ, IL-6 and IL-18 levels of mice with APAP-induced drug-induced liver injury, and has a preventive and protective effect on APAP-induced drug-induced liver injury.

[0052] Embodiment 3:

[0053] Effects of mannose on liver histopathology of mice with acetaminophen-induced drug-induced liver injury and its outcome

[0054] The animals were grouped and dosed according to Example 1. The livers of mice were taken 24 hours after modeling for H&E staining, and the pathological changes of liver tissue were observed under an optical microscope.

[0055] The results are as follows Figure 7 As shown: The APAP group mice had massive edema and flake necrosis of liver cells, nuclear fragmentation and nuclear dissolution, accompanied by infiltration of inflammatory cells, while the intervention group significantly reduced liver cell necrosis and inflammatory cell infiltration. The results suggest that feeding mannose for 4 weeks in advance can significantly reduce the liver pathological manifestations of APAP-induced drug-induced liver injury mice.

[0056] In this study, we detected the serum ALT and AST levels in mice with liver injury induced by liver ischemia-reperfusion, detected the serum TNF-α, IFN-γ, IL-6 and IL-18 levels in mice, and observed the pathological manifestations of liver tissue. We found that feeding mannose for 4 weeks in advance could reduce the serum ALT and AST of mice in the APAP-induced drug-induced liver injury model, reduce the serum TNF-α, IFN-γ, IL-6 and IL-18 levels, and improve the pathological manifestations of liver tissue, which confirmed that mannose can protect against APAP-induced drug-induced liver injury.

[0057] The preferred embodiments of the present invention are described in detail above; however, the present invention is not limited to the specific details in the above embodiments. Within 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 belong to the protection scope of the present invention.

[0058] 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 describe various possible combinations. In addition, the various different embodiments of the present invention can also be combined arbitrarily, 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. Use of mannose in the preparation of a drug for preventing acetaminophen-induced drug-induced liver injury, wherein: The mass concentration of mannose is 20%.

2. The use according to claim 1, wherein: The liver damage medication is used to: Reduce liver inflammation; and / or, Reduce the area of ​​liver necrosis.

3. The use according to claim 1, wherein: The liver damage drugs have the following effects: Reduce the levels of liver function transaminases ALT and AST; Reduce serum TNF-α, IFN-γ, IL-6 and IL-18 levels.

4. The use according to claim 1, wherein: The mannose administration method is free intake of drinking water.

5. The use according to claim 1, wherein: The mannose intervention time was 4 weeks.

6. The use according to claim 1, wherein: The intraperitoneal injection dose of acetaminophen is 300 mg / kg.

7. The use according to claim 1, wherein: The medicine also includes: Pharmaceutically acceptable adjuvants.

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