Application of Nepetoidin B in hepatic ischemia reperfusion injury

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to application of Nepetoidin B in hepatic ischemia reperfusion injury. The invention discloses application of Nepetoidin B as a target for screening prevention, relief or / and treatment of hepatic ischemia reperfusion injury. The invention researches the potential protection effect of NB in mouse liver I / R and AML12 H / R injury models. Results show that NB can relieve liver injury and inhibit oxidative stress, liver cell apoptosis and inflammatory response after liver I / R and H / R injury. Besides, NB can inhibit phosphorylation of JNK / P38 protein, the protective effect of NB is related to increase of MKP5 expression, and after MKP5 is knocked out, the liver protection effect and the inhibiting effect on a p38 / JNK pathway of NB are obviously weakened. The NB can regulate MKP5 mediated P38 / JNK signal transduction, so that liver I / R injury is relieved by inhibiting oxidative stress, cell apoptosis and inflammation. Therefore, NB is an effective candidate drug for treating liver I / R injury.

Description

Technical Field

[0001] The invention belongs to the field of medical biotechnology, and particularly relates to an application of Nepetoidin B in liver ischemia-reperfusion injury. Background Art

[0002] Hepatic ischemia-reperfusion (I / R) injury frequently occurs during liver surgery, such as liver transplantation and hepatectomy. In addition to directly damaging hepatocytes, hepatic I / R injury may also affect the regenerative capacity of hepatocytes, a key factor affecting the success rate and postoperative survival of liver surgery. Therefore, developing new treatments to reduce hepatic I / R injury is crucial.

[0003] Currently, measures to mitigate I / R injury include ischemic preconditioning, drug preconditioning, and gene-targeted strategies. Drug intervention is gaining increasing attention due to its potential clinical applications. Nepetoidin B (NB) is a compound extracted from plants of the genus Schizonepeta and belongs to the caffeic acid family. NB exhibits anti-inflammatory, antioxidant, and antimicrobial properties. Studies have shown that NB exerts anti-inflammatory effects by regulating the nuclear factor (N-kappa B) signaling pathway and also plays a regulatory role in insulin resistance and fat metabolism. To date, no studies have reported the potential role of NB in ​​liver I / R injury. Summary of the Invention

[0004] In view of the problems and shortcomings in the prior art, the present invention aims to provide a use of Nepetoidin B in liver ischemia-reperfusion injury.

[0005] Based on the above purpose, the present invention adopts the following technical solutions:

[0006] Application of Nepetoidin B as a target for screening the prevention, alleviation and / or treatment of liver ischemia-reperfusion injury.

[0007] Preferably, the Nepetoidin B is used as a target for screening the prevention, alleviation and / or treatment of liver injury, cell injury, oxidation and / or inflammation induced by liver ischemia-reperfusion injury.

[0008] Further preferably, the Nepetoidin B is positively correlated with the activities of SOD and GSH, and negatively correlated with the content of MDA and the mRNA expressions of cell apoptosis and inflammatory factors.

[0009] Application of Nepetoidin B in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

[0010] Preferably, the Nepetoidin B is used as an active ingredient in the preparation of a medicament for preventing, alleviating or / and treating liver damage, cell damage, oxidation or / and inflammation induced by liver ischemia-reperfusion injury.

[0011] Further preferably, the Nepetoidin B is positively correlated with the activities of SOD and GSH, and negatively correlated with the content of MDA and the mRNA expressions of cell apoptosis and inflammatory factors.

[0012] Use of a drug that promotes Nepetoidin B to inhibit JNK / P38 signal transduction in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

[0013] Preferably, the Nepetoidin B inhibits the phosphorylation levels of P38 and JNK proteins in liver ischemia-reperfusion injury.

[0014] Use of a drug that promotes Nepetoidin B to promote MKP5 mRNA or / and protein expression in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

[0015] Use of a drug that promotes Nepetoidin B to regulate the JNK / P38 pathway mediated by MKP5 in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This application studies the potential protective effects of NB in ​​mouse liver I / R and AML12 H / R injury models. The results showed that NB can alleviate liver damage and inhibit oxidative stress, hepatocyte apoptosis and inflammatory response after liver I / R and H / R injury. In addition, NB can inhibit the phosphorylation of JNK / P38 protein. The protective effect of NB is related to the increase in MKP5 expression. After MKP5 knockout, its hepatoprotective effect and inhibitory effect on the p38 / JNK pathway were significantly weakened. NB can regulate MKP5-mediated P38 / JNK signal transduction, thereby alleviating liver I / R injury by inhibiting oxidative stress, cell apoptosis and inflammation. Therefore, NB is an effective candidate drug for the treatment of liver I / R injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A and B are the serum ALT and AST levels in each group (n=6 / group); C is the effect of NB on the activity of AML12 cells under normoxia detected by CCK-8; D is the effect of NB on the activity of AML12 cells after H / R injury detected by CCK-8; E is H&E staining (the arrow indicates the necrotic area); F is the statistics of necrotic area in mouse liver tissue, **P<0.01 compared with the sham or control group; #P<0.05 and ##P<0.01 compared with the I / R or H / R group;

[0019] Figure 2 A is the MDA content in liver tissue (n=6 / group); B is the SOD activity in liver tissue; C is the GSH activity in liver tissue; D is DHE staining (red fluorescence indicates positive DHE staining); E is the fluorescence intensity analysis of liver tissue; F is the DHE staining (red fluorescence indicates positive DHE staining) and fluorescence intensity of AML12 cells (G), **P<0.01 vs. sham or control group; ##P<0.01 vs. I / R or H / R group;

[0020] Figure 3 In the figure, A shows TUNEL staining (green fluorescence indicates TUNEL-positive cells) and statistical analysis of mouse liver tissue (B) (n=6 / group); C-D shows the detection of cell apoptosis by flow cytometry (n=3);

[0021] Figure 4 In the figure, E is the detection of apoptotic proteins and (FH) statistical analysis of mouse liver tissues (n=3); I is the detection of apoptotic proteins in AML12 cells and (JL) statistical analysis (n=3), **P<0.01 compared with the sham or control group; ##P<0.01 compared with the I / R or H / R group;

[0022] Figure 5 Figure 5, AC, serum levels of Il-1β, Il-6, and TNF-α detected by ELISA (n=6 / group); D, Ly6g immunofluorescence staining (red fluorescence indicates Ly6g-positive cells) and (E) statistical analysis of mouse liver tissue; F, CD11b immunofluorescence staining (red fluorescence indicates CD11b-positive cells) and (G) statistical analysis of mouse liver tissue; HJ, Il-1β, Il-6, and Tnf-α mRNA expressions in AML12 cells (n=3), **P<0.01 compared with sham liver or control group; ##P<0.01 compared with I / R or H / R group;

[0023] Figure 6A shows the total protein, phosphorylated JNK protein, and P38 protein in mouse liver tissue (n=3); BC shows the statistical analysis; D shows the detection and (EF) statistical analysis of the total protein, phosphorylated JNK protein, and P38 protein in AML12 cells (n=3), **P<0.01 compared with the sham or control group; ##P<0.01 compared with the I / R or H / R group;

[0024] Figure 7 A is the mRNA expression of MKP5 in liver tissue (n=6 / group), B is the mRNA expression of MKP5 in AML12 cells (n=3), C is the protein detection and D is the statistical analysis of MKP5 in mouse liver tissue (n=3); E is the protein detection and F is the statistical analysis of MKP5 expression in AML12 cells (n=3), **P<0.01 compared with the sham or control group; ##P<0.01 compared with the I / R or H / R group;

[0025] Figure 8 In the figure, A and B are serum ALT and AST levels, respectively (n=6 / group); C is HE staining (the area indicated by the arrow indicates the necrotic area); D is the statistics of the necrotic area; E is DHE staining (red fluorescence indicates positive DHE staining); F is the fluorescence intensity analysis of liver tissue, G is TUNEL staining (green fluorescence indicates TUNEL-positive cells), and H is the statistical analysis;

[0026] Figure 9 In the figure, IK represents the levels of Il-1β, Il-6, and TNF-α in serum, and LM represents the protein expressions of MKP5 and JNK / P38 pathways. Statistical analysis of liver tissues, **P<0.01 vs. sham group; ##P<0.01 vs. I / R group; $$P<0.01 vs. I / R group; &&P<0.01 vs. I / R+NB group. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0028] 1. Materials and Methods

[0029] 1. Animals and Liver I / R Injury Model

[0030] Wild-type male C57BL / 6 mice, 6–8 weeks old and weighing 20–25 g, were purchased from the Experimental Animal Center of Zhengzhou University. Mice were housed in a pathogen-free environment with a 12-h light / dark cycle and free access to food and water. MKP5KO mice were provided by Xinxiang Medical College. Mice were intraperitoneally injected with 200 μL of NB one hour before I / R injury. A control group received an equal volume of saline. All experimental procedures adhered to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

[0031] Mouse model of liver I / R injury: Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital. The abdominal cavity was opened along the midline, and the vessels of the middle and left hepatic lobes were isolated and clamped with a noninvasive arterial clamp for 90 minutes to induce ischemia. The clamps were then removed to restore hepatic perfusion, and the abdominal incision was sutured with silk suture. Sham-operated mice were not subjected to vascular clamping, and the remaining procedures were the same as those in the I / R group. After reperfusion, blood and tissue samples were collected for analysis.

[0032] 2. Liver function measurement

[0033] After reperfusion, 0.2 mL of mouse blood was collected and centrifuged at 3000 rpm for 5 min, and the supernatant was collected for analysis. Serum AST and ALT levels were determined according to the manufacturer's instructions (Jianchen Bioengineering Institute, Nanjing, China).

[0034] 3. Hematoxylin-eosin Staining

[0035] Liver tissue was fixed with 10% formalin solution, embedded in paraffin, and cut into 5 μm thick sections. Paraffin sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E) according to the instructions of the Hematoxylin and Eosin Staining Kit (Servicebio, Wuhan). The sections were then dehydrated, transparentized, and sealed with neutral gum solution. Finally, photos were taken and analyzed under an optical microscope (Olympus, Japan).

[0036] 4. Cell Culture and H / R Model

[0037] The AML12 mouse hepatocyte H / R model was used to simulate in vitro I / R injury. AML12 cells were purchased from Wuhan Pronocell Biotechnology Co., Ltd. The cells were supplemented with 10% fetal bovine serum and 1x10 5 The cells were cultured in DMEM / F12 medium supplemented with 100 U / mL penicillin and 100 mg / mL streptomycin at 37°C in an incubator with 5% CO2.

[0038] Establishment of the H / R model for AML12 cells: Before hypoxia, cells were rinsed twice with PBS and placed in a sugar-free, serum-free medium (Procell, Wuhan) for hypoxia in a tri-gas incubator (5% CO2, 94% N2, 1% O2). After 12 hours of hypoxia, the medium was replaced with normal medium and cultured in a normal-pressure incubator for 6 hours to complete reoxygenation.

[0039] 5. Cell Counting Kit-8 Detection

[0040] AML12 cells were seeded at a density of 5,000 cells per well in a 100 μL volume in a 96-well plate and cultured in a normoxic incubator. When the cell density reached 70–80%, H / R treatment was performed according to the experimental group. The NB group was treated with NB 1 hour before hypoxia induction. After reoxygenation, 10 μL of CCK-8 reagent (Boster, Wuhan) was added to each well and the cells were cultured in a normoxic incubator for 1 hour. After incubation, the A450 absorbance was measured using a microplate reader (Thermo Fisher Scientific, Inc.).

[0041] 6. Oxidative stress index detection

[0042] 1 mL of the extract was added to every 100 mg of liver tissue for homogenization, followed by centrifugation at 12,000 × g for 10 min. The supernatant was collected and protein concentration was determined using a BCA kit. SOD, MDA, and GSH were detected according to the instructions provided by the corresponding kits (Solarbio, Beijing). Dihydroethidium (DHE) staining (Biyuntian, Shanghai) was used to detect reactive oxygen species (ROS) in liver tissue and AML12 cells. Liver tissue embedded in OTC was cut into 10 μm sections and stained with DHE (10 μM / L) fluorescent probe. The sections were incubated in the dark at 37°C for 30 min, washed three times with PBS (Solarbio, Beijing), and stained with DAPI (Biyuntian, Shanghai) for 10 min at room temperature before being photographed under a fluorescence microscope. For AML12 cells, after reoxygenation, 1 μL of 10 μM DHE staining solution was added per mL of culture medium, incubated for 20 min, rinsed three times with PBS, and photographed under a fluorescence microscope (Olympus, Japan).

[0043] 7. ELISA

[0044] The levels of IL-1β, IL-6, and TNF-α in serum were determined by ELISA, and all indicators were tested strictly according to the manufacturer's instructions (Sanying Biotechnology Co., Ltd., Wuhan).

[0045] 8. Real-time polymerase chain reaction (PCR) analysis

[0046] Total RNA was extracted from liver tissue and AML12 cells using TRIzol reagent (Solarbio, Beijing) according to the manufacturer's instructions. RNA concentration was determined using a micro-UV spectrophotometer (Thermo Fisher Scientific). One microgram of RNA was reverse transcribed into cDNA (Novagen, Nanjing) according to the transcription kit instructions, and cDNA was amplified using SYBR Green qPCR Mix (Novagen, Nanjing) on ​​a qPCR instrument. GAPDH was used as an internal control. Analysis was performed using the 2-ΔΔCT method. Primer sequences are shown in Table 1.

[0047] Table 1 Real-time polymerase chain reaction primer sequences

[0048]

[0049] 9. Immunofluorescence Staining

[0050] Paraffin-embedded liver tissue was cut into 5 μm sections, baked at 60°C for 2 h, then deparaffinized with xylene and hydrated with graded ethanol. Liver sections were fixed with 0.01 mol / L citrate buffer (pH 6.0), and endogenous enzymes were inactivated with 3% H₂O₂ for 10 min. After blocking with 10% goat serum for 1 h at room temperature, sections were incubated with primary antibodies against Ly6g (1:200; Seville, Wuhan) and CD11b (1:200; Seville, Wuhan) overnight at 4°C. The next day, sections were rinsed three times with PBS before and after the addition of fluorescent secondary antibodies (1:500, Seville, Wuhan), incubated at room temperature for 30 min, stained with DAPI for 10 min at room temperature, and mounted with anti-quencher (Seville, Wuhan). Sections were photographed under a fluorescence microscope (Olympus).

[0051] 10. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

[0052] Paraffin-embedded liver tissue was cut into 5 μm sections, dewaxed with xylene, and rehydrated with gradient ethanol. TUNEL staining was performed using a TUNEL kit (Seville, Wuhan). After staining, the sections were rinsed three times with PBS for 5 min each. The nuclei were then stained with DAPI, incubated at room temperature for 10 min, rinsed three times with PBS for 5 min each, and finally sealed with an anti-fluorescence quencher. The sections were photographed using a fluorescence microscope.

[0053] 11. Western blotting

[0054] Proteins from liver tissue and cells were extracted using RIPA buffer (Solerbo, Beijing) containing 1% PMSF and protein phosphatase inhibitors. Protein concentration was determined using a BCA kit (Solerbo, Beijing). Proteins were separated using 10% or 12% SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% skim milk powder for 1 h. The membranes were incubated overnight at 4°C with anti-BCL-2 (1:1000), anti-Cleaved caspase 3 (1:1000), anti-BAX (1:1000), anti-p-JNK (1:1000), anti-JNK (1:1000), anti-p-p38 (1:1000), anti-p38 (1:1000), anti-MKP5 (1:1000), and anti-GAPDH (1:5000) antibodies. The next day, the membranes were incubated with goat anti-rabbit (1:10,000, Mitsubishi Biotechnology Co., Ltd.) or goat anti-mouse secondary antibody (1:10,000, Mitsubishi Biotechnology Co., Ltd.) at room temperature for 1 h. After washing the membranes three times with TBST, ECL luminescent solution (New Cyme, Suzhou) was added and the membranes were developed using a gel imaging system (Cytiva, USA).

[0055] 12. Flow Cytometry Analysis

[0056] After reoxygenation, cells were digested with EDTA-free trypsin (Beyotime, Shanghai) and centrifuged at 200 g / min for 5 minutes. After rinsing twice with PBS, cells were resuspended in 195 μL of 1× binding buffer. Annexin V-FITC and PI (Beyotime, Shanghai) were added sequentially, and the cells were incubated at room temperature in the dark for 10 minutes before analysis using a flow cytometer (BD Biosciences, USA).

[0057] 13. Statistical Analysis

[0058] Experimental data were analyzed using SPSS software (version 22.0). Measurement data are expressed as mean ± standard deviation. Two-group comparisons were performed using the t-test, and multiple-group comparisons were performed using one-way analysis of variance. Statistical significance was considered to be P < 0.05.

[0059] 2. Results and Analysis

[0060] 1. Nepetoidin B alleviates I / R-induced liver and cell injury in vivo and in vitro

[0061] The determination of the NB concentration in mice and AML12 cells took into account both the lack of toxicity at low concentrations and the protective effect of NB on I / R injury in mouse liver and H / R injury in cells. Figure 1A shows that NB has no toxic effect on the mouse liver when the concentration is lower than 20 mg / kg. NB concentrations of 10, 15, and 20 mg / kg can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. 15 mg / kg and 20 mg / kg are more effective than 10 mg / kg, but there is no significant difference in effectiveness between 15 mg / kg and 20 mg / kg ( Figure 1 A-1B). Therefore, 15 mg / kg NB was selected for subsequent animal experiments.

[0062] In AML12 cells cultured under normal oxygen conditions, NB concentrations below 30 μM did not show any toxic effects ( Figure 1 C). NB increased the viability of AML12 cells after H / R injury in a concentration-dependent manner, however, there was no significant difference between 20 μM and 30 μM ( Figure 1 D). Therefore, 20 μM NB was selected for subsequent experiments.

[0063] HE staining showed that the hepatocytes in the I / R group had focal or large-area necrosis, and the structure of the liver lobule was also destroyed ( Figure 1 E). Compared with the I / R group, the area of ​​necrotic cells in the I / R+NB group was significantly reduced ( Figure 1 E-1F). Based on the above results, NB can alleviate I / R-induced liver injury and H / R-induced cell injury.

[0064] 2. Nepetoidin B reduces I / R-induced oxidative stress in vivo and in vitro

[0065] like Figure 2 As shown in AC, compared with the sham group, the MDA content in liver tissue after I / R injury was significantly increased, and the SOD and GSH activities were significantly decreased. Compared with the I / R group, NB pretreatment inhibited the MDA content while significantly increasing the SOD and GSH activities ( Figure 2 A-2C). Figure 2 D-2F showed that the ROS level in normal liver tissue and AML12 cells cultured under normal oxygen conditions was relatively low. However, after I / R and H / R, the ROS level increased significantly ( Figure 2 D-2F). NB can effectively inhibit ROS produced after liver I / R and AML12 cell H / R injury ( Figure 2 D-2F). The results showed that NB could alleviate I / R-induced oxidative stress both in vivo and in vitro.

[0066] 3. Nepetoidin B alleviates I / R-induced apoptosis in vivo and in vitro

[0067] After liver I / R injury, the number of TUNEL-positive cells in mouse liver tissue increased significantly ( Figure 3 A-3B), the apoptosis rate of AML12 cells increased significantly after H / R injury ( Figure 3 C-3D). Pretreatment with NB can significantly reduce the number of TUNEL-positive cells in liver tissue after I / R injury ( Figure 3 A-3B) and apoptosis rate of AML12 cells after H / R injury ( Figure 3 C-3D). Detection of apoptosis-related proteins showed that the levels of BAX and cleaved caspase 3 increased significantly in liver tissue after I / R injury in mice and in H / R injury in AML12 cells, while the expression of BCL-2 decreased significantly ( Figure 4 E-4L); NB pretreatment reversed the changes in the expression of apoptosis-related proteins. The results showed that NB pretreatment can reduce I / R-induced cell apoptosis in vivo and in vitro.

[0068] 4. Nepetoidin B can reduce I / R-induced inflammation in vivo and in vitro

[0069] After liver I / R injury, the levels of IL-1β, IL-6, and TNF-α in serum increased significantly ( Figure 5 A-5C), while NB pretreatment can significantly reduce the levels of IL-1β, IL-6 and TNF-α in the serum of mice after liver I / R injury. Figure 5 D-5G showed that the number of Ly6g and CD11b+ cells in liver tissue increased significantly after I / R injury, while the number of Ly6g and CD11b+ cells in the IR+NB group decreased significantly, indicating that NB reduced the tissue inflammatory response after liver I / R injury. In addition, the mRNA expression of IL-1β, IL-6 and TNF-α in AML12 cells after H / R injury was detected, and it was found that the mRNA expression of IL-1β, IL-6 and TNF-α increased significantly after H / R ( Figure 5 H-5J), while NB pretreatment significantly inhibited the mRNA expression of inflammatory factors. These results indicate that NB can inhibit I / R-induced inflammatory responses in vivo and in vitro.

[0070] 5. Nepetoidin B inhibits JNK / P38 signaling in vivo and in vitro

[0071] To detect changes in the JNK / P38 pathway, changes in JNK / P38 protein phosphorylation were detected in mice and cells. Compared with the sham operation group and the control group, JNK and P38 phosphorylation in liver tissue after I / R injury and AML12 cells after H / R injury were significantly increased ( Figure 6A-6F). However, NB pretreatment significantly reduced the phosphorylation of P38 and JNK proteins after liver I / R and H / R injury. These results suggest that NB pretreatment can reduce JNK / P38 activation in vitro and in vivo.

[0072] 6. Nepetoidin B promotes MKP5 expression in vivo and in vitro

[0073] like Figure 7 As shown in A and 7B, compared with the control mice and cells, the mRNA and protein expressions of MKP5 were significantly increased after NB pretreatment. In liver tissues after I / R injury and AML12 cells after H / R injury, the mRNA and protein expressions of MKP5 were significantly decreased ( Figure 7 A-7F). However, pretreatment with NB effectively reversed the decrease in MKP5 expression. In summary, NB pretreatment promoted MKP5 expression and reversed the downregulation of MKP5 expression induced by liver I / R injury and H / R injury in AML12 cells.

[0074] 7. Knockout of MKP5 weakens the hepatoprotective effect of Nepetoidin B

[0075] To investigate how NB protects the liver from I / R injury, MKP5 knockout (KO) mice were used. Compared with wild-type (WT) mice, serum ALT and AST levels were significantly increased after I / R injury ( Figure 8 A-8B), liver necrosis area ( Figure 8 C-8D), DHE fluorescence intensity in liver tissue ( Figure 8 E-8F), hepatocyte apoptosis rate ( Figure 8 G-8H) and inflammatory factor expression levels ( Figure 9 I-9K) were significantly increased, and P38 / JNK protein phosphorylation was more obvious after MKP5 KO ( Figure 9 L-9M), indicating that MKP5 knockout exacerbates liver I / R injury. Compared with the I / R group, treatment with NB significantly alleviated liver damage, oxidative stress, apoptosis, and inflammation after I / R. In MKP5 knockout mice, the hepatoprotective effects of NB and its inhibitory effects on JNK / P38 proteins were significantly attenuated. In summary, MKP5 knockout weakens the protective effects of NB, and the hepatoprotective effects of NB are related to the promotion of MKP5 expression.

[0076] GSH, SOD, MDA and DHE staining are important indicators for evaluating oxidative stress during liver I / R injury. This application used a mouse liver I / R injury model and an AML12 H / R cell model to explore the potential role of NB in ​​liver I / R injury. Compared with the sham operation group, the levels of MDA and ROS in liver tissue increased significantly after liver I / R injury. In contrast, after NB pretreatment, the levels of MDA and ROS decreased significantly, while the levels of SOD and GSH increased significantly. The DHE staining results of liver tissue and AML12 washed cells showed that NB can inhibit the oxidative effects caused by I / R and H / R injury. In addition, NB can inhibit liver damage, oxidation, apoptosis and inflammation, increase cell activity, and inhibit JNK / P38 protein phosphorylation after liver I / R or cell H / R injury.

[0077] Aberrant activation of the mitogen-activated protein kinase (MAPK) pathway is closely associated with liver I / R injury, and inhibition of this pathway can significantly alleviate liver I / R injury. MAPK phosphatases (MKPs) can dephosphorylate JNK / P38 phosphotyrosine residues, thereby inhibiting MAPK signaling pathway activation. MKP5, a member of the MKP family, inhibits activation of the JNK / P38 signaling pathway. NB exerts anti-inflammatory effects by promoting MKP5 expression. Therefore, it is hypothesized that NB plays a protective role in liver I / R injury by promoting MKP5 expression and thereby inhibiting the JNK / P38 signaling pathway. The MAPK family plays a key role in mediating liver I / R injury and regulating hepatocyte death and survival by mediating signaling pathways related to oxidative stress, apoptosis, and inflammatory responses.

[0078] In the present application, it was observed that the P38 / JNK signaling pathway was activated in mouse livers after I / R injury and H / R injury in AML12 cells, as manifested by increased phosphorylation of P38 and JNK. However, NB pretreatment significantly inhibited the activation of the JNK / p38 pathway after I / R injury in mouse livers and H / R injury in AML12 cells. The protective effect of NB was related to the promotion of MKP5 expression, and after MKP5 knockout, its hepatoprotective effect and inhibitory effect on the p38 / JNK pathway were significantly weakened. Therefore, NB can regulate MKP5-mediated P38 / JNK signal transduction, thereby alleviating liver I / R injury by inhibiting oxidative stress, cell apoptosis and inflammation.

[0079] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Application of Nepetoidin B as a target for screening the prevention, alleviation and / or treatment of hepatic ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that The nepetoidin B is used as a target for screening the prevention, alleviation and / or treatment of liver injury, cell injury, oxidation and / or inflammation induced by liver ischemia-reperfusion injury.

3. The use according to claim 2, characterized in that The nepetoidin B is positively correlated with the activities of SOD and GSH, and negatively correlated with the content of MDA and the mRNA expressions of cell apoptosis and inflammatory factors.

4. Application of Nepetoidin B in the preparation of drugs for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

5. The use according to claim 4, characterized in that The Nepetoidin B is used as an active ingredient in preparing a drug for preventing, alleviating or / and treating liver damage, cell damage, oxidation or / and inflammation induced by liver ischemia-reperfusion injury.

6. The use according to claim 5, characterized in that The nepetoidin B is positively correlated with the activities of SOD and GSH, and negatively correlated with the content of MDA and the mRNA expressions of cell apoptosis and inflammatory factors.

7. Use of drugs that promote Nepetoidin B to inhibit JNK / P38 signal transduction in the preparation of drugs for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

8. The use according to claim 7, characterized in that Nepetoidin B inhibits the phosphorylation levels of P38 and JNK proteins in liver ischemia-reperfusion injury.

9. Use of drugs that promote Nepetoidin B to promote MKP5 mRNA and / or protein expression in the preparation of drugs for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

10. Use of drugs that promote Nepetoidin B to regulate the JNK / P38 pathway mediated by MKP5 in the preparation of drugs for preventing, alleviating or / and treating liver ischemia-reperfusion injury.

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