WO2022012067A1

WO2022012067A1 - Use of fibroblast growth factor 6 in preparation of medicine for relieving liver damage of non-alcoholic steatohepatitis

Info

Publication number: WO2022012067A1
Application number: PCT/CN2021/080713
Authority: WO
Inventors: 万健
Original assignee: 上海市浦东新区人民医院
Priority date: 2020-07-15
Filing date: 2021-03-15
Publication date: 2022-01-20
Also published as: CN111714619A; CN111714619B

Abstract

Disclosed in the present invention is use of a fibroblast growth factor. The fibroblast growth factor is fibroblast growth factor 6, and the use is in the preparation of a medicine for relieving liver damage of non-alcoholic steatohepatitis.

Description

成纤维生长因子6在缓解非酒精性脂肪性肝炎肝损伤的药物中的应用Application of fibroblast growth factor 6 in drugs for alleviating liver injury in nonalcoholic steatohepatitis

技术领域technical field

本发明涉及医药生物领域，具体为成纤维生长因子6在缓解非酒精性脂肪性肝炎肝损伤的药物中的应用。The invention relates to the field of medicine and biology, in particular to the application of fibroblast growth factor 6 in a medicine for relieving liver damage of non-alcoholic steatohepatitis.

背景技术Background technique

随着生活水平的提高、生活方式的改变，非酒精性脂肪性肝病(Non-alcoholicFattyLiverdisease，NAFLD)的患病率逐年递增，已成为全球最主要的非感染性肝病。在全球范围内，NAFLD的患病率约为25％，在中东和南美最高，在非洲最低。在美国，NAFLD病例数预计将从2015年的8310万(占人口的25％)增加到2030年的1.009亿。在北美和欧洲，NAFLD通常伴有中枢性肥胖症(约占83％)，而在亚洲，尽管体重指数(BMI)为正常，但仍有相当比例的“瘦型NASH”患者。我国NAFLD的发病率亦不断攀升，约12-15％，在部分地区其患病率甚至高达30％。With the improvement of living standards and changes in lifestyle, the prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing year by year, and it has become the most important non-infectious liver disease in the world. Globally, the prevalence of NAFLD is approximately 25%, highest in the Middle East and South America and lowest in Africa. In the United States, the number of NAFLD cases is projected to increase from 83.1 million in 2015 (25% of the population) to 100.9 million in 2030. In North America and Europe, NAFLD is commonly associated with central obesity (approximately 83%), while in Asia, despite a normal body mass index (BMI), there is a significant proportion of "lean NASH" patients. The incidence of NAFLD in my country is also rising, about 12-15%, and its prevalence is even as high as 30% in some areas.

NAFLD疾病谱包括非酒精性单纯脂肪肝(Non-alcoholicfattyliver，NAFL)、非酒精性脂肪性肝炎(Non-alcoholicHepatitis，NASH)及其相关肝纤维化、肝硬化，少数可进展为肝癌。NASH是单纯脂肪肝向肝纤维化及肝硬化发展的重要阶段，以肝细胞脂肪变性、炎细胞浸润和肝小叶纤维化为主要特征。NASH往往同时伴随肝细胞凋亡和坏死增多、血浆谷丙转氨酶ALT和谷草转氨酶AST升高，炎症细胞浸润和炎性因子表达和释放增多，且通常伴随胶原纤维的沉积。The disease spectrum of NAFLD includes non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and its related liver fibrosis, liver cirrhosis, and a few can progress to liver cancer. NASH is an important stage in the development of simple fatty liver to liver fibrosis and cirrhosis, and is mainly characterized by hepatocyte steatosis, inflammatory cell infiltration and hepatic lobular fibrosis. NASH is often accompanied by increased apoptosis and necrosis of hepatocytes, increased plasma alanine aminotransferase ALT and aspartate aminotransferase AST, increased inflammatory cell infiltration and increased expression and release of inflammatory factors, and is usually accompanied by the deposition of collagen fibers.

NAFLD是一种复杂的疾病，其致病因素和不同个体之间的临床表现高度异质。学术界对NASH的关键发病机制并不清楚，目前NASH的发病机理以1998年提出的“二次打击”学说为主流。它提出脂肪变性是NAFL的第一次打击，而向NASH和晚期纤维化的发展则需要第二个打击。因此，导致NASH发生的所有事件都是潜在的治疗靶点，包括胰岛素抵抗，脂毒性，氧化应激，内质网(ER)应激，线粒体功能障碍，脂肪组织功能异常，先天免疫力调节改变，细胞因子分泌和肠肝轴等。临床上针对上述发病机制所使用的胰岛素增敏剂、抗氧化剂及其联合用药，并不能完全阻断NASH的进展。由于目前尚无批准用于NASH的特异性治疗方案，这更加突显了针对这种情况制定有效干预策略的迫切需要。NAFLD is a complex disease with a high degree of heterogeneity in causative factors and clinical manifestations among different individuals. The key pathogenesis of NASH is not clear in the academic community. At present, the pathogenesis of NASH is dominated by the "second hit" theory proposed in 1998. It proposes that steatosis is the first blow to NAFL, and that progression to NASH and advanced fibrosis requires a second. Therefore, all events leading to the development of NASH are potential therapeutic targets, including insulin resistance, lipotoxicity, oxidative stress, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, adipose tissue dysfunction, and altered regulation of innate immunity , cytokine secretion and the gut-liver axis, etc. Insulin sensitizers, antioxidants and their combination drugs used clinically for the above pathogenesis cannot completely block the progression of NASH. As there are currently no specific treatment options approved for NASH, this underscores the urgent need to develop effective intervention strategies for this condition.

成纤维细胞生长因子(fibroblastgrowthfactor，FGF)又称肝素结合因子，是一个多基因家族。FGF是一类通过与细胞膜表面酪氨酸激酶受体(fibroblastgrowthfactorreceptor，FGFR)结合发挥其生物学效应、调节细胞生长的肽类分子。FGF在促进胚胎发育、组织形成与修复、血管生成以及肿瘤发生等生理及病理过程中发挥着重要作用。FGF家族(FGFs)有23个成员，大多数都能特异性结合并启动FGFR的下游信号转导途径，从而发挥相应的生物学功能。Fibroblast growth factor (fibroblast growth factor, FGF), also known as heparin-binding factor, is a multi-gene family. FGF is a kind of peptide molecule that exerts its biological effect and regulates cell growth by binding with fibroblast growth factor receptor (FGFR) on the cell membrane surface. FGF plays an important role in promoting embryonic development, tissue formation and repair, angiogenesis and tumorigenesis and other physiological and pathological processes. There are 23 members of the FGF family (FGFs), most of which can specifically bind to and initiate the downstream signal transduction pathway of FGFR, thereby exerting corresponding biological functions.

成纤维细胞生长因子-21(fibroblastgrowthfactor21，FGF-21)是一个代谢调节因子，属于成纤维细胞生长因子家族，其特异性作用于肝脏、脂肪、胰岛细胞且不依赖于胰岛素有效安全地调节血糖血脂的能力深得研究人员青睐，也有报道成纤维细胞生长因子-21(FGF21)可以有效的防治体外诱导的NAFLD。公开号为CN103193878A的发明专利公开了突变hFGF-21蛋白成熟肽及其与聚乙二醇的交联物以及它们的应用，聚乙二醇交联物的蛋白质结构为序列4，发明人将其命名为脂糖素。公开号为的发明专利公开了一种长效化突变的人源成纤维生长因子的新用途，具体涉及一种长效化的突变的人源成纤维细胞生长子-21在治疗非酒精性脂肪性肝炎药物中的应用。Fibroblast growth factor-21 (fibroblast growth factor 21, FGF-21) is a metabolic regulator, belonging to the fibroblast growth factor family, which specifically acts on liver, fat, islet cells and is independent of insulin to effectively and safely regulate blood sugar and blood lipids The ability of fibroblast growth factor-21 (FGF21) has been favored by researchers, and it has also been reported that fibroblast growth factor-21 (FGF21) can effectively prevent and treat NAFLD induced in vitro. The invention patent with publication number CN103193878A discloses the mutant hFGF-21 protein mature peptide and its cross-linking product with polyethylene glycol and their application. The protein structure of the polyethylene glycol cross-linking product is sequence 4, and the inventors put Named as liposacin. The invention patent publication No. discloses a new use of a long-acting mutant human fibroblast growth factor, and specifically relates to a long-acting mutant human fibroblast growth factor-21 in the treatment of non-alcoholic fat. Application of Hepatitis Drugs.

人FGF6基因定位于染色体12p13，编码一个由208个氨基酸残基组成单链多肽，包括N端37个氨基酸残基组成的疏水信号肽。成熟的FGF6需要切除其N端信号肽，生成一个理论分子质量为19kDa的肽类分子。FGF6与FGFR结合后促进下游细胞信号转导，参与调控肌肉再生、心肌发育、血管形成和骨生成。同时，异常的FGF6-FGFR信号可能在包括***癌和乳腺癌在内的多种肿瘤的发生中起着重要作用。但是截至目前，FGF6和NASH发生发展的关系，国内外尚无报道。The human FGF6 gene is located on chromosome 12p13 and encodes a single-chain polypeptide consisting of 208 amino acid residues, including a hydrophobic signal peptide consisting of 37 amino acid residues at the N-terminus. Mature FGF6 needs to excise its N-terminal signal peptide to generate a peptide molecule with a theoretical molecular mass of 19kDa. The combination of FGF6 and FGFR promotes downstream cell signal transduction and participates in the regulation of muscle regeneration, myocardial development, angiogenesis and osteogenesis. Meanwhile, abnormal FGF6-FGFR signaling may play an important role in the occurrence of various tumors including prostate and breast cancer. But so far, there is no report at home and abroad about the relationship between FGF6 and NASH development.

发明内容SUMMARY OF THE INVENTION

本发明提供了成纤维生长因子6在缓解非酒精性脂肪性肝炎肝损伤的药物中的应用。The invention provides the application of fibroblast growth factor 6 in the medicine for relieving liver damage of non-alcoholic steatohepatitis.

所述成纤维生长因子6的氨基酸序列为：见序列表。The amino acid sequence of the fibroblast growth factor 6 is: see the sequence listing.

所述应用为成纤维生长因子6可以缓解非酒精性脂肪性肝炎肝损伤缓解非酒精性脂肪性肝炎的相关症状。The application is that fibroblast growth factor 6 can alleviate the liver damage of non-alcoholic steatohepatitis and alleviate the related symptoms of non-alcoholic steatohepatitis.

所述相关症状包括：肝脏损伤、炎症反应、胶原纤维沉积。The associated symptoms include: liver damage, inflammatory response, and deposition of collagen fibers.

所述的非酒精性脂肪性肝炎包括：高脂饮食引起的非酒精性脂肪性肝炎、肝炎引起的非酒精性脂肪性肝炎、肥胖症引起的非酒精性脂肪性肝炎、糖尿病引起的非酒精性脂肪性肝炎、胰岛素抵抗引起非酒精性脂肪性肝炎、高甘油三酯血症引起的非酒精性脂肪性肝炎、无β脂蛋白血症引起的非酒精性脂肪性肝炎、糖原贮积病引起的非酒精性脂肪性肝炎、韦克病引起的非酒精性脂肪性肝炎、沃尔曼病引起的非酒精性脂肪性肝炎、脂肪营养不良所引起的非酒精性脂肪性肝炎。The non-alcoholic steatohepatitis includes: non-alcoholic steatohepatitis caused by high-fat diet, non-alcoholic steatohepatitis caused by hepatitis, non-alcoholic steatohepatitis caused by obesity, and non-alcoholic steatohepatitis caused by diabetes. Steatohepatitis, nonalcoholic steatohepatitis caused by insulin resistance, nonalcoholic steatohepatitis caused by hypertriglyceridemia, nonalcoholic steatohepatitis caused by abetalipoproteinemia, and glycogen storage disease nonalcoholic steatohepatitis caused by Wake's disease, nonalcoholic steatohepatitis caused by Wolman's disease, and nonalcoholic steatohepatitis caused by lipodystrophy.

所述药物是含有成纤维生长因子6作为药物活性成分的药物组合物，所述药物组合物可以被制备成任何一种可药用的剂型，这些剂型包括：片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、丹剂、粉剂、溶液剂、注射剂、栓剂、喷雾剂、滴剂、贴剂，本发明的药物优选注射用药物，如制备成粉针或液体针，所述液体针如水针，有机溶剂针，混悬液针等。The medicine is a pharmaceutical composition containing fibroblast growth factor 6 as a pharmaceutical active ingredient, and the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form, and these dosage forms include: tablets, capsules, granules, Pills, powders, ointments, elixirs, powders, solutions, injections, suppositories, sprays, drops, patches, the drug of the present invention is preferably a drug for injection, such as being prepared into powder injection or liquid injection, the liquid injection such as water Needle, organic solvent needle, suspension needle, etc.

本发明提供的成纤维生长因子6(FGF6)在缓解非酒精性脂肪性肝炎肝损伤的药物中的应用，具有以下优点：The application of the fibroblast growth factor 6 (FGF6) provided by the present invention in the medicine for relieving nonalcoholic steatohepatitis liver injury has the following advantages:

1.FGF6作用后无明显毒性作用，且可有效缓解NASH肝脏损伤；1. FGF6 has no obvious toxic effects, and can effectively relieve NASH liver damage;

2.FGF6可以有效缓解炎性细胞聚集和胶原纤维沉积；2. FGF6 can effectively relieve inflammatory cell aggregation and collagen fiber deposition;

3.FGF6可以有效抑制巨噬细胞激活；3. FGF6 can effectively inhibit the activation of macrophages;

4.FGF6可以有效降低肝脏中炎症因子的表达。4.FGF6 can effectively reduce the expression of inflammatory factors in the liver.

附图说明Description of drawings

图1为本发明健康PBS组和健康FGF6组中健康小鼠的ALT含量数据对比图；Fig. 1 is the ALT content data comparison diagram of healthy mice in healthy PBS group of the present invention and healthy FGF6 group;

图2为本发明健康PBS组和健康FGF6组中健康小鼠的AST含量数据对比图；Fig. 2 is the AST content data comparison diagram of healthy mice in healthy PBS group of the present invention and healthy FGF6 group;

图3为本发明NASH PBS组和NASH FGF6组中NASH小鼠的ALT含量数据对比图；Fig. 3 is the comparison chart of the ALT content data of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图4为本发明NASH PBS组和NASH FGF6组中NASH小鼠的AST含量数据对比图；Fig. 4 is the comparison chart of the AST content data of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图5为本发明NASH PBS组和NASH FGF6组中NASH小鼠的Ly6g免疫组织化学染色对比图；Figure 5 is a comparison diagram of Ly6g immunohistochemical staining of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图6为本发明NASH PBS组和NASH FGF6组中NASH小鼠的天狼星红染色对比图；Fig. 6 is the contrast diagram of Sirius red staining of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图7为本发明NASH PBS组和NASH FGF6组中NASH小鼠的F4/80蛋白免疫荧光染色对比图；Fig. 7 is a comparison diagram of F4/80 protein immunofluorescence staining of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图8为本发明NASH PBS组和NASH FGF6组中NASH小鼠的TNFα炎症因子实时定量PCR数据图；Figure 8 is a graph of real-time quantitative PCR data of TNFα inflammatory factors in NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图9为本发明NASH PBS组和NASH FGF6组中NASH小鼠的IL-1β炎症因子实时定量PCR数据图；Fig. 9 is the real-time quantitative PCR data graph of IL-1β inflammatory factor of NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图10为本发明NASH PBS组和NASH FGF6组中NASH小鼠的iNOS炎症因子实时定量PCR数据图；Figure 10 is a graph of real-time quantitative PCR data of iNOS inflammatory factors in NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention;

图11为本发明NASH PBS组和NASH FGF6组中NASH小鼠的IL6炎症因子实时定量PCR数据图。Figure 11 is a graph of real-time quantitative PCR data of IL6 inflammatory factors in NASH mice in the NASH PBS group and the NASH FGF6 group of the present invention.

具体实施方式detailed description

实施例1Example 1

1、构建高脂饮食(Highfatdiet，HFD)诱导的非酒精性脂肪性肝炎(NASH)小鼠模型(简称：NASH小鼠模型)：参考相关文献(ImajoK，FujitaK，YonedaM，etal.Hyperresponsivitytolow-doseendotoxinduringprogressiontononalcoholicsteatohepatitisisregulatedbyleptin-mediatedsignaling.CellMetab.2012；16(1):44-54)，通过对C57BL/6J小鼠长时程的高脂饮食后，再给予低剂量LPS刺激，以模拟NASH相关的肝损伤。小鼠购自上海斯莱克实验动物有限责任公司。1. Construction of a high-fat diet (Highfatdiet, HFD)-induced non-alcoholic steatohepatitis (NASH) mouse model (abbreviation: NASH mouse model): refer to relevant literature (ImajoK, FujitaK, YonedaM, et al.Hyperresponsivitytolow-doseendotoxinduringprogressiontononalcoholicsteatohepatitisisregulatedbyleptin- mediatedsignaling. Cell Metab. 2012; 16(1): 44-54), by giving C57BL/6J mice a long-term high-fat diet followed by low-dose LPS stimulation to simulate NASH-related liver injury. Mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.

具体地，选取20只健康雄性8周龄C57BL6小鼠，给予12周长程高脂饮食，然后给予腹腔注射LPS(0.25mg/kg)，每天一次，持续4周，构建成NASH小鼠模型。在LPS注射的后两周，NASH小鼠构建完成。LPS购自美国Sigma-aldrich公司，货号:L2630-100MG。Specifically, 20 healthy male 8-week-old C57BL6 mice were selected, given a 12-week long-term high-fat diet, and then intraperitoneally injected with LPS (0.25 mg/kg) once a day for 4 weeks to construct a NASH mouse model. Two weeks after LPS injection, NASH mice were constructed. LPS was purchased from Sigma-aldrich Company in the United States, item number: L2630-100MG.

小鼠FGF6基因的mRNA序列：1-44位碱基为5’-不翻译区，45-671位碱基为氨基酸编码区；672-1232位碱基为3’-不翻译区。The mRNA sequence of mouse FGF6 gene: 1-44 bases are 5'-untranslated region, 45-671 bases are amino acid coding region; 672-1232 bases are 3'-untranslated region.

2、小鼠FGF6蛋白购自美国R&DSystems公司，货号:5750-F6/CF。2. Mouse FGF6 protein was purchased from R&D Systems, USA, item number: 5750-F6/CF.

3、FGF6注射剂制备：将小鼠FGF6蛋白溶解于PBS试剂中作为FGF6注射剂。PBS试剂购自上海碧云天生物技术有限公司。3. Preparation of FGF6 injection: The mouse FGF6 protein was dissolved in PBS reagent as FGF6 injection. PBS reagent was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

实验例1：FGF6在有效剂量范围内对高脂饮食诱导的非酒精性脂肪性肝炎小鼠模型的治疗作用。Experimental Example 1: Therapeutic effect of FGF6 on a high-fat diet-induced nonalcoholic steatohepatitis mouse model within an effective dose range.

1、实验方法：1. Experimental method:

(1)选取20只健康雄性8周龄C57BL6小鼠，随机分为两组，每组10只，分别给予腹腔注射PBS试剂(对照溶剂)或FGF6注射剂(1mg/kg)，每天注射一次(上午10点注射)。(1) 20 healthy male 8-week-old C57BL6 mice were selected and randomly divided into two groups, 10 mice in each group, and were given intraperitoneal injection of PBS reagent (control solvent) or FGF6 injection (1 mg/kg), once a day (morning 10 o'clock injection).

(2)选取20只NASH小鼠，随机分为两组，每组10只，分别给予腹腔注射PBS试剂(对照溶剂)或FGF6注射剂(1mg/kg)，每天注射一次(上午10点注射)。(2) 20 NASH mice were selected and randomly divided into two groups, 10 mice in each group, and were given intraperitoneal injection of PBS reagent (control solvent) or FGF6 injection (1 mg/kg), once a day (10 am injection).

动物分组及给药情况具体情况见表1所示：The details of animal grouping and administration are shown in Table 1:

表1：动物分组及给药情况Table 1: Animal grouping and administration

组别group	动物数目(只)Number of animals (only)	剂量dose	给药周期(天)Dosing cycle (days)
健康PBS组 Healthy PBS Group	1010	同体积对照溶剂The same volume of control solvent	14天14 days

健康FGF6组 Healthy FGF6 group	1010	1mg/kg/d1mg/kg/d	14天14 days
NASH PBS组 NASH PBS group	1010	同体积对照溶剂The same volume of control solvent	14天14 days
NASH FGF6组 NASH FGF6 group	1010	1mg/kg/d1mg/kg/d	14天14 days

实验结束后，安乐死小鼠，取血浆置于-80度冰箱保存备用，取部分肝脏组织置于-80度冰箱保存备用，取部分肝脏置于4％多聚甲醛中固定制作石蜡切片，并进行后续分析。After the experiment, the mice were euthanized, the plasma was taken and placed in a -80 degree refrigerator for future use, a part of the liver tissue was taken in a -80 degree refrigerator for future use, and a part of the liver was fixed in 4% paraformaldehyde to make paraffin sections. subsequent analysis.

2、检测指标2. Detection indicators

将安乐死后小鼠摘眼球取全血，离心后得到血浆，通过美国强生VITROS5.1FS生化分析仪分析血浆谷丙转氨酶(ALT)和谷草转氨酶(AST)含量，检测小鼠的肝脏损伤情况；After euthanasia, the whole blood of mice was collected by removing the eyeballs, and the plasma was obtained after centrifugation. The contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma were analyzed by Johnson & Johnson VITROS5.1FS biochemical analyzer, and the liver damage of the mice was detected;

将小鼠肝脏组织经4％多聚甲醛溶液固定后，制作石蜡切片，进行Ly6g免疫组织化学染色和DAPI复染，检测NASH小鼠的炎性细胞聚集情况；After fixation of mouse liver tissue with 4% paraformaldehyde solution, paraffin sections were made, and Ly6g immunohistochemical staining and DAPI counterstaining were performed to detect the aggregation of inflammatory cells in NASH mice;

将小鼠肝脏组织经4％多聚甲醛溶液固定后，制作石蜡切片，进行天狼星红染色，检测NASH小鼠的胶原纤维沉积情况；After the mouse liver tissue was fixed with 4% paraformaldehyde solution, paraffin sections were made and stained with Sirius red to detect the deposition of collagen fibers in NASH mice;

将小鼠肝脏组织经4％多聚甲醛溶液固定后，制作石蜡切片，进行F4/80免疫荧光染色和DAPI复染，检测NASH小鼠的巨噬细胞激活情况；After fixation of mouse liver tissue with 4% paraformaldehyde solution, paraffin sections were made, and F4/80 immunofluorescence staining and DAPI counterstaining were performed to detect the activation of macrophages in NASH mice;

提取小鼠肝脏的mRNA，反转成cDNA，通过实时定量PCR实验，检测肿瘤坏死因子(TNFα)，白介素1β(IL-1β)，诱导性一氧化氮合成酶(iNOS)和白介素6(IL6)等炎症因子的表达，检测NASH小鼠的炎症因子表达情况；The mRNA of mouse liver was extracted, reversed into cDNA, and tumor necrosis factor (TNFα), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS) and interleukin 6 (IL6) were detected by real-time quantitative PCR experiment. The expression of inflammatory factors was detected, and the expression of inflammatory factors in NASH mice was detected;

3、数据统计和分析，图1、图2、图3、图4、图8、图9、图10、图11的绘制由Graphpad软件完成(版本号6.01)。***表示两组之间P值<0.001；****表示两组之间P值<0.0001，以P<0.05为差异有统计学意义。3. Data statistics and analysis, the drawing of Figure 1, Figure 2, Figure 3, Figure 4, Figure 8, Figure 9, Figure 10, and Figure 11 was done by Graphpad software (version number 6.01). *** indicates the P value between the two groups < 0.001; **** indicates the P value between the two groups < 0.0001, with P < 0.05 as a statistically significant difference.

4、实验结果：4. Experimental results:

(1)FGF6对小鼠血浆中ALT含量和AST含量的影响(1) The effect of FGF6 on the content of ALT and AST in mouse plasma

ALT和AST是肝脏损伤的特异性指标，并被用于辅助诊断NASH。图1中，健康PBS组中健康小鼠的ALT含量和健康FGF6组中健康小鼠的ALT含量基本相等；图2中，健康PBS组中健康小鼠的AST含量和健康FGF6组中健康小鼠的AST含量基本相等；图1和图2数据表明FGF6对健康小鼠无毒性作用。图3中，NASH FGF6组中NASH小鼠的ALT含量明显低于NASH PBS组NASH小鼠的ALT含量；图4中，NASH FGF6组中NASH小鼠的AST含量也明显低于NASH PBS组NASH小鼠的AST含量，图3和图4数据表明FGF6可以有效降低NASH小鼠的血浆ALT和AST值，说明FGF6可以有效缓解NASH小鼠的肝损伤。ALT and AST are specific indicators of liver damage and are used to aid in the diagnosis of NASH. In Figure 1, the ALT content of the healthy mice in the healthy PBS group and the ALT content of the healthy mice in the healthy FGF6 group were basically equal; in Figure 2, the AST content of the healthy mice in the healthy PBS group and the healthy mice in the healthy FGF6 group were basically the same. The AST content of FGF6 was basically equal; the data in Figures 1 and 2 indicated that FGF6 had no toxic effect on healthy mice. In Figure 3, the ALT content of the NASH mice in the NASH FGF6 group was significantly lower than that of the NASH mice in the NASH PBS group; in Figure 4, the AST content of the NASH mice in the NASH FGF6 group was also significantly lower than that of the NASH PBS group. The AST content of mice, the data in Figure 3 and Figure 4 show that FGF6 can effectively reduce the plasma ALT and AST values of NASH mice, indicating that FGF6 can effectively alleviate liver damage in NASH mice.

(2)FGF6对NASH小鼠炎性细胞聚集的影响(2) The effect of FGF6 on the aggregation of inflammatory cells in NASH mice

图5中棕黄色：Ly6g染色；蓝色：DAPI。A：PBS试剂注射；B：FGF6注射剂注射。两组Ly6g染色显著差异，表明FGF6可以有效降低NASH小鼠的炎性细胞聚集。In Figure 5, tan: Ly6g staining; blue: DAPI. A: PBS reagent injection; B: FGF6 injection injection. Ly6g staining was significantly different between the two groups, indicating that FGF6 could effectively reduce inflammatory cell aggregation in NASH mice.

(3)FGF6对NASH小鼠胶原纤维沉积的影响(3) The effect of FGF6 on the deposition of collagen fibers in NASH mice

图6中A：PBS试剂注射；B：FGF6注射剂注射。两组染色显著差异，表明FGF6可以有效改善NASH小鼠的胶原纤维沉积。In Figure 6, A: PBS reagent injection; B: FGF6 injection injection. The staining was significantly different between the two groups, indicating that FGF6 could effectively improve collagen fiber deposition in NASH mice.

(4)FGF6对NASH小鼠巨噬细胞激活的影响(4) The effect of FGF6 on the activation of macrophages in NASH mice

图7中绿色：F4/80染色；蓝色：DAPI。A：PBS试剂注射；B：FGF6注射剂注射。两组染色显著差异，表明FGF6可以有效降低NASH小鼠的巨噬细胞激活。Green in Figure 7: F4/80 staining; blue: DAPI. A: PBS reagent injection; B: FGF6 injection injection. The staining was significantly different between the two groups, indicating that FGF6 could effectively reduce macrophage activation in NASH mice.

(5)FGF6对NASH小鼠炎症因子表达的影响(5) Effects of FGF6 on the expression of inflammatory factors in NASH mice

图8中，NASH FGF6组中NASH小鼠的TNFα炎症因子的表达明显低于NASH PBS组中NASH小鼠的TNFα炎症因子的表达；图9中，NASH FGF6组中NASH小鼠的IL-1β炎症因子的表达明显低于NASH PBS组中NASH小鼠的IL-1β炎症因子的表达；图10中，NASH FGF6组中NASH小鼠的iNOS炎症因子的表达明显低于NASH PBS组中NASH小鼠的iNOS炎症因子的表达；图11中，NASH FGF6组中NASH小鼠的IL6炎症因子的表达明显低于NASH PBS组中NASH小鼠的IL6炎症因子的表达。图8-图11数据表明FGF6可以有效降低NASH小鼠肝脏组织中TNFα炎症因子，IL-1β炎症因子，iNOS炎症因子和IL6炎症因子的表达。In Figure 8, the expression of TNFα inflammatory factors in NASH mice in the NASH FGF6 group was significantly lower than the expression of TNFα inflammatory factors in NASH mice in the NASH PBS group; in Figure 9, the IL-1β inflammation in NASH mice in the NASH FGF6 group The expression of the factor was significantly lower than that of the IL-1β inflammatory factor in the NASH mice in the NASH PBS group; in Figure 10, the expression of the iNOS inflammatory factor in the NASH mice in the NASH FGF6 group was significantly lower than that in the NASH mice in the NASH PBS group. The expression of iNOS inflammatory factors; in Figure 11, the expression of IL6 inflammatory factors in NASH mice in the NASH FGF6 group was significantly lower than the expression of IL6 inflammatory factors in NASH mice in the NASH PBS group. Figures 8-11 data show that FGF6 can effectively reduce the expression of TNFα inflammatory factor, IL-1β inflammatory factor, iNOS inflammatory factor and IL6 inflammatory factor in liver tissue of NASH mice.

5、实验结果：5. Experimental results:

通过注射FGF6，检测小鼠的ALT和AST发现，FGF6对健康小鼠无明显毒性作用，且可以有效降低NASH小鼠的ALT和AST，缓解肝脏损伤。通过对小鼠肝脏切片染色发现，FGF6可以有效缓解NASH小鼠的炎性细胞聚集和胶原纤维沉积。对肝脏组织进行巨噬细胞染色后发现，FGF6可以有效抑制NASH小鼠的巨噬细胞激活。此外，在分子机制方面发现FGF6可以有效降低NASH小鼠肝脏中炎症因子的表达。综上所述，本发明通过实验动物研究的分析，证实了FGF6注射药物对小鼠非酒精性脂肪性肝炎肝损伤的缓解作用。By injecting FGF6 and detecting ALT and AST in mice, it was found that FGF6 has no obvious toxic effect on healthy mice, and can effectively reduce ALT and AST in NASH mice and alleviate liver damage. By staining mouse liver sections, it was found that FGF6 could effectively alleviate inflammatory cell aggregation and collagen fiber deposition in NASH mice. After macrophage staining of liver tissue, it was found that FGF6 can effectively inhibit macrophage activation in NASH mice. In addition, in terms of molecular mechanism, it was found that FGF6 could effectively reduce the expression of inflammatory factors in the liver of NASH mice. In conclusion, the present invention confirms the relieving effect of FGF6 injection drug on the liver injury of non-alcoholic steatohepatitis in mice through the analysis of experimental animal research.

结论：FGF6具有缓解非酒精性脂肪性肝炎肝损伤的作用。Conclusion: FGF6 has the effect of alleviating liver injury in nonalcoholic steatohepatitis.

Claims

成纤维生长因子6在缓解非酒精性脂肪性肝炎肝损伤的药物中的应用。Use of fibroblast growth factor 6 in a drug for alleviating liver injury in nonalcoholic steatohepatitis.
根据权利要求1所述的应用，其特征在于，所述应用为成纤维生长因子6缓解非酒精性脂肪性肝炎的相关症状的应用。The application according to claim 1, wherein the application is the application of fibroblast growth factor 6 to relieve symptoms related to nonalcoholic steatohepatitis.
根据权利要求2所述的应用，其特征在于，所述相关症状包括：肝脏损伤、炎症反应、胶原纤维沉积。The use according to claim 2, wherein the related symptoms include liver damage, inflammatory response, and collagen fiber deposition.
根据权利要求1所述的应用，其特征在于，所述的非酒精性脂肪性肝炎包括：高脂饮食引起的非酒精性脂肪性肝炎、肝炎引起的非酒精性脂肪性肝炎、肥胖症引起的非酒精性脂肪性肝炎、糖尿病引起的非酒精性脂肪性肝炎、胰岛素抵抗引起非酒精性脂肪性肝炎、高甘油三酯血症引起的非酒精性脂肪性肝炎、无β脂蛋白血症引起的非酒精性脂肪性肝炎、糖原贮积病引起的非酒精性脂肪性肝炎、韦克病引起的非酒精性脂肪性肝炎、沃尔曼病引起的非酒精性脂肪性肝炎、脂肪营养不良所引起的非酒精性脂肪性肝炎。The application according to claim 1, wherein the non-alcoholic steatohepatitis comprises: non-alcoholic steatohepatitis caused by high-fat diet, non-alcoholic steatohepatitis caused by hepatitis, and non-alcoholic steatohepatitis caused by obesity NASH NASH caused by nonalcoholic steatohepatitis.
根据权利要求1所述的应用，其特征在于，其中所述药物为含有成纤维生长因子6作为药物活性成分的药物组合物。The use according to claim 1, wherein the medicine is a pharmaceutical composition containing fibroblast growth factor 6 as a pharmaceutical active ingredient.
根据权利要求5所述的应用，其特征在于，所述药物组合物被制成任何一种可药用的剂型。The use according to claim 5, wherein the pharmaceutical composition is prepared into any pharmaceutically acceptable dosage form.
根据权利要求6所述的应用，其特征在于，所述剂型包括：片剂、胶囊剂、颗粒剂、丸剂、散剂、膏剂、丹剂、注射剂、栓剂、喷雾剂、滴剂、贴剂、滴丸剂。The application according to claim 6, wherein the dosage forms include: tablets, capsules, granules, pills, powders, ointments, elixirs, injections, suppositories, sprays, drops, patches, drops pill.
根据权利要求7所述的应用，其特征在于，所述注射剂包括：粉针、液体针。The application according to claim 7, wherein the injection comprises: powder injection and liquid injection.
根据权利要求8所述的应用，其特征在于，所述液体针包括：水针、有机溶剂针、混悬液针。The application according to claim 8, wherein the liquid needle comprises: a water needle, an organic solvent needle, and a suspension needle.