WO2023143513A1 - 一种多肽化合物及其应用 - Google Patents

一种多肽化合物及其应用 Download PDF

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WO2023143513A1
WO2023143513A1 PCT/CN2023/073556 CN2023073556W WO2023143513A1 WO 2023143513 A1 WO2023143513 A1 WO 2023143513A1 CN 2023073556 W CN2023073556 W CN 2023073556W WO 2023143513 A1 WO2023143513 A1 WO 2023143513A1
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reperfusion injury
ischemia
polypeptide compound
polypeptide
pharmaceutical composition
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WO2023143513A9 (zh
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羽晓瑜
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羽晓瑜
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to the technical field of cell biology, in particular to a polypeptide compound and its application.
  • Pannexin 1 (Pannexin 1, Panx1) is a gap junction protein, a member of the Pannexin glycoprotein family, and an important channel protein for the transport of metabolites and signaling molecules between cells and inside and outside cells. Panx1 is widely involved in cell physiological functions and disease occurrence, and also plays an important regulatory role in inflammatory response.
  • Panx1 plays an important immune defense and protective function in the host immune system during pathogen infection, stress response, body injury or cell death.
  • Panx1 channels are widely present on various cell membranes and participate in inflammatory response processes and immune responses. Opening of Panx1 in innate immunity releases ATP, prompting immune cell migration. Thirumala-Devi Kanneganti and others found that blocking Panx1 can inhibit the migration of neutrophils and macrophages, and high expression of Panx1 can also increase the migration of immune cells. The study also found that thymocytes extracted from Panx1 knockout mice failed to recruit macrophages compared with controls.
  • Panx1 channel is closely related to inflammasome activation, inflammatory cytokine release and inflammatory cell recruitment.
  • High concentrations of extracellular ATP act on PRs, activate inflammasomes, and promote cysteine Caspase-1 catalyzes the precursor of IL-1 ⁇ to the mature form of IL-1 ⁇ , which induces an inflammatory response.
  • the interaction between Panx1 and P2X7R can form a large and highly permeable pore on the cell membrane and activate the inflammasome.
  • Panx1 expression is associated with ischemia-reperfusion inflammatory injury.
  • the Panx1 channel is closed, and mechanical stress, ischemia, and hypoxia can directly act on the Panx1 channel to open it.
  • Reperfusion brings a large amount of [Ca 2+ ] with the blood, which enters the cell in large quantities, induces the opening of ion channels in the cell, releases ATP to the outside of the cell, recruits immune cells to migrate and aggregate, and induces an inflammatory response [6-7].
  • Inflammation is one of the important mechanisms of IRI. Inhibiting the opening of Panx1 channels can reduce the excessive stress response and inflammatory response induced by the recruitment of extracellular ATP to immune cells.
  • Carbenoxolone inhibits the release of ATP by blocking the main channel of Panx1, and is the strongest inhibitor of Panx1 channel reported so far.
  • CBX can effectively reduce cerebral and renal ischemia-reperfusion injury.
  • Panx1 channel inhibitors CBX, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), NPPB, IAA-94, and flufenamic acid.
  • DIDS 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid
  • NPPB 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid
  • IAA-94 IAA-94
  • flufenamic acid flufenamic acid
  • the technical problem to be solved by the present invention is to provide a polypeptide compound and its application in order to overcome the lack of polypeptide compounds that inhibit Panx1 channel in the prior art.
  • the present invention obtains the polypeptide compounds Pan23 and Pan23D that inhibit the opening of the Panx1 channel protein on the cell membrane by means of in vitro synthesis based on the polypeptide targeting the tryptophan gating loop regulatory site at position 74 of the extracellular end of the Panx1 protein.
  • the polypeptide design was compared with CBX (the strongest inhibitor of Panx1 reported), and the results showed that the inhibitory effects of the polypeptide compounds Pan23 and Pan23D of the present invention were both Stronger than CBX, and the inhibitory effect is best when the concentration of Pan23D is 50 ⁇ M.
  • the polypeptide compounds Pan23 and Pan23D of the present invention can reduce cardiac ischemia-reperfusion injury, cerebral ischemia-reperfusion injury, liver ischemia-reperfusion injury and kidney ischemia-reperfusion injury. The results showed that the cell damage was reduced after the intervention of Pan23 and Pan23D, and the serological test of mice indicated that the cell function was better protected.
  • Ruan, Z. published an article in "Nature", reporting that the human wild-type Panx1 was found to be a heptamer assembly mode through cryo-electron microscopy.
  • the main channel in the middle can pass through ATP macromolecules, and the seven small channels on the outside are important. ion channels.
  • Ruan, Z. also analyzed the restriction site of ATP permeability, revealing that tryptophan at the 74th position of the extracellular end forms a gating loop with a diameter less than 10 angstroms, and selectively regulates the mechanism of ATP permeability, suggesting that This site can become a Panx1-specific regulatory site.
  • the present invention designs the 23-amino acid polypeptide Pan23 targeting the gating loop, thereby preventing the opening of the Panx1 hole protein.
  • Pan23D was designed and synthesized by replacing natural amino acids with D-type amino acids.
  • D-type amino acids are unnatural amino acids in the human body, so they are not easily degraded in the body.
  • Panx1 Under physiological conditions, the entrance of the main channel of Panx1 is blocked by its C-terminal structure, and small anions can be exchanged through the small outer channel. These narrow small channels connect to the main pore and are controlled by a long linker between the N-terminal helix and the first transmembrane helix.
  • Panx1 and related various P2 receptors are activated, and Panx1 is open to varying degrees (5%, 25%, 30%, 90% and fully open, see Bao, L., S .Locovei, and G.Dahl, Pannexin membrane channels are mechanosensitive conduits for ATP.
  • Panx1 channel can also be regulated and closed, and its inhibitory factors and extracellular ATP concentration are the most important regulatory factors.
  • Carbenoxolone is currently the strongest inhibitor of Panx1 found, it binds to the channel protein W74, thereby blocking the main channel of Panx1 and inhibiting the ATP release function.
  • Other inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), NPPB, IAA-94, and flufenamic acid weakened in turn.
  • One aspect of the present invention provides a polypeptide compound, which includes the amino acid sequence shown in SEQ ID NO:1.
  • amino acids in the amino acid sequence are in the D form.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the above-mentioned polypeptide compound.
  • Another aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to the present invention.
  • the backbone of the recombinant expression vector is preferably a plasmid, cosmid, phage or viral vector.
  • Another aspect of the present invention provides a transformant comprising the recombinant expression vector of the present invention.
  • the host cell of the transformant is preferably a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cells are preferably yeast cells or mammalian cells.
  • Another aspect of the present invention provides an assembly, which includes cargo molecules and the polypeptide compound of the present invention.
  • the cargo molecule is preferably linked to the polypeptide compound via a non-covalent bond.
  • a recombinant protein which includes the polypeptide compound of the present invention.
  • Another aspect of the present invention provides a pharmaceutical composition, which includes the polypeptide compound of the present invention, the assembly of the present invention or the recombinant protein of the present invention; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition preferably also contains hormone agents, targeted small molecule agents, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, costimulatory molecules that activate One or more of the group consisting of agents, inhibitors of inhibitory molecules, and vaccines.
  • kits which includes the polypeptide compound of the present invention, the assembly of the present invention, the recombinant protein of the present invention or the pharmaceutical composition of the present invention.
  • the kit preferably further comprises (i) administering the polypeptide compound, the assembly, the the device of the recombinant protein or the pharmaceutical composition; and/or (ii) instructions for use.
  • Another aspect of the present invention provides a polypeptide compound as described in the present invention, an assembly as described in the present invention, a recombinant protein as described in the present invention or a pharmaceutical composition as described in the present invention in the preparation of ubiquitin 1 inhibitors in the application.
  • the pan-nexin 1 inhibitor is preferably a drug for preventing and treating ischemia-reperfusion injury.
  • the ischemia-reperfusion injury is preferably drugs for cardiac ischemia-reperfusion injury, liver ischemia-reperfusion injury, kidney ischemia-reperfusion injury and cerebral ischemia-reperfusion injury.
  • Another aspect of the present invention provides a method for preventing and treating diseases related to pan-nexin 1, the method comprising administering a therapeutically effective amount of the polypeptide compound of the present invention, the assembly of the present invention, The recombinant protein of the present invention, the pharmaceutical composition of the present invention.
  • the disease is preferably cardiac ischemia-reperfusion injury, cerebral ischemia-reperfusion injury or liver ischemia-reperfusion injury.
  • the concentration of the polypeptide compound is preferably 1-100 ⁇ M, such as 10 ⁇ M, 50 ⁇ M.
  • pan-nexin 1 of the present invention is Panx1.
  • this polypeptide design was compared with CBX (the strongest inhibitor of Panx1 reported), and the results showed that the inhibitory effects of the polypeptide compounds Pan23 and Pan23D of the present invention were stronger than those of CBX, and when the concentration of Pan23D was 50 ⁇ M, they inhibited Works best.
  • the polypeptide compounds Pan23 and Pan23D of the present invention can reduce cardiac ischemia-reperfusion injury, liver ischemia-reperfusion injury, kidney ischemia-reperfusion injury and cerebral ischemia-reperfusion injury. decreased, cell damage was reduced, and cell function was restored.
  • Figure 1 shows the ATP detection results of the regulation of Panx1 by the polypeptide compound Pan23 or Pan23D.
  • Figure 2A shows a physical picture of a mouse model of cardiac ischemia-reperfusion.
  • Fig. 2B is the detection result of the reduction of infarcted cells by the polypeptide compound Pan23 or Pan23D.
  • Figure 3A shows a physical picture of a mouse model of liver ischemia-reperfusion.
  • Figure 3B is the detection results of alanine transaminase (ALT) and aspartic transaminase (Aspartic transaminase, AST) in mouse serum after Pan23 polypeptide intervention.
  • ALT alanine transaminase
  • AS aspartic transaminase
  • Figure 3C is the detection results of serum ALT and AST in mice after Pan23D polypeptide intervention.
  • Figure 3D is the detection result of necrosis observed by HE staining of mouse liver tissue after polypeptide intervention.
  • Figure 4A shows the physical picture of the mouse model of renal ischemia-reperfusion.
  • Fig. 4B is the detection results of serum uric acid (uric acid, UA) and creatinine (Crea) in mice after Pan23 polypeptide intervention.
  • Figure 4C is the detection results of serum Crea and UA in mice after Pan23D polypeptide intervention.
  • Fig. 4D is the detection result of necrosis observed by HE staining of mouse kidney tissue after polypeptide intervention.
  • Figure 5A shows the physical picture of the cerebral ischemia-reperfusion mouse model.
  • Figure 5B is a slice of the perfused surface of the brain tissue.
  • Figure 5C shows the arrangement and distribution of Nissl bodies stained with Nissl.
  • FIG. 6 shows the HPLC results of Pan23.
  • FIG. 7 shows the HPLC results of Pan23D.
  • Figure 8 shows the MALDI-TOF molecular weight detection results of Pan23 (Main Peak: 2919.67; MW[M+H+]: 2919.67; MW: 2918.67; Theoretical MW: 2919.38).
  • Figure 9 shows the MALDI-TOF molecular weight detection results of Pan23D (Main Peak: 2988.46; MW[M+H+]: 2988.46; MW: 2987.46; Theoretical MW: 2919.38).
  • Embodiment 1 Pan23 synthesis
  • Pan23 was synthesized by Fmoc solid-phase synthesis. Synthetic raw materials and related reagents are as follows:
  • Resin 2-Chlorotrityl Chloride resin with a substitution degree of 1.1 mmol/g.
  • Detection reagents phenol reagent, pyridine reagent, ninhydrin reagent.
  • Resin swelling Put 2-Chlorotrityl Chloride Resin into the reaction tube, add DMF (15ml/g), shake for 60min.
  • Cutting Prepare cutting fluid (10/g): TFA 94.5%; water 2.5%; EDT 2.5%; TIS 1%. Cutting time: 180min.
  • Drying and washing Blow the lysate as dry as possible with nitrogen, precipitate ether, centrifuge to remove the supernatant, wash the precipitate with ether six times, and evaporate to dry at room temperature.
  • Identification Take a small amount of finished peptides respectively, and do molecular weight identification by MS and purity identification by HPLC analysis.
  • HPLC separation and purification conditions HPLC column: C18 (250 ⁇ 4.6mm ID); detection wavelength: 220nm; gradient 40-100% buffer B (0.05% TFA+90% CH 3 CN), 30min;
  • Buffer A 0.05% TFA + 2% CH 3 CN
  • Buffer B 0.05% TFA + 90% CH 3 CN.
  • the results of HPLC separation and purification are shown in Figure 6.
  • the synthesis of Pan23D refers to the synthesis method of Pan23 in Example 1. The difference is that the raw material for the synthesis of Pan23 is L amino acid, and the raw material for the synthesis of Pan23D is D amino acid.
  • HPLC separation and purification conditions HPLC column: C18 (250 ⁇ 4.6mm ID); detection wavelength: 220nm; gradient 40-100% buffer B (0.05% TFA+90% CH 3 CN), 30min;
  • Buffer A 0.05% TFA + 2% CH 3 CN
  • Buffer B 0.05% TFA + 90% CH 3 CN.
  • the results of HPLC separation and purification are shown in Figure 7.
  • Pan23 and Pan23D polypeptide compounds poorly soluble in water, ethanol, isopropanol, n-propanol, soluble in ammonia with a concentration of more than 10%, DMSO with a concentration of more than 50%, and methanol with a concentration of more than 10%.
  • the MALDI-TOF molecular weight detection results of Pan23 are shown in Figure 8.
  • the MALDI-TOF molecular weight detection results of Pan23D are shown in Figure 9.
  • D-I RAW264.7 cell line+POT GLUC (150mMol)+Pan23/Pan23D (1.25 ⁇ M/2.5 ⁇ M/5 ⁇ M/10 ⁇ M/50 ⁇ M/100 ⁇ M).
  • Collect cells Aspirate the cell culture medium, add trypsin to digest for 30s. Then add the original culture medium to the original well and gently blow it several times with a pipette gun, collect the cells and centrifuge at 12000g for 5min at 4°C in the EP tube. Remove the medium, add 150 ⁇ l of lysate for lysis (1-5min), and repeatedly tap with a pipette gun to speed up cell lysis. After lysis, centrifuge at 12,000 g for 5 min at 4°C, and take the supernatant for subsequent experiments. test.
  • Example 4 verified the effectiveness of the polypeptide in vivo
  • mice In order to better verify the effect of the peptide compound, the effectiveness of the peptide was verified in mice.
  • mice were fixed with adhesive tape in a natural supine position.
  • the mouse nasal cavity was always placed in the mask of the gas anesthesia machine to maintain anesthesia.
  • the skin of the mouse incision site was disinfected with alcohol, and the skin was prepared.
  • the mouse heart was frozen in a -80°C refrigerator for more than 30 minutes, and cut into tissue pieces of about 2-3 mm along the short axis of the heart. Put it into the TTC working solution and incubate in a 37°C water bath for 10 minutes. Slices of heart tissue were arranged in a certain order, placed on glass slides, and photographed. The non-infarcted area was stained red, and the infarcted area was colored white, and the infarct area was calculated. The results are shown in FIG. 2B , the percentage of infarcted cells was reduced after the administration of the polypeptide compound Pan23 or Pan23D, and the effect was better than that of CBX.
  • Liver disease is a common disease in our country. All liver operations need to block the blood supply to the liver and complete medical intervention. At the same time, hepatic ischemia-reperfusion injury will inevitably be caused. At present, hormone preperfusion can be used in clinical practice to appropriately reduce liver tissue damage. We hope that through preperfusion of peptides, block Panx1 membrane channel, reduce liver tissue damage caused by ischemia-reperfusion.
  • Ward R500 general-purpose small animal anesthesia machine anesthetized the mice, took a midline incision in the upper abdomen, dissected out the middle lobe of the liver and the left portal vein, and ligated the junction of the left lobe of the liver and the middle lobe of Glisson with a slipknot of 6/0 silk surgical thread, resulting in 70
  • the abdominal incision was sutured in layers and continuously with 4/0 silk suture, and the vascular ligature was left outside the body (as shown in Figure 3A). After 1 hour, the vascular ligature was loosened to restore the blood supply.
  • solvent control group 5% methanol solution
  • mice were randomly selected from each group after anesthesia, blood samples were collected from fundus veins to separate serum, and liver function biochemical indicators ALT and AST were detected; left and middle lobe samples of liver tissue were collected, and right liver tissue samples were collected at the same time. Leaves were used as control specimens of the sham operation group.
  • the experimental results showed that after the Pan23 polypeptide intervention, the serum ALT and AST of the mice decreased significantly (the result is shown in Figure 3B), and the area of liver cell necrosis was reduced, suggesting that the Pan23 polypeptide can reduce liver ischemia-reperfusion injury (the result is shown in Figure 3D).
  • Pan23D implemented the same experimental method, and the experimental results showed that after the intervention of Pan23D polypeptide, serum ALT and AST decreased significantly (results shown in Figure 3C), and the area of liver cell necrosis was reduced, suggesting that Pan23D polypeptide can reduce liver ischemia-reperfusion injury (results are shown in Figure 3C). 3D).
  • Kidney disease is a common disease in our country. All kidney operations need to block the blood supply to the liver to complete medical intervention. At the same time, it will inevitably cause renal ischemia-reperfusion injury. We hope to reduce the renal tissue damage caused by ischemia-reperfusion through preperfusion of polypeptides and block Panx1 membrane channel.
  • FIG. 4A The physical picture of the renal ischemia-reperfusion mouse model is shown in Figure 4A.
  • Ward R500 general-purpose small animal anesthesia machine anesthetized the mice, cut the skin at 1 cm from the left side of the back spine and 2 cm from the lower edge of the ribs, and the yellow fat tissue was visible, then cut the fascia along the middle of the yellow fat and pushed the fat away. Kidneys are visible.
  • solvent control group 5% methanol solution
  • mice were randomly selected from each group after anesthesia, and blood samples were collected from the fundus vein to separate serum, and the biochemical indicators of liver function, uric acid (UA) and creatinine (Crea), were detected; kidney tissue was collected.
  • UA uric acid
  • Crea creatinine
  • HE staining observation showed: ischemia-reperfusion led to tubular necrosis, interstitial blood vessel congestion, and a small amount of scattered renal tubular cells detached.
  • serum UA and Crea in mice decreased significantly Significantly (the result is shown in Figure 4B), the area of tubular necrosis was reduced, and the injury of the renal tubule was alleviated, (the result was shown in Figure 4D).
  • FIG. 5A The physical picture of the renal ischemia-reperfusion mouse model is shown in Figure 5A.
  • Ward R500 general-purpose small animal anesthesia machine anesthetizes SD rats in a supine position on the operating table, and disinfects the operation area. Open the middle of the neck, free fat and connective tissue and stretch to expose the right common carotid artery (CCA) and free peripheral nerves (without damaging the vagus nerve), and continue to separate and expose the "Y"-shaped bifurcation. Place three 6/0 silk wires under the CCA and make sure that the CCA is not twisted. Slip knots at the proximal end and "Y" bifurcation, and keep the opening in the middle. Guide the wire bolts above the CCA and make an incision.
  • CCA right common carotid artery
  • Y free peripheral nerves
  • solvent control group 5% methanol solution
  • the effect of drugs on the neurological function after brain injury was evaluated by scoring the neurological deficit of rats with cerebral ischemia-reperfusion in each group.
  • the neurological deficit score the higher the score, the more severe the injury.
  • the scoring criteria are as follows: 0 points, normal, no symptoms of neurological impairment; 1 point, the animal cannot fully extend the left forelimb when the tail is raised; 2 points, the animal turns to the left in a circle, and there is a rear-end phenomenon; 3 points, the animal falls to the left or Rolling; 4 points, unable to walk spontaneously, and there is a disturbance of consciousness. Animals with scores of 0 and 4 were eliminated, and mice with scores of 1 to 3 were included in the statistical standard.
  • the neurological function score of the rats in the sham operation group was 0, and there was no symptom of neurological deficit; compared with the sham operation group, the neurological function score of the model group was significantly higher (P ⁇ 0.05), and the rats could not fully extend the contralateral forepaw, Different degrees of neurological deficit symptoms such as walking in circles on the contralateral side, falling to the contralateral side, etc.; compared with the model group, the neurological function scores of the Pan23 and Pan23D groups were lower (P ⁇ 0.05), and the neurological deficits were improved. Statistical significance.
  • Vultaggio-Poma V., et al. Extracellular ATP: AFeasible Target for Cancer Therapy. Cells. 2020, 9(11): 2496.

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Abstract

本发明公开了一种多肽化合物及其应用。一种多肽化合物，其包括如SEQ ID NO:1所示的氨基酸序列。在细胞实验中，本多肽设计与CBX进行了对比，结果显示本发明的多肽化合物Pan23与Pan23D的抑制效果都强于CBX。本发明的多肽化合物Pan23与Pan23D能减少心脏缺血再灌注损伤、肝脏缺血再灌注损伤、肾脏缺血再灌注损伤和脑缺血再灌注损伤，结果显示Pan23与Pan23D干预后细胞损伤减少，细胞功能得到恢复。

Description

一种多肽化合物及其应用
本申请要求申请日为2022/1/26的中国专利申请2022100949143的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及细胞生物学技术领域，具体涉及一种多肽化合物及其应用。
背景技术
泛连接蛋白1(Pannexin 1,Panx1)是缝隙连接蛋白，Pannexin糖蛋白家族成员之一，是细胞间和细胞内外代谢产物及信号分子转运的重要通道蛋白。Panx1广泛参与细胞生理功能及疾病发生，在炎症反应中也发挥着重要的调节作用。
具体来说，根据现有文献，在病原体的感染、应激反应、机体损伤或细胞死亡过程中，Panx1在宿主免疫系统都发挥着重要的免疫防御及保护功能。
此外，根据现有文献，Panx1通道广泛存在各种细胞膜上，参与炎症反应过程和免疫反应。先天性免疫中Panx1的开放释放ATP,促使免疫细胞迁移。Thirumala-Devi Kanneganti等研究发现，阻断Panx1可以抑制中性粒细胞和巨噬细胞的迁移，高表达Panx1也可以增加免疫细胞的迁移。该研究还发现与对照组相比，Panx1基因敲除小鼠中提取出胸腺细胞无法募集巨噬细胞。
越来越多的证据表明，Panx1通道与炎症小体激活，炎性细胞因子释放和炎性细胞募集关系密切。半胱氨酸天冬氨酸蛋白水解酶1型(cysteinyl aspartate specific proteinase-1,Caspase-1)组成的蛋白复合体，简称NLRP3炎性小体，其中效应蛋白Caspase-1的激活可以进一步促进细胞因子IL-1β和IL-18的释放。高浓度的细胞外ATP作用于PRs，激活炎性小体，促使半胱 氨酸天冬氨酸蛋白水解酶1型(Caspase-1)催化IL-1β前体成有成熟形式的IL-1β，诱发炎症反应。Panx1与P2X7R相互作用可以在细胞膜上形成一个大的高通透性孔道，激活炎性小体。
此外，还有现有文献报道了Panx1表达与缺血再灌注炎症损伤相关。正常生理条件下，Panx1通道呈关闭状态，机械应力、缺血、缺氧可直接作用于Panx1通道使其开放。再灌注随血运来大量[Ca2+]，大量进入细胞内，诱导细胞离子通道开放，释放ATP到细胞外，招募免疫细胞迁移聚集并诱导炎症反应[6-7]。炎症反应是IRI发生的重要机制之一。抑制Panx1通道开放，可以减少细胞外ATP对免疫细胞的招募所诱发的过度应激反应和炎症反应。生胃酮(Carbenoxolone，CBX)通过封闭Panx1主通道，抑制ATP释放，是目前报道的最强的Panx1通道抑制剂。研究发现CBX可以有效减轻脑和肾缺血再灌注损伤。Panx1通道抑制剂：CBX、4,4′-二异硫氰基芪-2,2′-二磺酸(DIDS)、NPPB、IAA-94、氟灭酸的相关研究提示：抑制Panx1通道开放可作为治疗感染、IRI、肿瘤等多种疾病的靶点。
尽管多肽化合物的研究取得了一定的突破和发展，但要涉及获得一个纯度高、分布单一的多肽化合物实际仍有较多的问题需要解决，而如何使得该多肽化合物能够成功用于治疗人类疾病，其稳定性、特异性、靶向性等仍需要攻克。
发明内容
本发明要解决的技术问题是为了克服现有技术中缺乏抑制Panx1通道的多肽化合物的缺陷，提供一种多肽化合物及其应用。本发明通过基于靶向Panx1蛋白细胞外端74位的色氨酸门控环调控位点的多肽，采用体外合成的方式，获得抑制细胞膜上Panx1通道蛋白开放的多肽化合物Pan23以及Pan23D。在细胞实验中，本多肽设计与CBX(已报道Panx1最强抑制剂)进行了对比，结果显示本发明的多肽化合物Pan23与Pan23D的抑制效果都 强于CBX，且当Pan23D的浓度为50μM时抑制效果最佳。本发明的多肽化合物Pan23与Pan23D能减少心脏缺血再灌注损伤、脑缺血再灌注损伤、肝脏缺血再灌注损伤和肾缺血再灌注损伤。结果显示Pan23与Pan23D干预后细胞损伤降低，且小鼠血清学检测提示细胞功能得到较好保护。
2020年Ruan,Z.在《自然》杂志发表文章，报道通过低温冷冻电镜发现人源野生型Panx1是七聚体的组装模式，中间的主通道可以通过ATP大分子，外侧七个小通道是重要的离子通道。Ruan,Z.同时解析了ATP通透性的限制性位点，揭示了细胞外端74位的色氨酸形成一个直径小于10埃的门控环，选择性调控ATP通透性的机制，提示该位点可以成为Panx1特异性调控位点。
本发明设计了靶向该门控环的23个氨基酸的多肽Pan23，从而可以阻止Panx1孔道蛋白开放。并在此基础上将天然氨基酸替换为D型氨基酸设计合成Pan23D，D型氨基酸是人体内非自然氨基酸，因而在体内不易被降解。
生理条件下，Panx1主通道内入口被其C末端结构封闭，小的阴离子可通过外侧小通道进行离子交换。这些狭窄小通道与主孔相连，并受N端螺旋结构和第一跨膜螺旋之间的长连接体控制。在炎症、再生和机械信号传导中，Panx1及相关的各种P2受体被激活，Panx1呈不同程度开放(5％、25％、30％、90％和完全开放，参见Bao,L.,S.Locovei,and G.Dahl,Pannexin membrane channels are mechanosensitive conduits for ATP.FEBS Lett,2004.572(1-3):p.65-8)，参与机体生理和病理过程。大量研究证明Panx1通道也可以被调控关闭，其抑制因子和细胞外ATP浓度是最主要的调控因素。
生胃酮(Carbenoxolone，CBX)是当前发现Panx1最强的抑制剂，它通过与通道蛋白W74结合，从而封闭Panx1主通道，抑制ATP释放功能。其他抑制剂4,4’-二异硫氰基芪-2,2’-二磺酸(DIDS)、NPPB、IAA-94、氟灭酸抑制作用依次减弱。
本发明是通过下述技术方案来解决上述技术问题：
本发明一方面提供了一种多肽化合物，其包括如SEQ ID NO:1所示的氨基酸序列。
在某一较佳实施方案中，所述氨基酸序列中的氨基酸为D型。
本发明另一方面提供了一种分离的核酸，其编码如上述的多肽化合物。
本发明另一方面提供一种重组表达载体，所述重组表达载体包含如本发明所述的分离的核酸。
所述重组表达载体的骨架优选为质粒、粘粒、噬菌体或病毒载体。
本发明另一方面提供一种转化体，所述转化体包含本发明所述的重组表达载体。
所述转化体的宿主细胞优选地为真核细胞或原核细胞。
所述真核细胞优选为酵母细胞或哺乳动物细胞。
本发明另一方面提供一种组装体，所述组装体包括货物分子以及本发明所述的多肽化合物。
所述货物分子与所述多肽化合物较佳地通过非共价键连接。
一种重组蛋白，所述重组蛋白包括本发明所述的多肽化合物。
本发明另一方面提供一种药物组合物，所述药物组合物包括本发明所述的多肽化合物、本发明所述的组装体或本发明所述的重组蛋白；以及药学上可接受的载体。
所述药物组合物较佳地还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明另一方面提供一种试剂盒，其包括本发明所述的多肽化合物、本发明所述的组装体、本发明所述的重组蛋白或本发明所述的药物组合物。
所述试剂盒优选地还包括(i)施用所述的多肽化合物、所述的组装体、所 述的重组蛋白或所述的药物组合物的装置；和/或(ii)使用说明。
本发明另一方面提供一种如本发明所述的多肽化合物、本发明所述的组装体、本发明所述的重组蛋白或者如本发明所述的药物组合物在制备泛连接蛋白1抑制剂中的应用。
所述泛连接蛋白1抑制剂较佳地为防治缺血再灌注损伤的药物。
所述缺血再灌注损伤较佳地为心脏缺血再灌注损伤、肝脏缺血再灌注损伤、肾脏缺血再灌注损伤和脑缺血再灌注损伤的药物。
本发明另一方面提供一种防治与泛连接蛋白1相关的疾病的方法，所述方法包括向有需要的患者施用治疗有效量的本发明所述的多肽化合物、本发明所述的组装体、本发明所述的重组蛋白、本发明所述的药物组合物。
所述疾病较佳地为心脏缺血再灌注损伤、脑缺血再灌注损伤或肝脏缺血再灌注损伤。
本发明中，所述多肽化合物浓度优选1-100μM，例如10μM、50μM。
本发明所述的泛连接蛋白1即为Panx1。
本发明的积极进步效果在于：
在细胞实验中，本多肽设计与CBX(已报道Panx1最强抑制剂)进行了对比，结果显示本发明的多肽化合物Pan23与Pan23D的抑制效果都强于CBX，且当Pan23D的浓度为50μM时抑制效果最佳。本发明的多肽化合物Pan23与Pan23D能减少心脏缺血再灌注损伤、肝脏缺血再灌注损伤、肾脏缺血再灌注损伤和脑缺血再灌注损伤，结果显示Pan23与Pan23D干预后梗死细胞百分比有所降低，细胞损伤减少，细胞功能得到恢复。
附图说明
图1显示了多肽化合物Pan23或Pan23D调控Panx1的ATP检测结果。
图2A显示了心脏缺血再灌注小鼠模型实物图。
图2B为多肽化合物Pan23或Pan23D减少梗死细胞的检测结果。
图3A显示肝脏缺血再灌注小鼠模型实物图。
图3B为Pan23多肽干预后小鼠血清丙氨酸氨基转移酶(alanine transaminase,ALT)和天门冬氨酸氨基转移酶(Aspartic transaminase，AST)的检测结果。
图3C为Pan23D多肽干预后小鼠血清ALT、AST的检测结果。
图3D为多肽干预后小鼠肝脏组织HE染色观测坏死的检测结果。
图4A显示了肾脏缺血再灌注小鼠模型实物图。
图4B为Pan23多肽干预后小鼠血清尿酸(uric acid，UA)和肌酐(Crea)的检测结果。
图4C为Pan23D多肽干预后小鼠血清Crea、UA的检测结果。
图4D为多肽干预后小鼠肾脏组织HE染色观测坏死的检测结果。
图5A显示了脑缺血再灌注小鼠模型实物图。
图5B为脑组织灌注面切片。
图5C为尼氏染色尼氏体排列分布。
图6显示了Pan23的HPLC结果。
图7显示了Pan23D的HPLC结果。
图8显示了Pan23的MALDI-TOF分子量检测结果(Main Peak:2919.67；MW[M+H+]:2919.67；MW:2918.67；Theoretical MW:2919.38)。
图9显示了Pan23D的MALDI-TOF分子量检测结果(Main Peak:2988.46；MW[M+H+]:2988.46；MW:2987.46；Theoretical MW:2919.38)。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。
实施例1 Pan23合成
采用Fmoc固相合成法合成Pan23。合成原料及相关试剂如下：
1：保护氨基酸原料。
2：缩合试剂：HBTU、DIEA。
3：溶剂：DMF、DCM、甲醇、乙腈。
4：树脂：取代度为1.1mmol/g的2-Chlorotrityl Chloride resin。
5：脱保护试剂：哌啶。
6：检测试剂：苯酚试剂、吡啶试剂、茚三酮试剂。
7：切割试剂：TFA、TIS、EDT、无水乙醚。
仪器设备：十二通道半自动多肽合成仪、高效液相色谱仪、冻干机、离心机。
合成过程：
1.树脂溶涨：将2-Chlorotrityl Chloride Resin放入反应管中，加DMF(15ml/g)，振荡60min。
2.接第一个氨基酸：通过沙芯抽滤掉溶剂，加入3倍摩尔过量的Fmoc-D-Asp(tbu)-OH氨基酸(C端第一个氨基酸)，再加入10倍摩尔过量的DIEA，最后加入DMF溶解，振荡30min。甲醇封头，30min。
3.脱保护：去掉DMF，加20％哌啶DMF溶液(15ml/g)，5min，去掉再加20％哌啶DMF溶液(15ml/g)，15min。
4.检测：抽掉哌啶溶液，取十几粒树脂，用乙醇洗三次，加入茚三酮，KCN，苯酚溶液各一滴，105℃-110℃加热5min，变深蓝色为阳性反应。
5.洗：DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次。
6.缩合：加入3倍摩尔过量Fmoc保护氨基酸，3倍摩尔过量HBTU，再加入10倍摩尔过量的DIEA，最后加入DMF溶解，振荡45min。
7.检测：取十几粒树脂，用乙醇洗三次，加入茚三酮，吡啶，苯酚溶液各一滴，105℃-110℃加热5min，无色为阴性反应。
8.洗：DMF(10ml/g)一次，甲醇(10ml/g)两次，DMF(10ml/g)两次。
9.重复三至八步操作，从右到左依次连接如SEQ ID NO:1序列中的氨 基酸。
10.按照下列方法洗树脂，抽干：DMF(10ml/g)两次，DCM(10ml/g)三次，甲醇(10ml/g)四次，抽干10min。
11.切割：配制切割液(10/g)：TFA 94.5％；水2.5％；EDT 2.5％；TIS1％。切割时间：180min。
12.吹干洗涤：将裂解液用氮气尽量吹干，乙醚析出，离心去除上清，沉淀用乙醚洗六次，然后常温挥干。
13.纯化制备：
①取少许粗品，H2O/ACN溶解。
②取少量样品在HPLC分析仪器上进行分析判断目标峰对应出峰时间。
③利用C18反相色谱制备系统：波长：220nm；流速：15ml/min；注射体积：20mL；柱温：25℃；Buffer A：0.1％TFA溶于水；Buffer B：0.1％TFA溶于乙腈；收集目标峰溶液。
④用1.5ml离心管取少许目标峰溶液进行质谱确认及纯度检测。
14.将合格的目标峰溶液进行冻干。
15.利用C18反相色谱制备系统转盐：波长：220nm；流速：15ml/min；进液量：20mL；柱温：25℃；Buffer A：0.05％HCl溶于水；Buffer B：0.05％HCl溶于乙腈；收集目标峰溶液。
16.将目标峰溶液进行冻干，即得成品。
17.鉴定：分别取少量的成品多肽，做MS的分子量鉴定和HPLC分析的纯度鉴定。
18.将白色粉末状的多肽密封包装，-20℃保存。
HPLC分离纯化条件：HPLC柱：C18(250×4.6mm I.D.)；检测波长为：220nm；梯度40-100％缓冲液B(0.05％TFA+90％CH3CN)、30min；
缓冲液A：0.05％TFA+2％CH3CN；缓冲液B：0.05％TFA+90％CH3CN。HPLC分离纯化结果见图6。
实施例2 Pan23D合成
Pan23D的合成参考实施例1中关于Pan23的合成方法，区别在于Pan23合成原料为L氨基酸，Pan23D的合成原料为D氨基酸。
HPLC分离纯化条件：HPLC柱：C18(250×4.6mm I.D.)；检测波长为：220nm；梯度40-100％缓冲液B(0.05％TFA+90％CH3CN)、30min；
缓冲液A：0.05％TFA+2％CH3CN；缓冲液B：0.05％TFA+90％CH3CN。HPLC分离纯化结果见图7。
Pan23以及Pan23D多肽化合物的理化性质：难溶于水，乙醇、异丙醇、正丙醇，可溶于10％浓度以上氨水，50％以上DMSO，10％浓度以上甲醇。
Pan23的MALDI-TOF分子量检测结果见图8。Pan23D的MALDI-TOF分子量检测结果见图9。
实施例3 Pan23以及Pan23D抑制Panx1的效果验证
1.小鼠巨噬细胞细胞株RAW264.7，每孔2×106个细胞铺六孔板设三复孔，过夜培养。实验分十组进行给药处理：
A:RAW264.7细胞株；
B：RAW264.7细胞株+POT GLUC(150mMol)；
C：RAW264.7细胞株+POT GLUC(150mMol)+CBX(100μM)；
D-I:RAW264.7细胞株+POT GLUC(150mMol)+Pan23/Pan23D(1.25μM/2.5μM/5μM/10μM/50μM/100μM)。
继续培养16h。
2.收集细胞：吸出细胞培养基，加入胰酶消化30s。然后将原培养基加入原孔用移液枪反复轻柔吹打几次，收集细胞至EP管内4℃、12000g离心5min。去除培养基，加入150μl裂解液进行裂解(1-5min)可用移液枪反复吹打加快细胞裂解。裂解后，4℃、12000g离心5min，取上清用于后续的实 验。
3.制备标曲：使用ATP检测裂解液把ATP标准溶液稀释成不同的浓度梯度(0.1、0.3、1、3、10)μM，配制ATP检测工作液：每孔100μl共15个孔(7+6+3)共计1.5ml，按照1:9的比例用ATP检测试剂稀释液稀释ATP检测试剂，冰上暂存。
4.测定ATP的浓度：取新的96孔板，做好标记，设置好间隔孔。每孔加入100μl ATP检测工作液，室温放置3-5min消除本底ATP。按照顺序依次快速加入20μl标准品和20μl样品，用枪混匀避免气泡的产生。随后立即用多功能酶标仪进行检测。Pan23共孵育16小时，ATP检测显示本发明的多肽Pan23比CBX具有更好的抑制效果(结果见图1)。ATP检测显示Pan23D有良好的抑制效果(结果见图1)。同时由于Pan23D由非天然氨基酸组成，不被体内酶降解，具有更好的稳定性。
实施例4体内验证了多肽的有效性
为了更好地验证多肽化合物的效果，在小鼠体内验证了多肽的有效性。
1.心脏缺血再灌注小鼠模型
心血管疾病发病不断攀升，所有心脏手术和心脏介入操作，都需要阻断心脏血流供应，完成医疗干预。心脏缺血再灌注损伤成为医疗常见损伤，目前尚无特效药和有效干预手段。我们希望通过多肽的干预治疗，block细胞Panx1膜通道，减少缺血再灌注引起的心肌组织损伤。
开启小动物气体麻醉机(麻醉气体浓度2％，气体流量1L/min)，将小鼠放入气体麻醉机诱导盒中，待小鼠全身放松昏迷后，将小鼠取出，固定在小鼠手术台上面，用胶布固定小鼠呈自然仰卧位。将小鼠鼻腔始终置于气体麻醉机的面罩之中，维持麻醉状态。酒精消毒小鼠切口部位皮肤，备皮。用持针器夹持3/0手术线和5/0手术线(带针)备用。在小鼠心脏部位第3、4肋间隙位置沿着腋窝与胸骨下端连线做1.5cm的切口。钝性分离胸大肌与肋骨 外肌。于第3、4肋间穿破肋间隙，左手迅速将心脏挤出，剥开心包。在光源下，于左心耳右下缘可见左冠状动脉。以左心耳下缘水平线为标志，在线下2mm处以5-0线结扎左冠脉前降支，进针深度1mm左右，避免刺破心脏。结扎完毕，快速将心脏推入胸腔。挤出胸内气体，用3-0线打结关胸。此时将心电图导联固定在小鼠四肢根部肌肉，测量结扎后心电图，确认小鼠心脏缺血-再灌注损伤模型建立成功。摘除麻醉面罩。整个过程在1分钟之内完成(如图2A所示)。面罩摘除后，小鼠会在3～5分钟内苏醒。结扎35min时，经尾静脉给药。5min后松开结扎线，颈动脉取血1mL，小鼠心脏-80℃冰箱中冷冻30min以上，沿心脏短轴面将其切成2-3mm左右的组织片。放入TTC工作液中，37℃水浴锅孵育10min。将心脏组织切片，按照一定顺序排列，放在载玻片上，拍照。非梗死区染成红色，梗死区呈白色，计算心脏梗死面积。结果如图2B所示，施用多肽化合物Pan23或Pan23D后梗死细胞的百分比有所减少，且效果优于施用CBX时。
2.肝脏缺血再灌注小鼠模型
肝脏疾病是我国常见疾病，所有肝脏手术，都需要阻断肝脏血流供应，完成医疗干预。同时不可避免引起肝脏缺血再灌注损伤，目前临床可通过激素预灌注，适当减少肝脏组织损伤。我们希望通过多肽的预灌注，block Panx1膜通道，减少缺血再灌注引起的肝脏组织损伤。
沃德R500通用型小动物麻醉机麻醉小鼠，取上腹部正中切口，解剖出肝脏中叶、左的门静脉，应用6/0丝线外科手术线活结结扎肝脏左叶和中叶Glisson交汇处，造成肝脏70％缺血，用4/0丝线分层连续缝合腹部切口，结扎血管线留置体外(如图3A所示)，1h后松开血管结扎线，恢复血流供应。
手术前30分钟尾静脉注射药物干预：
(1)溶剂对照组：5％甲醇溶液；
(2)(CBX+1/R)组CBX：100μM、200μL；
(3)(Pan23+1/R)组：Pan23 50μM、200μL。
再灌注3后，每组随机各取8只小鼠麻醉后，眼底静脉收集血液标本分离血清，检测肝功能生化指标ALT和AST；留取肝组织左叶和中叶标本，同时留取肝脏组织右叶作为假手术组对照标本。实验结果显示Pan23多肽干预后小鼠血清ALT、AST下降明显(结果如图3B)，肝脏细胞坏死面积减少，提示Pan23多肽可以减少肝脏缺血再灌注肝脏损伤(结果如图3D)。
Pan23D实施相同实验方法，实验结果显示Pan23D多肽干预后小鼠血清ALT、AST下降明显(结果见图3C)，肝脏细胞坏死面积减少，提示Pan23D多肽可以减少肝脏缺血再灌注肝脏损伤(结果见图3D)。
3.肾脏缺血再灌注小鼠模型
肾脏疾病是我国常见疾病，所有肾脏脏手术，都需要阻断肝脏血流供应，完成医疗干预。同时不可避免引起肾脏缺血再灌注损伤，我们希望通过多肽的预灌注，block Panx1膜通道，减少缺血再灌注引起的肾脏组织损伤。
肾脏缺血再灌注小鼠模型实物图如图4A所示。沃德R500通用型小动物麻醉机麻醉小鼠，在背部脊椎左侧旁1cm、肋骨下缘2cm处剪开皮肤，可见到黄色脂肪组织，再沿黄色脂肪中间剪开筋膜并拨开脂肪，可见到肾脏。调整体位为右侧卧位，挤出肾脏并剥离肾脏两极组织(勿损伤肾上腺)，分离肾蒂周围及肾脏下级与输尿管之间结缔组织，完全游离肾脏；同样操作完全游离右侧肾脏，血管夹夹闭两侧肾蒂45min后松开，手术前30分钟尾静脉注射药物干预：
(1)溶剂对照组：5％甲醇溶液；
(2)(CBX+1/R)组CBX：100μM、200μL；
(3)(Pan23+1/R)组：Pan23 50μM、200μL。
再灌注24后，每组随机各取8只小鼠麻醉后，眼底静脉收集血液标本分离血清，检测肝功能生化指标尿酸(uric acid，UA)和肌酐(Crea)；留取肾脏组织。HE染色观察显示：缺血再灌注导致肾小管带状坏死，间质血管充血，散在少量肾小管细胞脱落。Pan23多肽干预后小鼠血清UA、Crea下降明 显(结果如图4B)，肾小管带状坏死区域减少，肾小管损伤减轻，(结果如图4D所示)。
Pan23D实施相同实验方法，实验结果显示Pan23D多肽干预后小鼠血清UA、Crea下降明显(结果见图4C)，肾小管带状坏死面积减少，提示Pan23D多肽可以减少肝脏缺血再灌注肝脏损伤(结果见图4D)。
4.脑缺血再灌注小鼠模型
脑部手术及脑部血管梗死后再通，都是缺血再灌注过程，因而会发生缺血再灌注损伤，我们希望通过多肽的预灌注，阻断Panx1膜通道，减少缺血再灌注引起的脑组织损伤。
肾脏缺血再灌注小鼠模型实物图如图5A所示。沃德R500通用型小动物麻醉机麻醉SD大鼠仰卧位置于操作台上，消毒手术区域。颈部正中开口，游离脂肪和结缔组织并牵拉暴露右颈总动脉(Common carotid artery，CCA)和游离周围神经(勿损伤迷走神经)，继续分离暴露“Y”型分叉。CCA下方置三条6/0丝线并确保CCA没有扭曲，近心端和“Y”分叉处活结、中间位置活结并保持开口，线栓引导至CCA上方并做切口，快速插入切口(如插入受阻，扩张切口重新插入)，解开“Y”分叉处活结并顺势插入ICA，线栓硅胶与丝线衔接处打结，限制血液流出，但保证线栓自由移动。插入到标记长度时，轻微推入线栓，遇到轻微阻力即停止并固定。梗阻120min后，解开固定线，线栓出“Y”分叉处时并在分叉处打活结，10-0缝合线缝合CCA处切口，去除所有活结，恢复血流供应。手术前30分钟鞘内注射20Ul药物干预：
(1)溶剂对照组：5％甲醇溶液；
(2)(Pan23+1/R)组：Pan23 50μM、20μL。
Pan23D实施相同实验方法，神经功能学打分降低，提示神经损伤减轻(结果见表1)。
表1 Pan23多肽干预后大鼠神经功能评分
本实验通过对各组脑缺血再灌注大鼠进行神经功能缺损评分评估药物对脑损伤后神经功能的影响。在神经功能缺损评分中，得分越高，损伤越重。参考Longa[1]5级4分法，分别评分观察术后各组动物神经功能。评分标准如下：0分，正常，无神经功能损伤症状；1分，提尾时动物不能完全伸展左前肢；2分，动物向左侧转圈，出现追尾现象；3分，动物向左侧倾倒或打滚；4分，不能自发行走，存在意识障碍。剔除0分和4分动物，1～3分小鼠计入统计标准。
假手术组大鼠神经功能评分为0，无神经功能缺损症状；与假手术组相比，模型组神经功能评分明显升高(P<0.05)，大鼠出现对侧前爪不能伸展完全、向对侧转圈行走、向对侧行走倾倒等不同程度的神经功能缺损症状；与模型组相比，Pan23及Pan23D组神经功能评分有所降低(P<0.05)，神经功能缺损有所改善，差异有统计学意义。
大鼠做完神经功能评分后，用2％戊巴比妥钠腹腔注射麻醉大鼠，断头取脑。脑组织冠状面切片(参见图5B)，梯度酒精脱水，1％甲苯胺蓝溶液染色，置于60℃恒温箱反应40min，蒸馏水冲洗3次(每次3min)，95％酒精分色，梯度酒精脱水，二甲苯透明，封片，倒置显微镜观察、拍照。正常神经元中尼氏体数量丰富；当神经元受损时，尼氏体可减少、解体或消失。尼氏染色显示(图5C)，IRI组神经细胞出现变形，细胞排列混乱，尼氏体数量明显减少(P<0.05)；与模型组相比，多肽治疗组和CBX组细胞状态得到改善，尼氏体数量增多(P<0.05)。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理 解，这仅是举例说明，本发明的保护范围是由所附权利要求书限定的。
本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更和修改均落入本发明的保护范围。
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Claims (11)

  1. 一种多肽化合物，其特征在于，所述多肽化合物包括如SEQ ID NO:1所示的氨基酸序列。
  2. 如权利要求1所述的多肽化合物，其特征在于，所述氨基酸序列中的氨基酸为D型。
  3. 一种分离的核酸，其编码如权利要求1或2所述的多肽化合物。
  4. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求3所述的分离的核酸；优选地，所述重组表达载体的骨架为质粒、粘粒、噬菌体或病毒载体。
  5. 一种转化体，其特征在于，所述转化体包含如权利要求4所述的重组表达载体；优选地，所述转化体的宿主细胞为真核细胞或原核细胞；更优选地，所述真核细胞为酵母细胞或哺乳动物细胞。
  6. 一种组装体，其特征在于，所述组装体包括货物分子以及如权利要求1或2所述的多肽化合物；
    较佳地，所述货物分子与所述多肽化合物通过非共价键连接。
  7. 一种重组蛋白，其特征在于，所述重组蛋白包括如权利要求1或2所述的多肽化合物。
  8. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1或2所述的多肽化合物、如权利要求6所述的组装体或如权利要求7所述的重组蛋白；以及药学上可接受的载体；
    较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  9. 一种试剂盒，其包括如权利要求1或2所述的多肽化合物、如权利要 求6所述的组装体、如权利要求7所述的重组蛋白或如权利要求8所述的药物组合物；
    优选地，所述试剂盒还包括(i)施用所述的多肽化合物、所述的组装体、所述的重组蛋白或所述的药物组合物的装置；和/或(ii)使用说明。
  10. 一种如权利要求1或2所述的多肽化合物、如权利要求6所述的组装体、如权利要求7所述的重组蛋白或者如权利要求8所述的药物组合物在制备泛连接蛋白1抑制剂中的应用；
    较佳地，所述泛连接蛋白1抑制剂为防治缺血再灌注损伤的药物；
    更佳地，所述缺血再灌注损伤为心脏缺血再灌注损伤、脑缺血再灌注损伤、肾脏缺血再灌注损伤和/或肝脏缺血再灌注损伤的药物。
  11. 一种防治与泛连接蛋白1相关的疾病的方法，所述方法包括向有需要的患者施用治疗有效量的如权利要求1或2所述的多肽化合物、权利要求6所述的组装体、如权利要求7所述的重组蛋白、如权利要求8所述的药物组合物；
    较佳地，所述疾病为缺血再灌注损伤；
    更佳地，所述缺血再灌注损伤为心脏缺血再灌注损伤、肝脏缺血再灌注损伤、肾脏缺血再灌注损伤和/或脑缺血再灌注损伤。
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