WO2021244010A1 - 热休克因子2结合蛋白在肝脏缺血再灌注损伤、药物性肝损伤中的应用 - Google Patents

热休克因子2结合蛋白在肝脏缺血再灌注损伤、药物性肝损伤中的应用 Download PDF

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WO2021244010A1
WO2021244010A1 PCT/CN2020/137834 CN2020137834W WO2021244010A1 WO 2021244010 A1 WO2021244010 A1 WO 2021244010A1 CN 2020137834 W CN2020137834 W CN 2020137834W WO 2021244010 A1 WO2021244010 A1 WO 2021244010A1
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hsf2bp
liver
drug
mice
injury
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French (fr)
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吴荣谦
张佳
毕建斌
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西安交通大学医学院第一附属医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis

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  • the invention belongs to the technical field of gene function and application, and particularly relates to the application of HSF2BP (MEILB2) as a drug target in the screening of drugs for the treatment of liver ischemia-reperfusion injury and drug-induced liver injury, and the preparation of HSF2BP agonists for the treatment of liver ischemia Application of drugs for reperfusion injury and drug-induced liver injury.
  • HSF2BP HSF2BP
  • Liver damage caused by ischemia reperfusion is an important cause of severe trauma, burns, blood loss, septic shock, liver resection, liver insufficiency and failure after liver transplantation.
  • liver I/R injury involves a series of complex pathophysiological processes, including intracellular ion balance disorder, mitochondrial damage, oxygen free radical production, endothelial damage, inflammatory cell activation, and inflammatory factor release.
  • intracellular ion balance disorder including intracellular ion balance disorder, mitochondrial damage, oxygen free radical production, endothelial damage, inflammatory cell activation, and inflammatory factor release.
  • Acetaminophen is a commonly used analgesic and antipyretic drug. It is safe and effective to use the recommended dose, and excessive use may cause liver toxicity and acute liver failure (ALF). It is generally believed that APAP-induced oxidative stress and mitochondrial dysfunction play a central role in the pathogenesis of ALF.
  • N-acetylcysteine NAC is the only treatment option for patients with APAP overdose; however, due to factors such as adverse reactions and narrow treatment windows, the clinical use of this drug is limited. Therefore, the study of new therapeutic targets for drug-induced liver injury is of great significance for the clinical treatment of drug-induced liver injury.
  • Heat shock factor 2 binding protein (Heat Shock Factor 2 Binding Protein, HSF2BP) is a protein isolated from human testis cDNA library that can bind to heat shock factor 2 (HSF2).
  • HSF2BP Heat shock Factor 2 Binding Protein
  • the purpose of the present invention is to determine the relationship between the expression of HSF2BP gene and ischemia-reperfusion and drug-induced liver injury. It also provides an application of HSF2BP as a drug target in the screening of drugs for the prevention and treatment of liver ischemia-reperfusion injury and drug-induced liver injury, and further provides a HSF2BP agonist in the preparation of drugs for the prevention and treatment of liver ischemia-reperfusion injury and drug-induced liver injury In the application.
  • HSF2BP can be used as a drug target for screening drugs and/or biological reagents for protecting liver function or preventing, alleviating and/or treating liver ischemia-reperfusion injury or drug-induced liver injury.
  • HSF2BP agonists can be used in the preparation of drugs and/or biological preparations for protecting liver function or preventing, alleviating and/or treating liver ischemia-reperfusion injury or preventing, alleviating and/or treating drug-induced liver injury.
  • the present invention also claims drugs and/or biological agents that protect liver function including HSF2BP agonists; drugs and/or biological agents that prevent, alleviate and/or treat liver ischemia-reperfusion injury; and prevent and alleviate And/or drugs and or biological agents for the treatment of drug-induced liver injury diseases.
  • the HSF2BP agonist is preferably an overexpression plasmid of HSF2BP gene, virus-mediated HSF2BP transfection, or other drugs or biological agents that can promote HSF2BP expression.
  • the present invention discovers the new function of HSF2BP gene, that is, HSF2BP gene has the effect of protecting liver from ischemia-reperfusion injury and drug-induced liver injury.
  • HSF2BP liver ischemia-reperfusion injury and drug-induced liver injury
  • HSF2BP agonists can be used to prepare drugs and/or biological preparations for protecting liver function and treating liver ischemia-reperfusion injury and drug-induced liver injury.
  • Figure 1 shows the construction and identification results of hepatocyte-specific HSF2BP overexpression and knockout mice.
  • A is the identification result diagram of hepatocyte-specific HSF2BP overexpressing mice
  • B is the identification result diagram of hepatocyte-specific HSF2BP knockout mice.
  • FIG. 2 is a schematic diagram of the role of HSF2BP in liver ischemia-reperfusion injury and drug-induced liver injury.
  • A is the effect of HSF2BP overexpression and HSF2BP knockout on liver ischemia-reperfusion injury
  • B is the effect of HSF2BP overexpression and HSF2BP knockout on drug-induced liver injury.
  • FIG. 3 is a schematic diagram of the liver injury caused by ischemia-reperfusion in HSF2BP-TG and NTG mice.
  • A is the HE staining image of HSF2BP-TG and NTG mice;
  • B is the statistics of necrotic area of HSF2BP-TG and NTG mice;
  • C is the histological score of HSF2BP-TG and NTG mice;
  • D is HSF2BP-TG and NTG Mouse serum AST;
  • E is the serum ALT of HSF2BP-TG and NTG mice.
  • FIG. 4 is a schematic diagram of the liver injury caused by ischemia-reperfusion in HSF2BP-KO and WT mice.
  • A is the HE staining image of HSF2BP-KO and WT mice;
  • B is the statistics of necrotic area of HSF2BP-KO and WT mice;
  • C is the histological score of HSF2BP-KO and WT mice;
  • D is HSF2BP-KO and WT Mouse serum AST;
  • E is the serum ALT of HSF2BP-KO and WT mice.
  • FIG. 5 is a schematic diagram of the liver injury induced by drugs in HSF2BP-TG and NTG mice.
  • A is the HE staining image of HSF2BP-TG and NTG mice;
  • B is the statistics of necrotic area of HSF2BP-TG and NTG mice;
  • C is the histological score of HSF2BP-TG and NTG mice;
  • D is HSF2BP-TG and NTG Mouse serum AST;
  • E is the serum ALT of HSF2BP-TG and NTG mice.
  • FIG. 6 is a schematic diagram of the liver injury induced by drugs in HSF2BP-KO and WT mice.
  • A is the HE staining image of HSF2BP-KO and WT mice;
  • B is the statistics of necrotic area of HSF2BP-KO and WT mice;
  • C is the histological score of HSF2BP-KO and WT mice;
  • D is HSF2BP-KO and WT Mouse serum AST;
  • E is the serum ALT of HSF2BP-KO and WT mice.
  • the present invention takes C57 mice with hepatocyte-specific HSF2BP gene overexpression and knockout as the experimental object.
  • liver ischemia-reperfusion injury and drug-induced liver injury models the results show that HSF2BP gene overexpression is compared with non-overexpression mice.
  • the liver damage in mice was significantly inhibited and liver function was significantly improved, while the liver damage in HSF2BP knockout mice was significantly increased and liver function was significantly worsened.
  • the HSF2BP gene can be used as a drug target to construct an in vitro cell model or animal model of HSF2BP gene overexpression for screening drugs that protect liver function; or for screening prevention, alleviation and/or treatment of liver ischemia-reperfusion injury Drugs; or used to screen drugs to prevent, alleviate and/or treat drug-induced liver injury.
  • the HSF2BP gene can also be used as a target gene in gene therapy to design and prepare drugs and/or biological reagents to prevent, alleviate and/or treat liver ischemia-reperfusion injury, drug-induced liver injury, and achieve the prevention, alleviation and prevention through genetic engineering technology. / Or the purpose of treating liver ischemia-reperfusion injury and drug-induced liver injury.
  • HSF2BP can also be used as a target to design agonists. Through screening, it is found that the molecules that can specifically agonize HSF2BP can provide new therapeutic molecules for the treatment of liver ischemia-reperfusion injury and drug-induced liver injury. That is, HSF2BP agonists can be used in the preparation of drugs for protecting liver function or drugs for preventing, alleviating and/or treating liver ischemia-reperfusion injury or drugs for preventing, alleviating and/or treating drug-induced liver injury.
  • the HSF2BP agonist is preferably an overexpression plasmid of HSF2BP gene, virus-mediated HSF2BP transfection, or other agonist drugs or biological agents that can promote HSF2BP expression. One of them.
  • mice 8-12 weeks old, 20-25g in weight, male C57BL/6 strain in the background, including hepatocyte-specific HSF2BP gene overexpression (TG) and non-overexpression (NTG) mice , Hepatocyte-specific HSF2BP gene knockout mice (KO) and wild-type mice (WT, purchased from the SPF Laboratory Animal Center of the Medical Department of Xi’an Jiaotong University).
  • TG hepatocyte-specific HSF2BP gene overexpression
  • NTG non-overexpression mice
  • KO Hepatocyte-specific HSF2BP gene knockout mice
  • WT wild-type mice
  • mice All experimental mice are raised in the SPF Laboratory Animal Center of the Medical Department of Xi'an Jiaotong University.
  • the rearing temperature is 22-24°C
  • the humidity is 40-70%
  • the alternating light and dark lighting time is 12 hours, and they are free to drink and eat.
  • mice male 8-12 weeks old TG, NTG, KO and WT mice weighing 20-25g were the research objects, divided into 8 groups, namely HSF2BP overexpression and non-overexpression mice.
  • Surgery NTG-Sham and TG-Sham
  • operation group NTG-I/R and TG-I/R
  • sham operation of HSF2BP knockout mice and wild-type mice WT-Sham and KO-Sham
  • Surgery group WT-I/R and KO-I/R
  • mice were fasted 12h before the operation, and they were allowed to drink freely.
  • mice male 8-12 weeks old TG and NTG, KO and WT mice weighing 20-25g were the research objects, divided into 8 groups, namely HSF2BP overexpression and non-overexpression mice.
  • Surgery NTG-Sham and TG-Sham
  • model NTG-APAP and TG-APAP
  • sham operation WT-Sham and KO-Sham
  • model for HSF2BP knockout mice and wild-type mice WT-APAP and KO-APAP
  • mice were fasted 12h before the administration, and they were allowed to drink freely.
  • APAP 500mg/kg
  • the evaluation indicators of the severity of liver ischemia-reperfusion injury mainly include determining the area of liver necrosis and/or histological score based on HE staining, and determining the recovery of liver function by detecting serum AST and ALT. These indicators are positively correlated with the severity of liver damage. Specifically, liver specimens were obtained 24 hours after liver I/R and 6 hours after APAP treatment, fixed in 10% neutral formalin for 24 hours, dehydrated, embedded, and paraffin sectioned and then HE stained; at the same time, blood samples were obtained and centrifuged The supernatant was taken, and the mouse serum ALT (C009-2-1, built in Nanjing) and AST (C010-2-1, built in Nanjing) were detected according to the kit instructions.
  • FIG. 2A shows that HSF2BP overexpression reduces liver ischemia-reperfusion injury, while HSF2BP knockout aggravates liver ischemia-reperfusion injury. Perfusion injury.
  • Figure 2B shows that HSF2BP overexpression reduced drug-induced liver injury, while HSF2BP knockout aggravated drug-induced liver injury.
  • Figure 3A shows that the liver tissues of the two groups of mice in the Sham group are basically normal. In the liver I/R group, the liver cells of the two groups of mice had obvious necrosis, edema, and inflammatory cell infiltration.
  • Figure 4A shows that the liver tissues of the two groups of mice in the Sham group are basically normal. In the liver I/R group, the liver cells of the two groups of mice had obvious necrosis, edema, and inflammatory cell infiltration.
  • Figure 4B the infarct area of liver tissue in KO mice after liver I/R It was significantly higher than that of the WT group ( Figure 4B), and the histological score was also lower ( Figure 4C); the liver function AST ( Figure 4D) and ALT ( Figure 4E) of the same KO mice after I/R were also significantly lower than that of WT Mice. Therefore, it further verifies that HSF2BP has a protective effect on liver ischemia-reperfusion injury.
  • FIG. 5A shows that after APAP treatment, the liver of the NTG group mice showed obvious inflammatory cell infiltration, hepatocellular edema, and extensive liver necrosis.
  • the necrotic area and histological scores of TG mice in the drug-induced liver injury group were significantly lower than those in the NTG group ( Figure 5B-C); the serum AST ( Figure 5D) and ALT ( Figure 5E) of the same TG mice after APAP treatment It is also significantly lower than NTG mice. Therefore, HSF2BP has a protective effect in drug-induced liver injury.
  • FIG. 6A shows that after APAP treatment, the liver of the WT group mice showed obvious inflammatory cell infiltration, hepatocellular edema, and extensive liver necrosis.
  • the necrotic area and histological scores of KO mice in the drug-induced liver injury group were significantly higher than those in the WT group ( Figure 6B-C); the serum AST ( Figure 6D) and ALT ( Figure 6E) of the KO mice after APAP treatment were also significantly higher. It is also significantly higher than that of KO mice. Therefore, it is further verified that HSF2BP has a protective effect in drug-induced liver injury.
  • hepatocyte-specific HSF2BP gene can improve liver ischemia-reperfusion injury and drug-induced liver injury, including reducing the area of necrosis, histological score, and promoting the recovery of liver function.
  • Hepatocyte-specific HSF2BP gene knockout can aggravate liver ischemia-reperfusion injury and drug-induced liver injury, including increasing necrosis area, histological score, and delaying liver function recovery.
  • HSF2BP has a protective effect on liver ischemia-reperfusion injury and drug-induced liver injury.
  • HSF2BP can be used as a drug target to prepare HSF2BP agonists for the prevention, alleviation and/or treatment of liver ischemia-reperfusion injury or drug-induced liver injury, including HSF2BP gene overexpression plasmid, virus-mediated HSF2BP transfection or other capable Drugs or biological agents that promote the expression of HSF2BP.

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Abstract

一种热休克因子2结合蛋白（HSF2BP）在肝脏缺血再灌注损伤、药物性肝损伤中的应用，以肝脏特异性HSF2BP基因过表达和敲除小鼠为实验对象，通过肝脏缺血再灌注损伤、药物性肝损伤模型研究表明与野生型C57小鼠对比，HSF2BP基因过表达小鼠中肝脏损伤明显被抑制，肝功能明显好转，而HSF2BP基因敲除小鼠中肝脏损伤则明显增加，肝功能明显恶化。HSF2BP基因具有保护缺血再灌注诱导的肝脏损伤、药物性诱导的肝损伤的作用，可通过其特异性激动剂促进HSF2BP基因表达来对肝脏缺血再灌注损伤、药物性肝损伤进行干预。

Description

热休克因子2结合蛋白在肝脏缺血再灌注损伤、药物性肝损伤中的应用 技术领域
本发明属于基因的功能与应用技术领域，特别涉及HSF2BP(MEILB2)作为药物靶标在筛选治疗肝脏缺血再灌注损伤、药物性肝损伤药物中的应用，以及HSF2BP的激动剂在制备治疗肝脏缺血再灌注损伤、药物性肝损伤药物中的应用。
背景技术
缺血再灌注(Ischemia Reperfusion，I/R)所导致的肝脏损伤是严重创伤、烧伤、失血、脓毒性休克、及肝切除、肝移植术后肝功能不全、衰竭的重要原因。近年来，随着肝切除和肝移植手术在临床的日益普及，肝脏I/R损伤已成为制约肝脏外科发展的重要瓶颈。肝脏I/R损伤涉及到一系列复杂的病理生理过程，包括细胞内离子平衡紊乱、线粒体损伤、氧自由基产生、内皮受损、炎性细胞激活和炎症因子释放等。目前，临床上对肝脏I/R损伤尚无有效的预防及治疗方法。因此，研究肝脏I/R损伤新的治疗靶点，对促进肝切除及肝移植术后肝脏功能恢复有重要意义。
对乙酰氨基酚(acetaminophen，APAP)是一种常用的镇痛解热药物。按推荐剂量使用是安全有效的，而过量使用可能导致肝毒性和急性肝衰竭(acute liver failure，ALF)。一般认为，APAP诱导的氧化应激和线粒体功能障碍在ALF的发病机制中起核心作用。目前N-乙酰半胱氨酸(N-acetylcysteine，NAC)是APAP过量患者的唯一治疗选择；然而，由于不良反应和狭窄的治疗窗口等因素，限制了这种药物的临床使用。因此，研究药物性肝损伤新的治疗靶点，对临床治疗药物性肝损伤具有重要意义。
热休克因子2结合蛋白(Heat Shock Factor 2 Binding Protein，HSF2BP)是 从人类睾丸cDNA文库中分离出来的一种可以和热休克因子2(HSF2)结合的蛋白。目前关于HSF2BP的研究不多，现有的研究显示HSF2BP在多发性硬化患者的脑部病变中表达增高，可与乳腺癌相关蛋白2相互作用，参与精子的形成，并可能与冠状动脉疾病的发生有关，但其在肝脏疾病中的作用目前仍不清楚。
发明内容
为解决临床防治肝脏缺血再灌注损伤、药物性肝损伤现有技术的缺陷和不足，本发明的目的是确定HSF2BP基因的表达与缺血再灌注及药物诱导的肝损伤之间的相互关系，并提供一种HSF2BP作为药物靶标在筛选防治肝脏缺血再灌注损伤、药物性肝损伤药物中的应用，进而提供一种HSF2BP的激动剂在制备防治肝脏缺血再灌注损伤、药物性肝损伤药物中的应用。
为了实现上述目的，本发明采用的技术方案是：
本发明提出HSF2BP可作为药物靶标应用在筛选保护肝脏功能或预防、缓解和/或治疗肝脏缺血再灌注损伤或药物性肝损伤的药物和/或生物试剂中。
本发明提出HSF2BP的激动剂可应用在制备保护肝脏功能或者预防、缓解和/或治疗肝脏缺血再灌注损伤或者预防、缓解和/或治疗药物性肝损伤的药物和/或生物制剂中。
相应地，本发明还要求保护包含HSF2BP的激动剂的保护肝脏功能的药物和/或生物制剂；预防、缓解和/或治疗肝脏缺血再灌注损伤疾病的药物和或生物制剂；以及预防、缓解和/或治疗药物性肝损伤疾病的药物和或生物制剂。
所述的HSF2BP的激动剂优选为HSF2BP基因的过表达质粒、病毒介导的HSF2BP转染或者其他能够促进HSF2BP表达的药物或者生物制剂。
与现有技术相比，本发明的有益效果是：
1.本发明发现HSF2BP基因的新功能，即HSF2BP基因具有保护肝脏缺血再灌注损伤、药物性肝损伤的作用。
2.基于HSF2BP在肝脏缺血再灌注损伤、药物性肝损伤中的保护功能，为 肝脏缺血再灌注损伤、药物性肝损伤的药物和/或生物制剂提供靶标。
3.HSF2BP的激动剂可用于制备保护肝脏功能和治疗肝脏缺血再灌注损伤、药物性肝损伤的药物和/或生物制剂。
附图说明
图1为肝细胞特异性HSF2BP过表达和敲除小鼠的构建及鉴定结果图。其中A为肝细胞特异性HSF2BP过表达小鼠的鉴定结果图；B为肝细胞特异性HSF2BP敲除小鼠的鉴定结果图。
图2为HSF2BP在肝脏缺血再灌注损伤和药物性肝损伤中的作用示意图。其中A为HSF2BP过表达和HSF2BP敲除对肝脏缺血再灌注损伤的作用效果；B为HSF2BP过表达和HSF2BP敲除对药物性肝损伤的作用效果。
图3为HSF2BP-TG和NTG小鼠缺血再灌注的肝脏损伤情况示意图。其中A为HSF2BP-TG和NTG小鼠的HE染色图；B为HSF2BP-TG和NTG小鼠的坏死面积统计；C为HSF2BP-TG和NTG小鼠的组织学评分；D为HSF2BP-TG和NTG小鼠的血清AST；E为HSF2BP-TG和NTG小鼠的血清ALT。
图4为HSF2BP-KO和WT小鼠缺血再灌注的肝脏损伤情况示意图。其中A为HSF2BP-KO和WT小鼠的HE染色图；B为HSF2BP-KO和WT小鼠的坏死面积统计；C为HSF2BP-KO和WT小鼠的组织学评分；D为HSF2BP-KO和WT小鼠的血清AST；E为HSF2BP-KO和WT小鼠的血清ALT。
图5为HSF2BP-TG和NTG小鼠药物诱导的肝脏损伤情况示意图。其中A为HSF2BP-TG和NTG小鼠的HE染色图；B为HSF2BP-TG和NTG小鼠的坏死面积统计；C为HSF2BP-TG和NTG小鼠的组织学评分；D为HSF2BP-TG和NTG小鼠的血清AST；E为HSF2BP-TG和NTG小鼠的血清ALT。
图6为HSF2BP-KO和WT小鼠药物诱导的肝脏损伤情况示意图。其中A为HSF2BP-KO和WT小鼠的HE染色图；B为HSF2BP-KO和WT小鼠的坏死面积统计；C为HSF2BP-KO和WT小鼠的组织学评分；D为HSF2BP-KO和WT小鼠的血清AST；E为HSF2BP-KO和WT小鼠的血清ALT。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
本发明以肝细胞特异性HSF2BP基因过表达和敲除的C57小鼠为实验对象，通过肝脏缺血再灌注损伤、药物性肝损伤模型，结果表明与非过表达小鼠对比，HSF2BP基因过表达小鼠肝脏损伤明显被抑制，肝功能明显好转，而HSF2BP基因敲除小鼠的肝脏损伤则明显增加，肝功能明显恶化。
因此，HSF2BP基因可作为药物靶点，构建HSF2BP基因过表达的体外细胞模型或动物模型，用于筛选保护肝脏功能的药物；或者用于筛选预防、缓解和/或治疗肝脏缺血再灌注损伤的药物；或者用于筛选防、缓解和/或治疗药物性肝损伤的药物。
HSF2BP基因也可作为基因治疗中的靶基因，设计并制备预防、缓解和/或治疗肝脏缺血再灌注损伤、药物性肝损伤的药物和/或生物试剂，通过基因工程技术达到预防、缓解和/或治疗肝脏缺血再灌注损伤、药物性肝损伤的目的。
此外，还可以以HSF2BP为靶点设计激动剂，通过筛选，发现其中能够特异性激动HSF2BP的分子，从而为肝脏缺血再灌注损伤、药物性肝损伤的治疗提供新的治疗性分子。即，HSF2BP的激动剂可应用在制备保护肝脏功能的药物或者预防、缓解和/或治疗肝脏缺血再灌注损伤的药物或者预防、缓解和/或治疗药物性肝损伤的药物中。所述的HSF2BP的激动剂优选为HSF2BP基因的过表达质粒、病毒介导的HSF2BP转染或者其他能够促进HSF2BP表达的激动剂药物或者生物制剂。中的一种。
以下是本发明的详细实验过程和数据。
实验用动物及饲养
实验动物：选用8-12周龄、体重在20-25g，背景为雄性C57BL/6品系的小鼠，包括肝细胞特异性HSF2BP基因过表达小鼠(TG)和非过表达(NTG)小鼠，肝细胞特异性HSF2BP基因敲除小鼠(KO)和野生型小鼠(WT，购自 西安交通大学医学部SPF级实验动物中心)。
饲养环境：所有实验小鼠均饲养在西安交通大学医学部SPF级实验动物中心。饲养温度在22-24℃，湿度在40-70％，明暗交替照明时间为12h，自由饮水摄食。
【实施例1】肝细胞特异性HSF2BP过表达和敲除小鼠的构建：
为进一步研究HSF2BP过表达对于肝脏缺血再灌注损伤的影响，我们构建了肝脏特异性HSF2BP过表达(TG)和敲除(KO)小鼠。通过实时定量PCR(q-PCR)实验发现过表达小鼠肝脏中HSF2BP基因表达明显增高，而敲除小鼠肝脏中HSF2BP基因表达明显降低(如图1中A、B)。
【实施例2】小鼠肝缺血再灌注损伤模型的构建：
1.实验动物分组：分别以雄性8-12周龄，体重20-25g的TG和NTG、KO和WT小鼠为研究对象，分为8组，即HSF2BP过表达和非过表达小鼠的假手术(NTG-Sham和TG-Sham)和手术组(NTG-I/R和TG-I/R)；HSF2BP敲除小鼠和野生型小鼠的假手术(WT-Sham和KO-Sham)和手术组(WT-I/R和KO-I/R)，之后建立肝脏缺血再灌注损伤模型。
2.肝缺血再灌注损伤模型操作流程：
1)手术前12h给小鼠禁食，可自由饮水。
2)异氟烷吸入麻醉后仰卧固定，剃掉腹部毛发，消毒。
3)取腹正中3cm左右切口进腹，暴露肝脏左、中叶之肝蒂。
4)用无创血管夹夹闭中叶和左叶的门静脉和肝动脉，使约70％的肝脏缺血。期间用湿的盐水纱布覆盖切口，而Sham组的小鼠采用了除夹闭中叶和左叶的门静脉和肝动脉以外的所有操作。
5)60分钟后开放血流，开始再灌注，然后分两层关闭腹腔，将术后的小鼠置于干净的笼子中单独饲养，观察。
【实施例3】小鼠药物性肝损伤模型的构建：
1.实验动物分组：分别以雄性8-12周龄，体重20-25g的TG和NTG、KO 和WT小鼠为研究对象，分为8组，即HSF2BP过表达和非过表达小鼠的假手术(NTG-Sham和TG-Sham)和造模组(NTG-APAP和TG-APAP)；HSF2BP敲除小鼠和野生型小鼠的假手术(WT-Sham和KO-Sham)和造模组(WT-APAP和KO-APAP)，之后建立药物性肝损伤模型。
2.药物性肝损伤模型操作流程：
1)给药前12h给小鼠禁食，可自由饮水。
2)注射部位消毒后，通过腹腔注射APAP(500mg/kg)。
3)于给药后6h收集小鼠血清和肝脏标本。
【实施例4】肝脏损伤的测定：
肝脏缺血再灌注损伤严重程度的评估指标主要包括根据HE染色确定肝脏坏死面积和/或组织学评分，通过检测血清AST和ALT确定肝功能恢复情况。这些指标均与肝脏损伤严重程度正相关。具体来说，肝脏I/R术后24h和APAP处理6h时获取肝脏标本，于10％中性福尔马林中固定24h后脱水，包埋，进行石蜡切片后进行HE染色；同时获取血液标本，离心取上清，根据试剂盒说明书检测小鼠血清ALT(C009-2-1，南京建成)和AST(C010-2-1，南京建成)。
实验结果如下：
总的来说，HSF2BP在肝脏缺血再灌注损伤和药物性肝损伤中的作用见图2，图2A可见HSF2BP过表达减轻了肝脏缺血再灌注损伤，而HSF2BP敲除加重了肝脏缺血再灌注损伤。图2B可见HSF2BP过表达减轻了药物性肝损伤，而HSF2BP敲除加重了药物性肝损伤。NTG和TG小鼠在肝脏I/R后结果见图3，图3A可见两组小鼠Sham组肝组织均基本正常。而在肝脏I/R组，两组小鼠肝细胞均发生了明显的坏死、水肿、炎症细胞浸润等表现，然而相比于NTG小鼠，TG小鼠在肝脏I/R后肝组织梗死面积明显低于NTG小鼠(图3B)，组织学评分也更低(图3C)；同样TG小鼠肝脏I/R后的肝功AST(图3D)和ALT(图3E)也明显低于NTG组。因此，HSF2BP在肝脏缺血再灌注损伤中 具有保护作用。
WT和KO小鼠在肝脏I/R后结果见图4：图4A可见两组小鼠Sham组肝组织均基本正常。而在肝脏I/R组，两组小鼠肝细胞均发生了明显的坏死、水肿、炎症细胞浸润等表现，然而相比于WT小鼠，KO小鼠在肝脏I/R后肝组织梗死面积明显高于WT组小鼠(图4B)，组织学评分也更低(图4C)；同样KO小鼠I/R后的肝功AST(图4D)和ALT(图4E)也明显低于WT小鼠。因此进一步反面验证了HSF2BP在肝脏缺血再灌注损伤中具有保护作用。
NTG和TG小鼠在药物性肝损伤后结果见图5，图5A可见APAP处理后，NTG组小鼠肝脏出现明显的炎细胞浸润，肝细胞水肿，肝脏大面积坏死。然而，TG小鼠在药物性肝损伤组坏死面积和组织学评分显著低于NTG组(图5B-C)；同样TG小鼠在APAP处理后的血清AST(图5D)和ALT(图5E)也明显低于NTG小鼠。因此，HSF2BP在药物性肝损伤中具有保护作用。
WT和KO小鼠在药物性肝损伤后结果见图6，图6A可见APAP处理后，WT组小鼠肝脏出现明显的炎细胞浸润，肝细胞水肿，肝脏大面积坏死。然而，KO小鼠在药物性肝损伤组坏死面积和组织学评分显著高于WT组(图6B-C)；同样KO小鼠在APAP处理后的血清AST(图6D)和ALT(图6E)也明显高于KO小鼠。因此进一步反面验证了HSF2BP在药物性肝损伤中具有保护作用。
以上研究结果表明，肝细胞特异性HSF2BP基因过表达可以改善肝脏缺血再灌注损伤、药物性肝损伤，包括减小坏死面积、组织学评分，促进肝功能恢复。肝细胞特异性HSF2BP基因敲除可以加重肝脏缺血再灌注损伤、药物性肝损伤，包括增加坏死面积、组织学评分，延缓肝功能恢复。证明HSF2BP在肝脏缺血再灌注损伤、药物性肝损伤中具有保护作用。
鉴于此，可以HSF2BP作为药物靶标制备预防、缓解和/或治疗肝脏缺血再灌注损伤或药物性肝损伤的HSF2BP激动剂，包括HSF2BP基因的过表达质粒、病毒介导的HSF2BP转染或者其他能够促进HSF2BP表达的药物或者生物制剂。
上述实施例为本发明较佳的实施方式，单本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围内。

Claims (10)

  1. HSF2BP作为药物靶标在筛选保护肝脏功能的药物和/或生物制剂中的应用。
  2. HSF2BP作为药物靶标在筛选或制备预防、缓解和/或治疗肝脏缺血再灌注损伤或药物性肝损伤的药物和/或生物试剂中的应用。
  3. HSF2BP的激动剂在制备保护肝脏功能的药物和/或生物制剂中的应用。
  4. HSF2BP的激动剂在在筛选或制备预防、缓解和/或治疗肝脏缺血再灌注损伤的药物和/或生物制剂或者预防、缓解和/或治疗药物性肝损伤的药物和/或生物制剂中的应用。
  5. 根据权利要求3或4所述应用，其特征在于，所述HSF2BP的激动剂为能够促进HSF2BP表达的药物和/或生物制剂。
  6. 根据权利要求3或4所述应用，其特征在于，所述HSF2BP的激动剂为HSF2BP基因的过表达质粒或病毒介导的HSF2BP转染。
  7. 一种保护肝脏功能的药物和/或生物制剂，其特征在于，包含HSF2BP的激动剂。
  8. 一种预防、缓解和/或治疗肝脏缺血再灌注损伤疾病或预防、缓解和/或治疗药物性肝损伤疾病的药物和/或生物制剂，其特征在于，包含HSF2BP的激动剂。
  9. 根据权利要求7或8所述的药物，其特征在于，所述HSF2BP的激动剂为能够促进HSF2BP表达的药物或者生物制剂。
  10. 根据权利要求7或8所述的药物，其特征在于，所述HSF2BP的激动剂为HSF2BP基因的过表达质粒或病毒介导的HSF2BP转染。
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