Polypeptide for preventing and / or relieving hepatic ischemia-reperfusion injury, and use thereof

Abstract

The present invention provides a use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or an inverse peptide thereof in the preparation of a drug for preventing and / or relieving hepatic ischemia-reperfusion injury, wherein the polypeptide can be modified.

Description

Polypeptide for preventing and / or alleviating liver ischemia-reperfusion injury and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a polypeptide for preventing or alleviating liver ischemia-reperfusion injury and an application thereof. Background Art

[0002] Ischemia-reperfusion injury (IRI) refers to organ damage caused when blood supply is temporarily blocked and then restored. Liver ischemia-reperfusion injury usually occurs during liver transplantation, liver surgery, or in the case of liver trauma, shock or ischemic hepatitis. The pathological process of ischemia-reperfusion injury is complex and the exact mechanism is not yet fully understood. During ischemia-reperfusion, factors such as cellular adenosine triphosphate (ATP) depletion, intracellular calcium overload, increase in oxygen free radicals, enhanced inflammatory response, mitochondrial dysfunction, and endoplasmic reticulum stress response lead to tissue damage (Rampes, S. and Ma, D., Hepatic ischemia-reperfusion injury in liver transplant setting: mechanisms and protective strategies. J Biomed Res. 2019 Jul 28; 33(4): 221-234.). On the contrary, improvement of the above processes can help prevent or alleviate ischemia-reperfusion injury. The measures taken include reducing the time of organ hypoxia during surgery, optimizing blood flow restoration techniques, inhibiting harmful pro-inflammatory responses or promoting beneficial anti-inflammatory responses, alleviating oxidative stress, reducing ATP depletion, and reducing calcium overload.

[0003] Pannexin-1 channel protein (PANX1) is a transmembrane protein involved in the release of adenosine triphosphate (ATP) to the extracellular space. PANX1 can be activated by a variety of factors, such as mechanical stimulation, membrane depolarization, increased intracellular calcium levels, increased extracellular potassium levels, cleavage of the C-terminal region of the PANX1 protein, receptor protein activation, Src family kinase (SFK)-mediated phosphorylation, and proinflammatory cytokine TNFα. Activated PANX1 releases ATP to the extracellular space, participating in inflammation and injury responses (Koval, M., et al. Pannexin 1 as a driver of inflammation and ischemia-reperfusion injury. Purinergic Signal. 2021 Dec; 17(4): 521-531.). The proinflammatory cytokine TNFα activates PANX1, which induces an increase in intracellular calcium levels (Yang Y. et al. Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium. J Immunol. 2020 Jun 1; 204(11): 2995-3007.).

[0004] Studies have shown that genetic deletion of endothelial cell PANX1 can reduce the size of ischemic stroke infarcts (Good, ME, et al. Endothelial cell Pannexin1 modulates severity of ischemic stroke by regulating cerebral inflammation and myogenic tone. JCI Insight. 2018 Mar 22; 3(6): e96272.). The PANX1 inhibitor carbenoxolone (CBX) has a protective effect against renal ischemia-reperfusion injury in mice (Jankowski, J. et al. Epithelial and Endothelial Pannexin1 Channels Mediate AKI. J Am Soc Nephrol. 2018 Jul; 29(7): 1887-1899.). Clinical trial results show that patients taking the PANX1 inhibitor probenecid have significantly reduced vascular disease, especially myocardial infarction and stroke (Kim, SC et al. Cardiovascular Risks of Probenecid Versus Allopurinol in Older Patients With Gout. J Am Coll Cardiol. 2018 Mar 6; 71(9): 994-1004.). PANX1 inhibitors carbenoxolone and probenecid significantly reduce lung dysfunction, edema, cytokine production, and neutrophil infiltration in mice with lung ischemia-reperfusion injury (Sharma, AK et al. Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2018 Aug 1; 315(2): L301-L312.). These data provide strong evidence for preventing or alleviating ischemia-reperfusion injury by intervening in PANX1.

[0005] Although there are many small molecule compounds that can inhibit PANX1, such as probenecid, carbenoxolone, and Brilliant Blue FCF, most PANX1 antagonists also bind to unrelated channel proteins, transport proteins, and non-membrane proteins. 10Panx is a specific peptide antagonist of PANX1, but its inhibitory effect is not as good as that of small molecule antagonists (Navis, KE, et al., Pannexin 1 Channels as a Therapeutic Target: Structure, Inhibition, and Outlook. ACS Chem Neurosci. 2020 Aug 5; 11(15): 2163-2172.). Therefore, the development of highly effective and specific therapeutic drugs targeting PANX1 is highly desirable.

[0006] Summary of the Invention

[0007] Based on the structure of PANX1 protein, two new mimetic peptides were invented (Chinese patent application number 202211496945.8, PCT application number PCT / CN2022 / 134256). Cell experiments have shown that the new mimetic peptides are more effective in inhibiting PANX1 than carbenoxolone and 10 The present inventors used one of the mimetic peptides, QE20, in a mouse experiment to demonstrate that the peptide has the effect of preventing and alleviating liver ischemia-reperfusion injury.

[0008] The present invention provides a use of a polypeptide in the preparation of a medicament for preventing and / or alleviating liver ischemia-reperfusion injury, wherein the polypeptide is a polypeptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 is as follows:

[0009] In a specific embodiment, one or more amino acids in the polypeptide of the present invention may be in the form of the D-enantiomer. More preferably, all amino acids in the polypeptide are in the form of the D-enantiomer.

[0010] In specific embodiments, the polypeptides of the present invention may be further modified. The polypeptides of the present invention may be modified using various well-known techniques. Polypeptide modifications include, but are not limited to, amino acid substitutions, N-terminal modifications, C-terminal modifications, side chain modifications, amino acid modifications, peptide backbone modifications, and conjugation to other polypeptides or proteins. Polypeptide modification techniques can optimize the physicochemical properties of the polypeptide, increase its water solubility, prolong its duration of action in vivo, alter its distribution in vivo, eliminate immunogenicity, and reduce toxic side effects.

[0011] In a specific embodiment, the polypeptide of the present invention is linked to the N-terminus of the polypeptide via stearic acid, and the C-terminus of the polypeptide is modified by amination.

[0012] In a specific embodiment, the polypeptide of the present invention may be a fragment of a polypeptide in the opposite direction (ie, a reverse polypeptide) consisting of the amino acid sequence shown in SEQ ID NO: 1. Beneficial effects

[0013] This application explores new pharmaceutical uses for the peptide QE20. This application demonstrates for the first time that the peptide QE20 is highly effective in preventing and inhibiting ischemia-reperfusion injury. Therefore, it has potential application value in preventing and alleviating ischemia-reperfusion injury in liver, heart, kidney, and lung transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG1 is a graph showing that the polypeptide of the present invention does not cause toxicity to normal human liver cells L-02 at a concentration of 50 μM.

[0015] Figure 2 shows the degree of liver damage at different times after liver ischemia-reperfusion in mice, A: effect on ALT; B: effect on AST.

[0016] FIG3 is a graph showing that the polypeptide of the present invention prevents and alleviates liver ischemia-reperfusion injury in mice by intraperitoneal injection, A: effect on ALT; B: effect on AST.

[0017] FIG4 is a graph showing that the polypeptide of the present invention prevents and alleviates liver ischemia-reperfusion injury in mice by intravenous injection, A: effect on ALT; B: effect on AST. DETAILED DESCRIPTION

[0018] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] Example

[0022] Peptide synthesis

[0023] The peptide was synthesized by a peptide synthesis company (Zhejiang Paipeptide Biological Co., Ltd., No. 291, Fucheng Road, Xiasha Street, Jianggan District, Hangzhou) through the solid phase method. Its amino acid sequence is as follows:

[0024] Stearic acid is conjugated to the N-terminus of the polypeptide SEQ ID NO: 1 (FKHLWLPLNGSESQLSNKQE), and the C-terminus is aminated to become the polypeptide of the present application (designated QE20). The steps are as follows: First, an aminated polypeptide is synthesized by a solid-phase method, and then stearic acid is conjugated to the N-terminus of the polypeptide through a chemical reaction. The stearic acid polypeptide is released from the solid phase through a chemical reaction. The synthesized polypeptide is purified by reverse-phase chromatography to a purity of >95%. The purity of the polypeptide is determined by high-performance liquid chromatography (HPLC), and the molecular weight of the polypeptide is determined by mass spectrometry (MALDI-TOF).

[0025] Preparation of experimental peptides

[0026] First, the peptide QE20 prepared above was dissolved in 2.5% volume of DMSO (D2650, Sigma-Aldrich), and then 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.2-7.4) (absin, abs962, Shanghai, China) buffer was added to a final concentration of 1 mM.

[0027] Experimental steps and results

[0028] (1) Cell hypoxia and reoxygenation experiment

[0029] Normal human hepatocytes L-02 (ATCC, HL-7702, BFN608006124, BLUEFBIO, Shanghai, China) were cultured in DMEM medium (Gibco, C11995500BT) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) (hereinafter referred to as complete medium) at 37°C in a humidified incubator containing 5% CO2 (20% O2, 5% CO2 and 94% N2, hereinafter referred to as normal incubator) until the logarithmic growth phase and the cell density reached more than 80%. After separation, 5×10 cells were plated per well. 5 The cells were seeded into 3.5 cm culture dishes at a density of 10 cells / mL.

[0030] Culture the cells for 24 to 48 hours until the cell growth density reaches more than 80%. Gently aspirate the culture medium and wash the cells once with 1× PBS preheated at 37°C. In the hypoxia-reoxygenation experiment group, add 1 mL of sugar-free and serum-free DMEM culture medium (Gibco, 11966025) to each culture dish, and then add 50 μL of 1× PBS without or with the following test samples to the final concentrations: 150 μM carbenoxolone (Sigma-Aldrich, C4790), 200 μM 10Panx (Tocris Bioscience, Cat#3348), 50 μM QE20. Incubate in a normal incubator for 30 minutes, then transfer to a 37°C hypoxic incubator containing 1.0% O2, 5% CO2, and 94% N2 for 12 hours (this is the hypoxic stage). After the incubation period, remove the cell culture medium from the hypoxic incubator, wash once with 1× PBS incubated at 37°C, and replace with 1 mL of fresh complete culture medium incubated at 37°C. Add 1× PBS, 150 μM carbenoxolone, and 200 μM acetaminophen to the corresponding wells. 10 Panx, 50 μM QE20, transferred to a normal incubator and cultured for 4 hours (this is the reoxygenation stage).

[0031] After the cell culture was completed, the culture medium was discarded, and the cells were washed once with 1× PBS. Then, 0.25% trypsin solution without EDTA (Beyotime, C0205) was added and incubated at 37°C for 2 minutes. Then, complete culture medium was added to neutralize the reaction. After the cells were collected, they were centrifuged (1000 rpm) for 3 minutes, the supernatant was discarded, and the cells were washed twice with 1× PBS pre-cooled at 4°C. Then, the apoptosis detection kit (BD Pharmingen) was used. TM Antibody staining was performed using the FITC Annexin V Apoptosis Detection Kit I, 556547. After staining, the cells were analyzed by flow cytometry (BD LSRFortessa TM ) for testing.

[0032] The results are shown in Figure 1. Compared with the control group (PBS), 150 μM carbenoxolone, 200 μM 10 Panx, 50μM QE20 did not cause obvious toxicity to cells.

[0033] (2) Detection of liver damage at different times after liver ischemia-reperfusion in mice

[0034] Eight-week-old male C57BL / 6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and raised with SPF rat and mouse growth and breeding feed (Beijing Keao Xieli Feed Co., Ltd.) under pathogen-free conditions. They were fasted for 12 hours before surgery.

[0035] Mice in the experimental group were anesthetized with an intraperitoneal injection of 1.25% avertin. The limbs of the mice were then immobilized, the abdomen disinfected with alcohol, and the laparotomy was performed layer by layer along the midline to expose the hilum of the liver. The hilum was ligated with 4-0 non-absorbable surgical sutures (Jiangsu Yangzhou Fuda Medical Instrument Co., Ltd.), creating a model of 70% hepatic blood flow occlusion (the liver discolors grayish white) in the left and middle lobes. The incision was then covered with wet gauze and placed under a warming lamp. After one hour, the ligature was loosened, and the ligature was gently massaged with a cotton swab. Once blood flow to the liver was restored, the abdominal cavity was closed, and the mice were then placed under a warming lamp to recover. The sham group (4 mice) underwent the same surgical procedure, but without ligating the hilum. Blood was collected from the eyeballs 3 hours (4 mice), 6 hours (4 mice), 12 hours (4 mice), and 24 hours (5 mice) after surgery. The blood was placed in a 1.5 mL Eppendorf tube and allowed to stand at room temperature for 30 minutes. The blood was then centrifuged at 5000 rpm at 4°C. The serum was transferred to another 1.5 mL Eppendorf tube and tested for alanine aminotransferase (ALT) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) and aspartate aminotransferase (AST) (Jiangsu Kangshang Biomedical Technology Co., Ltd.) according to the procedures of the kit.

[0036] Mouse liver function (ALT, AST) tests showed that the liver damage was most severe after 1 hour of ischemia and 6 hours of reperfusion in mice, as shown in Figure 2.

[0037] (3) Mouse liver ischemia-reperfusion injury experiment (intraperitoneal injection)

[0038] According to the above experimental results, the mouse liver was ischemic for 1 hour and then reperfused for 6 hours to establish the mouse ischemia-reperfusion model. The experimental groups were divided into NC (Normal Control) group (5 mice), Panx1 - / - group [5 male mice with Panx1 gene deficiency at 8 weeks old, Saiye Biotechnology Co., Ltd. (Suzhou, Jiangsu)] and QE20 group (6 mice). - / - The control group was injected intraperitoneally with an equal volume (200 μl) of sterile 1×PBS, and the QE20 group was injected intraperitoneally with 50 mg / kg QE20. The subsequent experimental steps were the same as above (2).

[0039] Sham operation group (NC Sham group, 4 mice, Panx1 - / - The sham group (5 mice) underwent the same surgical procedure, but without ligating the hepatic hilum.

[0040] Figure 3 shows that Panx1 gene deletion can reduce liver ischemia-reperfusion injury (reduced ALT by 76%, reduced AST by 76%), and after intraperitoneal injection of polypeptide QE20, the degree of liver ischemia-reperfusion injury is even lower than that of Panx1 gene deletion (reduced ALT by 91%, reduced AST by 91%).

[0041] (4) Mouse liver ischemia-reperfusion injury experiment (intravenous injection)

[0042] The average weight of 8-week-old mice was 22 g. The mouse liver ischemia-reperfusion model was constructed as described above (3), i.e., the mouse liver was ischemic for 1 hour and then reperfused for 6 hours. The experimental groups were divided into the NC (Normal Control) group (3 mice) and the QE20 group. One hour before surgery, the NC group was intravenously injected with 100 μl of sterile 1× PBS, and the QE20 group was intravenously injected with the same volume of 34 μg of peptide (3 mice, the final peptide blood concentration was approximately 10 μM) and 169 μg of peptide (4 mice, the final peptide blood concentration was approximately 50 μM). The subsequent experimental steps were the same as described above (2).

[0043] The results in Figure 4 show that the effectiveness of the peptide QE20 in preventing and inhibiting liver ischemia-reperfusion injury in mice depends on the peptide's blood concentration. Low concentrations of the peptide (10 μM) were already effective in preventing and inhibiting liver ischemia-reperfusion injury in mice (reducing ALT by 42% and AST by 34%), while high concentrations of the peptide (50 μM) had an increasing effect (reducing ALT by 83% and AST by 85%).

[0044] The above results indicate that the polypeptide QE20 of the present application has the effect of inhibiting and alleviating liver ischemia-reperfusion injury. Therefore, the polypeptide of the present application has potential application value in preventing and alleviating ischemia-reperfusion injury in the fields of liver transplantation, heart transplantation, kidney transplantation, lung transplantation, etc.

Claims

1. Use of a polypeptide in the preparation of a medicament for preventing and / or alleviating liver ischemia-reperfusion injury, wherein: The polypeptide is a polypeptide fragment consisting of the amino acid sequence shown in SEQ ID NO: 1, or the polypeptide is a reverse peptide fragment of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein One or more amino acids in the polypeptide are in the D-enantiomer form.

3. The use according to claim 1, wherein All amino acids of the polypeptide are in the D-enantiomer form.

4. The use according to claim 1, wherein The polypeptide is modified.

5. The use according to claim 4, wherein The modifications include amino acid substitution, N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and conjugation with other polypeptides or proteins.

6. The use according to claim 1, wherein The polypeptide is linked to the N-terminus of the polypeptide via stearic acid, and the C-terminus of the polypeptide is modified by amination.

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