Application of Peli1 as target spot for relieving and / or treating acute liver injury

Abstract

The invention discloses an application of E3 ubiquitin protein ligase Pellino (Peli1) in preparation of a medicine for relieving and / or treating acute liver injury. The research finds that when an organism is in acute liver injury (ALI), the Peli1 gene expression level in liver cells is obviously reduced. A (murine) overexpression plasmid containing a Peli1 coding region sequence is used for transfecting a murine hepatocyte (AML12 cell line), an in-vitro overexpression Peli1 model is constructed, an in-vitro ALI cell model is induced by APAP, and the result shows that after overexpression of Peli1, hepatocyte apoptosis is remarkably inhibited, and hepatocyte proliferation is promoted. Therefore, Peli1 can be used for preparing medicines for relieving and / or treating acute liver injury related diseases. The application of Peli1 as a drug target for relieving and / or treating the diseases has an important clinical prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of Peli1 as a target for alleviating and / or treating acute liver injury. Background Art

[0002] Acute liver injury (ALI) refers to a clinical disease in which patients without pre-existing liver disease or with stable liver disease are directly or indirectly exposed to various liver injury risk factors, resulting in a rapid deterioration of liver function within 2 weeks, manifested as symptoms such as fatigue, decreased appetite, jaundice, etc., and accompanied by abnormal laboratory indicators related to liver enzymes, bilirubin, coagulation function, etc.; if ALI is not corrected in time, it can progress to acute liver failure (ALF) in the short term, seriously affecting the prognosis of patients (Liu Shuangqing et al., Expert consensus on the diagnosis and treatment of adult acute liver injury in the emergency department, Chinese Journal of Emergency Medicine, 2024, 44(01): 5-12).

[0003] Since infections, traumas, drugs, poisons, and various physical and chemical factors can all cause varying degrees of liver damage, considering that the liver has a certain self-repair and regeneration ability, in clinical treatment of various types of ALI, in addition to removing the cause, adjuvant treatments mainly include liver protection, anti-inflammatory, and antioxidant therapies, and individualized treatment plans are emphasized according to the specific type of liver injury. However, there are still several limitations in this clinical treatment mode: (1) Limitations in removing the cause: For some irreversible or unknown causes, such as certain drug reactions or toxicant exposures, the cause may not be completely removed; when the cause is complex, it may be difficult to determine the specific cause and take targeted measures, greatly delaying the treatment time and resulting in permanent liver function damage.

[0004] (2) Limitations in supportive treatment: In the treatment of ALI, the main treatments are liver protection, anti-inflammatory, and antioxidant therapies. Although certain clinical symptoms are alleviated, the function of damaged hepatocytes cannot be restored, and for critically ill patients, symptomatic supportive treatment alone cannot effectively prevent the occurrence of acute liver failure, seriously affecting the prognosis of patients.

[0005] (3) Lack of effective treatment technologies for ALI: In the symptomatic supportive treatment of ALI, there are limited drugs available clinically at present, and there is a lack of targeted drugs directly targeting damaged hepatocytes, greatly limiting the treatment effect.

[0006] E3 ubiquitin-protein ligase 1 (Pellino-like E3 ubiquitin protein ligase 1, Peli1), a member of the Pellino family, has biological functions such as negatively regulating necroptosis and protein polyubiquitination. Peli1 has been found to accelerate the occurrence and progression of various malignancies, but its specific role in ALI remains unclear. Summary of the Invention

[0007] Object of the Invention: The object of the present invention is to provide an application of Peli1 as a target for alleviating and / or treating acute liver injury in view of the deficiencies of the existing technology. Starting from the perspective of hepatocytes, the present invention hopes to widely resist liver injuries caused by various different etiologies by regulating the necrosis and apoptosis processes of hepatocytes.

[0008] In the present invention, acetaminophen (APAP) overdose was used to simulate the process of drug-induced acute liver injury. After overexpressing the Peli1 gene in hepatocytes, the results showed that various disease phenotypes of ALI were significantly alleviated, indicating that this gene has good application prospects in alleviating and even treating ALI.

[0009] Technical Solution: The object of the present invention is achieved by the following technical solutions: The present invention provides an application of Peli1 in the preparation of a drug for alleviating and / or treating acute liver injury.

[0010] The acute liver injury is drug-induced acute liver injury.

[0011] The drug alleviates and / or treats acute liver injury by overexpressing Peli1.

[0012] Overexpressing Peli1 can reduce the expression level of inflammatory factors in the ALI cell model.

[0013] Overexpressing Peli1 can increase the proliferation level of hepatocytes.

[0014] The drug includes a substance overexpressing Peli1 and a pharmaceutically acceptable carrier.

[0015] The drug includes small molecule chemical drugs, nucleic acid drugs or antibody drugs.

[0016] Means for preparing a drug with overexpressed Peli1 gene include, but are not limited to, mRNA delivery, Peli1 epigenetic modifiers, gene therapy using Peli1 overexpressing plasmids, etc.

[0017] The present invention also provides an application of the Peli1 gene as a target in screening products for alleviating and / or treating drug-induced acute liver injury.

[0018] The product includes reagents, reagent kits or drugs.

[0019] The present invention also provides a method for screening drugs for alleviating and / or treating drug-induced acute liver injury. Using the Peli1 gene as a drug target, substances capable of overexpressing Peli1 are sought as candidate drugs.

[0020] The NCBI ID of the human-derived Peli1 gene of the present invention is 57162, and the NCBI ID of the mouse-derived Peli1 gene is 67245.

[0021] The present invention uses an (mouse-derived) overexpression plasmid containing the Peli1 coding region sequence, which can specifically and efficiently increase the expression of the Peli1 gene in hepatocytes. The in vitro overexpression Peli1 model constructed in the present invention is based on plasmid transfection technology, and the constructed Peli1 overexpression plasmid is used to transfect mouse-derived hepatocytes (AML12 cell line).

[0022] The in vitro ALI model constructed in the present invention uses acetaminophen (APAP) at a concentration of 25 mmol / L to stimulate the above-mentioned AML12 cell line overexpressed with the Peli1 plasmid (APAP-Peli1 group) and the AML12 cells not treated with plasmid overexpression (APAP group). AML12 cells without APAP stimulation and without plasmid overexpression treatment are used as the blank control group (Con group). The APAP stimulation time is 24 hours, and after 24 hours, samples of the above three groups are collected for detection of relevant indicators.

[0023] The present invention uses RT-PCR reagents to detect the expression levels of the Peli1 gene and inflammation-related genes; uses immunoblotting reagents to evaluate the application of Peli1, inflammation and apoptosis-related proteins; uses a cell proliferation (Edu, 5-Ethynyl-2'-deoxyuridine) staining experiment to evaluate the hepatocyte proliferation level.

[0024] The present invention's research findings show that the Peli1 level in AML12 cells in the APAP group is significantly lower than that in the Con group. After the AML12 cells are overexpressed with the Peli1 plasmid (i.e., the APAP-Peli1 group) and then treated with liver injury drugs for 24 hours, compared with the hepatocytes in the Con group and the APAP group, the hepatocytes with increased Peli1 gene expression show a significant decrease in inflammatory factors and an increase in the hepatocyte proliferation rate. This verifies the effect of Peli1 gene overexpression on the alleviation and even treatment of ALI, and plays an important guiding role in the preparation of drugs for alleviating and / or treating ALI.

[0025] Beneficial effects: In the present invention, it was found that in an in vitro (cell) model of ALI, the expression level of the Peli1 gene was significantly down-regulated. After overexpressing the Peli1 gene, the levels of inflammatory factors in the ALI cell model decreased, the apoptosis situation improved, and the hepatocyte proliferation rate was significantly increased.

[0026] The present invention first discovered that the Peli1 overexpression plasmid can significantly inhibit the apoptosis rate of hepatocytes and promote the proliferation of hepatocytes. On this basis, preparing therapeutic drugs with the Peli1 gene as a target has great clinical significance and application prospects for alleviating and even treating ALI. Brief Description of the Drawings

[0027] Figure 1 It is a result diagram of the differential expression of the Peli1 gene and liver injury-related inflammatory factors in hepatocytes after APAP treatment.

[0028] Figure 2 It is a result diagram of the differential expression of the Peli1 gene and liver injury-related inflammatory factors in hepatocytes after APAP treatment after applying the Peli1 overexpression plasmid.

[0029] Figure 3 It is the hepatocyte proliferation situation after APAP treatment after applying the Peli1 overexpression plasmid. Detailed Embodiments

[0030] The technical solution of the present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the described embodiments.

[0031] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The cell lines and test materials used in the following embodiments are all commercially available products unless otherwise specified.

[0032] AML12 normal mouse hepatocytes, DEME / F12 (containing double antibodies) medium, fetal bovine serum (FBS), OPTI-MEM serum-free medium, and PBS phosphate buffer were purchased from Jiangsu KeyGen Biotech Co., Ltd.; Dexamethasone (50 mg) and insulin-transferrin-selenium medium supplement (100x) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0033] AML12 complete medium: DMEM / F12 (containing double antibodies) medium + 10% fetal bovine serum + 1% of 100x insulin-transferrin-selenium medium supplement + 40 ng / ml dexamethasone; The Edu kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; APAP, in powder form, was purchased from Aladdin Biochemical Co., Ltd.; it was dissolved in PBS to prepare an APAP solution with a concentration of 150 mmol / L, and then diluted to a concentration of 25 mmol / L with AML12 complete medium for stimulating cell experiments; The Peli1 overexpression plasmid was purchased from Anhui General Biotechnology Co., Ltd., and Lipofectamine 3000 and transfection enhancer required for plasmid transfection experiments were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0034] The specific reverse transcription reagent used in real-time fluorescence quantitative PCR (qPCR) experiments, HiScript II Q RTSuperMix for qPCR, and the template tracking dye-based quantitative PCR detection kit (ChamQ SYBR Color qPCR Master Mix) were both purchased from Nanjing Novozymes Biotechnology Co., Ltd.; the Peli1, IL-1, IL-6, TNF-α, 18s primers used for qPCR were purchased from Nanjing General Biotechnology Co., Ltd.

[0035] Example 1 Detection of Peli1 expression in an in vitro (cell) model of ALI induced by different concentrations of APAP using real-time fluorescence quantitative technology (qPCR) AML12 cells (normal murine hepatocytes), culture conditions: cultured with AML12 complete medium (i.e., DMEM / F12 medium containing double antibodies + 10% fetal bovine serum + 1% of 100x insulin-transferrin-selenium medium supplement + 40 ng / mL dexamethasone); the cells were placed in an incubator at 37°C with 5% carbon dioxide, and when the cells proliferated to 75% - 85%, they were routinely digested, passaged, and cryopreserved.

[0036] Preparation of different concentrations of APAP: It was dissolved in PBS to prepare an APAP solution with a concentration of 150 mmol / L, and then diluted to concentrations of 5, 10, 25 mmol / L with AML12 complete medium.

[0037] An in vitro (cell) model of ALI was constructed by stimulating AML12 cells with three different concentrations (5 mmol / L, 10 mmol / L, 25 mmol / L) of APAP for 24 hours. The qPCR method was used to detect the expression of inflammatory factors in cells after stimulation with different concentrations of APAP, and the stimulation concentration with the most obvious increase in inflammatory factors was selected as the optimal concentration of APAP used to simulate the in vitro (cell) model of ALI in this invention.

[0038] RNA was extracted from cells using the Trizol method. 500 μl of Trizol was added to the cell culture dish. After sufficient lysis, total mRNA was extracted. According to the instructions of Novoprotein HiScript II Q RT SuperMix for qPCR, a reverse transcription reaction solution with a volume of 20 μl was prepared, and 1000 ng of the RNA sample was pipetted for reverse transcription.

[0039] The expression levels of the Peli1 gene, inflammatory factors such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the cDNA samples after reverse transcription were detected by qPCR. The expression level of 18S mRNA was used as an internal reference. Table 1 shows the sequence information of the above primers.

[0040] Table 1 Sequence information of qPCR primers

[0041] The 2-ΔΔCT method was used to calculate the relative expression level of the Peli1 gene. The preparation of the 20-μl reaction system used in the specific qPCR experiment can be found in the instructions of Novoprotein ChamQ SYBR Color qPCR Master Mix.

[0042] The qPCR results showed that after stimulation with three different concentrations (5 mmol / L, 10 mmol / L, 25 mmol / L) of APAP, the ALI cell models all showed a significant decrease in Peli1 expression and an increase in the expression of inflammatory factors. After stimulation with 25 mmol / L of APAP for 24 hours, the increase in inflammatory factors in the ALI in vitro model was the most obvious. That is, 25 mmol / L was selected as the optimal concentration for the ALI in vitro model in the present invention. (The results are shown in Figure 1 )

[0043] It can be seen that the Peli1 gene is lowly expressed in the drug-induced ALI cell model.

[0044] Example 2 Overexpression of the Peli1 gene can alleviate hepatocyte damage induced by various drugs The AML12 cell line was divided into three groups: The control group (Con group) of cell lines was only routinely cultured using AML12 complete medium (the cells were placed in an incubator at 37°C with 5% carbon dioxide) without any treatment; the drug stimulation group (APAP group) of cell lines was stimulated with APAP (25 mmol / L) for 24 hours; the Peli1 overexpression group (APAP-Peli1 group) was first transfected with the Peli1 overexpression plasmid and then stimulated with APAP (25 mmol / L) for 24 hours.

[0045] 1. Cell transfection technology The Peli1 overexpression plasmid was constructed with the assistance of Anhui General Biotechnology Co., Ltd. First, solution A containing 5 μg of plasmid and 150 μL of OPTI-MEM serum-free medium was prepared; then, solution B containing 0.5 μL of Lipofectamine 3000 liposome and 5 μL of transfection enhancer in 150 μL of OPTI-MEM serum-free medium was prepared. After preparing solutions A and B, they were left standing at room temperature for 5 min. Subsequently, solution A was added to solution B, gently vibrated and mixed evenly, and after standing for 15 min, it was added to the cell culture dish. After 24 h, it was replaced with AML12 complete medium.

[0046] After the above steps were completed, the cells were stimulated with APAP at a concentration of 25 mmol / L for 24 h and then collected for subsequent experiments.

[0047] 2. Use qPCR technology to clarify the changes in inflammatory factors and hepatocyte apoptosis-related factors in hepatocytes after overexpression of the Peli1 gene The qPCR implementation scheme was the same as in Example 1. The expression level of Peli1 in AML12 cells after drug stimulation decreased compared with the Con group, and the levels of inflammatory factors and cell apoptosis-related factors increased significantly (the results are shown in Figure 2 ). When the Peli1 gene was overexpressed in AML12 cells, the levels of inflammatory factors in hepatocytes decreased significantly compared with the drug stimulation group (the results are shown in Figure 2 ).

[0048] 3. Use Edu staining technology to clarify the proliferation of hepatocytes after overexpression of the Peli1 gene AML12 cells in the logarithmic growth phase were seeded in 48-well plates at a density of 5000 cells per well, with 3 replicates in each group. After the Peli1 overexpression group was pre-transfected with the plasmid for 24 h, the cells in the Peli1 overexpression group and the drug stimulation group were stimulated with the drug for 24 h and then the stimulation was terminated. 200 μL of diluted EdU solution (diluted with AML12 complete medium at a ratio of 1:1000) was added to each well, and the cells were incubated for 2 h. Subsequently, the cells were washed with PBS phosphate buffer, fixed with 4% paraformaldehyde at room temperature for 30 min, washed again, and permeabilized with 1% polyethylene glycol octylphenyl ether (Triton X-100) for 10 min. After washing again, the Apollo staining solution was prepared according to the instructions of the Edu kit, and 70 μL was added to each well. After incubating in the dark at room temperature for 30 min, the cells were washed, and then the diluted 4',6-diamidino-2-phenylindole staining solution (PBS phosphate buffer, dilution ratio 1:5000) was added. After incubating in the dark at room temperature for 30 min, the cells were washed and photographed under a fluorescence microscope. The photographing results are shown in Figure 3 . According to Figure 3The results showed that the proliferative ability of hepatocytes after APAP stimulation was significantly weaker than that of the blank control group, while after overexpressing the Peli1 gene in AML12 cells, the proliferative ability of hepatocytes was significantly enhanced compared with the drug stimulation group.

[0049] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. Use of Peli1 in the preparation of a drug for relieving and / or treating acute liver injury.

2. The use according to claim 1, characterized in that, The acute liver injury is drug-induced acute liver injury.

3. The use according to claim 1, wherein The drug relieves and / or treats acute liver injury by overexpressing Peli1.

4. The use according to claim 1, characterized in that, Overexpression of Peli1 can reduce the expression level of inflammatory factors in the ALI cell model.

5. The use according to claim 1, characterized in that, Overexpression of Peli1 can increase the hepatocyte proliferation level.

6. The use according to claim 1, characterized in that, The drug comprises a substance overexpressing Peli1 and a pharmaceutically acceptable carrier.

7. The use according to claim 1, wherein, The drug comprises a small molecule chemical drug, a nucleic acid drug or an antibody drug.

8. Use of the Peli1 gene as a target in screening products for relieving and / or treating drug-induced acute liver injury.

9. The use according to claim 8, wherein, The products include reagents, kits or drugs.

10. A method for screening drugs for relieving and / or treating drug-induced acute liver injury, characterized in that, Using the Peli1 gene as a drug target, substances capable of overexpressing Peli1 are sought as candidate drugs.

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