CN114129556A - 野鸢尾黄素在治疗肝纤维化中的应用 - Google Patents
野鸢尾黄素在治疗肝纤维化中的应用 Download PDFInfo
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Abstract
本发明公开野鸢尾黄素在治疗肝纤维化中的应用。本发明通过体内外实验证明，野鸢尾黄素能抑制肝星状细胞活化，减少肝纤维化小鼠肝脏中胶原沉积，下调肝纤维化相关蛋白表达，缓解肝纤维化进程，说明野鸢尾黄素具有抗肝纤维化的作用。此外，野鸢尾黄素具有毒副作用小、用药剂量较小、原料来源广泛、生产成本低等优点。
Description
技术领域
本发明涉及医药领域，具体涉及野鸢尾黄素在治疗肝纤维化中的应用。
背景技术
肝纤维化是响应慢性肝损伤的愈合反应，它是由各种原因引起的，包括肝炎病毒感染、酗酒、胆汁淤积、自身免疫、药物、毒素和非酒精性脂肪肝，最终导致肝功能和肝脏结构破坏。如果不及时治疗，肝纤维化发展为肝硬化，导致正常肝功能进行性丧失，肝脏结构改变、血流异常，最终导致门静脉高压，出现腹水、肝性脑病和静脉曲张出血等并发症，进而发生肝功能衰竭甚至死亡。失代偿肝硬化是全球范围内因肝病死亡的主要原因，并且与肝细胞癌患病风险增加有关。由肝硬化引起的失代偿和肝细胞癌每年造成200万人死亡，占全世界死亡总数的3.5％以上。
近几年不断上升的发病率对世界人口健康造成了沉重负担，虽然肝病患者的数量不断增加，但早期和晚期肝纤维化的诊断和治疗选择仍然有限。目前，肝活组织检查仍然是肝纤维化诊断的“金标准”。虽然血清学诊断模型、瞬时弹性成像等一些无创检测方法对肝纤维化具有较高的诊断价值，但仍有待进一步完善。目前临床上尚无公认有效的抗肝纤维化治疗方法，主要通过对病因治疗缓解肝损伤和炎症，同时防治肝纤维化。基于肝星状细胞(hepatic starate cells，HSCs)的抗纤维化策略主要包括抑制HSCs活化相关的靶分子(PPARα、PPARγ和PPARδ、FXR、趋化因子受体CCR2和CCR5、HSP47和galectin 3)。但是这些药物仍然没有很好的治疗效果并且有不同程度的不良反应。肝移植是第二大最常见的实体器官移植，但以目前的疾病增加速度，满足全球移植需求的不到10％。目前尚没有理想的抗肝纤维化药物批准上市，因此寻找有效的治疗手段仍然迫在眉睫。
纤维化是由瘢痕组织(细胞外基质，ECM)的过度积累引起的，伴随免疫细胞组成和血管生成改变，最终导致严重的结构变化，危及器官功能。肝脏中ECM主要来源于激活的HSCs。HSCs位于肝窦状内皮细胞(LSECs)和肝细胞之间，在正常肝脏中，HSCs 作为视黄酯储存细胞维持非增殖、静止表型，在肝损伤后，HSCs被激活，转分化为肌成纤维样细胞，它具有增殖性、收缩性、炎性和趋化性，以及ECM生成增强。因此，针对活化HSCs的功能调节是可靠的抗纤维化治疗策略。
肝纤维化的发病机制与多因素、多环节有关，因此，一种有效的治疗药物必须有能力调节参与肝纤维化进展的多个途径。在可能的治疗方法中，天然产物通常具有结构多样性、低毒性和广泛可用性，因此在治疗肝纤维化中可能发挥独特的作用。已经有研究表明从Silybummarianum提取的水飞蓟宾(Silibinin，SIL)对于慢性肝病动物模型有抗肝纤维化的作用，并已被提出作为治疗各种肝脏疾病的抗肝毒性药物。
发明内容
本发明目的之一，提供野鸢尾黄素的新用途，即提供了野鸢尾黄素在预防或治疗肝纤维化的药物中的应用。
本发明目的之二，提供野鸢尾黄素在抑制肝星状细胞活化和抑制细胞外基质生成的抑制剂的应用。
本发明的目的可以通过以下技术方案实现：
野鸢尾黄素在制备治疗肝纤维化或肝纤维化相关疾病药物中的应用，所述野鸢尾黄素的结构式如式Ⅰ所示：
本发明技术方案中，所述野鸢尾黄素用于制备抑制肝星状细胞的活化的抑制剂的应用。
本发明技术方案中，所述野鸢尾黄素用于制备抑制细胞外基质生成的抑制剂的应用。
本发明技术方案中，所述肝纤维化疾病包括病毒性肝炎、酒精性肝病、非酒精性脂肪性肝病、自身免疫性肝病、胆汁淤积性肝病、中毒和药物性肝病。
本发明技术方案中，所述野鸢尾黄素的给药剂量为1mg/kg、5mg/kg、10mg/kg。
本发明技术方案中，所述野鸢尾黄素的给药剂型包括口服制剂和注射制剂。
本发明技术方案中，本发明利用四氯化碳诱导肝脏损伤构建小鼠肝纤维化模型，通过灌胃不同剂量的IRI，评估其对肝纤维化的治疗效果。经研究发现，IRI可以抑制肝星状细胞活化，下调肝纤维化相关基因和肝星状细胞活化标志物表达，减少肝脏中胶原纤维沉积。
本发明的有益效果：
本发明首次发现野鸢尾黄素具有预防或治疗肝纤维化的作用。具体来说，首先利用人肝星状细胞LX-2作为模式细胞，发现野鸢尾黄素对LX-2细胞肝纤维化相关基因COL1A1、COL3A1在转录水平下调，肝星状细胞活化标志物α-SMA蛋白表达降低，及纤维化肝脏主要胶原纤维COL1的蛋白表达降低，且具有剂量依赖性。同时对CCl4诱导的肝纤维化小鼠模型进行药物治疗，以评价野鸢尾黄素对肝纤维化的治疗作用。通过对肝脏组织形态学观察，我们发现野鸢尾黄素能缓解肝纤维化小鼠肝脏结节生成。通过组织病理学切片检测发现，与模型组相比，野鸢尾黄素能显著改善肝纤维化小鼠肝脏的损伤、炎性浸润和胶原沉积，抑制肝星状细胞活化标志物α-SMA蛋白表达。通过对肝脏羟脯氨酸和Ⅰ型胶原蛋白含量的测定，证实野鸢尾黄素具有改善肝脏纤维化的作用。在分子水平上，我们发现野鸢尾黄素能显著降低肝纤维化模型小鼠肝纤维化相关基因mRNA和蛋白表达水平，对肝纤维化具有一定的治疗作用。
附图说明
图1为IRI对LX-2细胞的安全性评价。用CCK-8试剂盒检测不同浓度(A)、不同处理时间IRI对LX-2细胞(B)的毒性作用。
图2为IRI对LX-2细胞肝纤维化相关基因表达的影响。A.Western Blot分析不同浓度IRI 治疗对LX-2细胞活化和肝纤维化相关基因α-SMA和COL1A1的蛋白表达水平；B. RT-qPCR分析不同浓度IRI治疗对LX-2细胞肝纤维化相关基因COL1A1、COL3A1的mRNA 表达水平；C.不同浓度IRI治疗对LX-2细胞活化标志物α-SMA免疫荧光染色结果。
图3为CCl4造模和IRI治疗期间小鼠体重变化情况。
图4为对照组(Control，CTL)、不同剂量IRI治疗组、SIL阳性药治疗组的小鼠肝组织形态学观察。
图5为CTL组、不同剂量IRI治疗组、SIL阳性药治疗组的小鼠肝组织H&E、天狼星红、Masson、α-SMA免疫组化染色。
图6为IRI对CCl4诱导的肝纤维化小鼠肝脏胶原纤维沉积的影响。A-B对照组、模型组、不同剂量IRI治疗组、SIL阳性药治疗组的肝脏羟脯氨酸(Hydroxyproline)和Ⅰ型胶原(collagenⅠ)含量。
图7为IRI对CCl4诱导的肝纤维化小鼠相关基因表达的影响。A.CTL组、不同剂量IRI 治疗组、SIL阳性药治疗组的肝纤维化相关基因α-SMA、Col1a1的蛋白表达；B.CTL组、不同剂量IRI治疗组、SIL阳性药治疗组的肝纤维化相关基因Col1a1、Col3a1的mRNA表达水平。
图8为RNA测序生物信息学分析结果。A.RNA测序显著变化的差异基因火山图；B.节点基因的蛋白互作分析；C.GO分析；D.KEGG分析；P<0.05为差异有统计学意义。
图9为LX-2细胞中验证RNA测序的结果。
图10为小鼠肝脏组织中验证RNA测序的结果。
图11为靶点预测结果。A.VENN图；B.13个交集基因名称.
图12为靶点预测结果分析。*P<0.05和**P<0.01vs.CTL组，#P<0.05和##P<0.01vs.CCl4组，*P<0.05和**P<0.01vs.IRI 0μM组。
具体实施方式
下面结合实施例对本发明做进一步说明，但本发明的保护范围不限于此：
实施例1野鸢尾黄素在抑制肝星状细胞活化中的应用
1.对LX-2细胞的药物处理
LX-2细胞购自中南大学湘雅医学院细胞库。IRI购自成都瑞芬思生物科技有限公司，为纯度100％的白色粉末。SIL购自成都瑞芬思生物科技有限公司，为纯度99.46％的白色粉末。LX-2细胞培养于37℃，体积百分数为5％CO2，体积百分数为10％FBS DMEM培养基条件下，分别加入不同浓度的IRI作为药物治疗组，另外设置不含药物的LX-2细胞作为对照组，在药物处理24h后收集细胞样品。
2.CCK-8
CCK-8试剂盒购自南京建成生物工程研究所。为了评估IRI的安全性，在本研究中选取LX-2细胞作为模型，将LX-2细胞以1×104/孔的密度接种到96孔板中，培养24h。配制含有不同浓度IRI(0、1、2.5、5、10、20、50、100μM)的完全培养基，然后将上述配制的培养基加入96孔板中，继续孵育24h。孵育结束后，通过CCK-8试剂盒检测细胞活力。或配制含有100μMIRI的完全培养基，将培养基加入96孔板中，继续孵育0、6、 12、24、36、48h，孵育结束后，通过CCK-8试剂盒检测细胞活力。并根据以下公式计算细胞存活率：
细胞存活率＝(A1-A0)/(A2-A0)
A1：实验组吸光值；A2：对照组吸光值；A0：空白组吸光值。
3.实时荧光定量聚合酶链式反应检测(RT-qPCR)
使用TRIzol裂解细胞，提取总RNA。使用Takara反转录试剂盒进行反转录。根据Primer 3软件设计目的基因引物，然后通过北京擎科生物科技有限公司合成相应引物。根据南京诺唯赞公司的Vazyme试剂盒的说明书制备反应体系，然后在qPCR仪上进行扩增反应。根据Ct值计算出目的基因的相对表达量。
4.Western Blot
药物处理LX-2细胞后，6孔板细胞每孔加100μL裂解液，充分裂解细胞后，于4℃，13000rpm离心10min，收集上清。使用BCA测定试剂盒测定蛋白浓度。调整蛋白浓度后，与6×Sample buffer混匀，置于100℃条件下加热7min，使蛋白质彻底变性，最后将提取好的蛋白样品放在-80℃冰箱中保存备用。使用质量百分数为10％或8％SDS-PAGE凝胶电泳，将目的条带转至PVDF膜上，在质量百分数为5％的脱脂牛奶中室温封闭1h。4℃孵育一抗过夜。孵育结束后，PBST清洗3次，每次10min。二抗在室温条件下孵育1h 后，PBST清洗3次，每次10min。将PVDF膜浸泡在显色液中，通过Tanon5200化学发光成像系统观察目的蛋白条带。
5.免疫荧光
将LX-2细胞接种到直径44mm的激光共聚焦皿中，待细胞培养24h贴壁后，分别加入0、10、50和100μM IRI处理24h。弃去培养基并用PBS轻轻摇晃清洗3次，避免细胞脱落。加入质量百分数为4％多聚甲醛室温固定30min，之后使用PBS用同样方法清洗 3次。加入体积百分数为0.5％TritonX-100的PBS溶液室温条件下孵育15min，接着用PBS 清洗3次，然后加入10％山羊血清在室温条件下封闭1h。之后使用α-SMA一抗4℃孵育过夜。孵育结束后，PBST清洗3次，每次10min。然后用山羊抗兔IgG H&L(Alexa
555)孵育1h。PBST清洗3次，每次10min。用DAPI对细胞核染色，最后在LSCM下观察荧光并拍照。
6.实验结果
6.1野鸢尾黄素在细胞水平上的安全性评价
如图1A所示，IRI处理LX-2细胞24h在100μM以内对细胞活力无显著改变。并且，如图1B所示，100μM IRI处理LX-2细胞在48h内对细胞活力也无显著影响。因此本研究使用的IRI剂量对LX-2细胞是安全的。
6.2野鸢尾黄素抑制肝星状细胞纤维化相关基因的表达
为了研究IRI对HSCs活化的影响。如图2A-C所示，与对照组相比，IRI显著抑制 LX-2细胞肝纤维化相关基因COL1A1、COL3A1的mRNA表达和α-SMA、COL1A1的蛋白表达，且呈剂量依赖性。与Western blot结果一致，免疫荧光染色结果显示随着IRI剂量增加，α-SMA荧光强度减弱，HSCs活化被抑制。
实施例2野鸢尾黄素在治疗CCl4诱导的小鼠肝纤维化中的应用
1.实验动物与试剂
选用实验动物为8周龄雄性C57BL/6J小鼠(购自江苏集萃药康生物科技股份有限公司)，按照小鼠标准饲养流程(美国国立卫生研究院和中国药科大学实验动物饲养及使用指导章程)进行饲养。所有小鼠均置于12小时光照/12小时黑暗、温度及湿度恒定的标准环境下饲养，待小鼠适应环境后，进行后续相关实验的操作。所有实验均已被批准并按照中国药科大学实验动物管理委员会的指导进行(许可证号SYXK-2021-0011)。四氯化碳购买自Sigma公司。
2.CCl4诱导小鼠肝纤维化模型构建及给药方案
为构建肝纤维化模型，实验中选取置于标准环境下饲养的8周龄雄性C57BL/6J小鼠，将所选小鼠随机分成六组。模型组每周按2mL/kg腹腔注射体积百分数为20％CCl4(橄榄油稀释)，对照组按2ml/kg腹腔注射橄榄油，一周两次，共8周。造模四周之后，给予IRI 或SIL药物治疗四周。对照组小鼠每天灌胃10mL/kg的助溶剂即质量百分数为0.5％羧甲基纤维素钠溶液。模型组每天灌胃10mL/kg的助溶剂，IRI低剂量治疗组小鼠每天灌胃 1mg/kgIRI溶液，IRI中剂量治疗组小鼠每天灌胃5mg/kg IRI溶液，IRI高剂量治疗组小鼠每天灌胃10mg/kg IRI溶液，SIL治疗组小鼠每天灌胃100mg/kgSIL溶液。
3.肝脏病理学检测
小鼠肝脏组织在4％多聚甲醛中固定过夜后，用石蜡包埋，切成5μm左右厚的切片脱蜡后，分别进行H&E、天狼星红、Masson、α-SMA免疫组化染色，在显微镜下观察病理学改变并进行图像采集。
4.肝脏羟脯氨酸含量测定
为了评价肝纤维化小鼠肝脏中胶原纤维的沉积，测定胶原蛋白特有的羟脯氨酸作为反应胶原蛋白含量的指标，具体步骤详见南京建成生物工程研究所的羟脯氨酸测定试剂盒(碱水解法)说明书。
5.肝脏Ⅰ型胶原蛋白含量测定
为了检测肝脏Ⅰ型胶原蛋白含量，使用泉州市睿信生物科技有限公司的小鼠Ⅰ型胶原 (ColⅠ)定量检测试剂盒。
6.实验结果
6.1野鸢尾黄素改善CCl4诱导的小鼠肝纤维化
如图3所示，IRI或SIL灌胃给药对小鼠体重无显著改变。如图4所示，CCl4注射后肝脏形态明显改变，给药治疗后，随着IRI药物剂量增多，肝脏损伤得到一定改善，肝脏结节变少，高剂量IRI与SIL阳性药相当。如图5所示H&E染色，对照组小鼠肝小叶结构完整，未见病理变化。与CCl4模型组小鼠相比，随着IRI给药剂量增多，小鼠肝细胞坏死和空泡化逐渐减少，炎性细胞浸润减少。SIL也能改善上述过程。天狼星红和Masson 染色结果显示模型组小鼠肝门静脉和中央静脉周围有明显的胶原沉积，在IRI和SIL治疗后，胶原沉积明显减少。为了研究肝纤维化小鼠HSCs的活化情况，我们通过免疫组化检查了HSCs活化标志物α-SMA蛋白表达情况，如图3所示α-SMA染色，模型组小鼠肝脏α-SMA阳性细胞明显增多，IRI给药后α-SMA阳性细胞显著减少，且存在剂量依赖性。 SIL给药也能减少肝脏α-SMA表达。
6.2野鸢尾黄素降低CCl4诱导的肝纤维化模型小鼠肝脏胶原纤维沉积
图6A所示，模型组小鼠肝脏胶原沉积增加，羟脯氨酸含量增加。IRI中剂量和高剂量治疗组羟脯氨酸含量显著减少，因此肝脏胶原沉积减少且与SIL治疗组相当。IRI低剂量治疗组与模型组相比无显著差异。如图6B所示，与模型组相比，IRI高剂量治疗组和 SIL治疗组小鼠肝脏Ⅰ型胶原蛋白含量明显减少，且IRI高剂量治疗组Ⅰ型胶原蛋白含量低于SIL治疗组。IRI低剂量、中剂量治疗组与模型组相比无显著差异。
6.3野鸢尾黄素下调CCl4诱导的肝纤维化模型小鼠肝纤维化相关基因表达
如图7A-B所示，与对照组相比，模型组α-SMA、Col1a1蛋白表达量显著升高，Col1a1和Col3a1的mRNA表达量显著升高，与模型组相比，IRI中剂量和高剂量治疗后显著抑制α-SMA、Col1a1蛋白以及Col1a1、Col3a1的mRNA表达，而且具有剂量依赖性，而 IRI低剂量治疗后能抑制α-SMA蛋白表达以及Col1a1、Col3a1的mRNA表达，而对Col1a1 蛋白表达水平无显著改变。SIL显著抑制α-SMA、Col1a1蛋白表达和Col3a1的mRNA 表达，而对Col1a1的mRNA表达无显著影响。
实施例3野鸢尾黄素调控肝星状细胞活化中的分子靶点预测
1.高通量RNA测序
为了研究活化的HSCs中，响应IRI给药信号调控的基因，我们收集了LX-2细胞 100μM和0μM IRI处理细胞样本，提取总RNA，并构建RNA-seq文库。使用Agilent2100 生物分析仪对RNA文库的质量进行评估。将构建好的RNA-seq文库在Illumina Novaseq6000平台上进行测序。然后，通过DESeq2(具有生物重复)或edgeR(无生物重复)R软件包，对LX-2细胞转录本差异性表达进行分析，当P<0.05和|Log2(Fold change)| ＞1则被认为具有统计学差异。在本研究中样本的高通量RNA测序工作委托广州基迪奥生物科技有限公司完成。
2.实验结果
为了研究活化的HSCs中，响应IRI给药信号调控的基因，我们收集了LX-2细胞 100μM和0μM IRI处理细胞样本，进行了高通量RNA测序。差异分析显示，IRI处理LX-2 细胞后，有249个基因改变，其中42个基因表达上调，207个基因下调(图8A)。根据生物信息学分析，GO(基因本体论，Gene Ontology)主要富集在细胞外基质、细胞外组织结构，以及白细胞趋化、髓系白细胞迁移、趋化调控、γ干扰素应答、粒细胞迁移等炎症过程(图8C)。KEGG结果显示也与预期表型符合(图8D)。为了对RNA测序结果的差异基因进行蛋白质相互作用(protein-protein interaction，PPI)分析，我们利用STRING 数据库(https://www.string-db.org)和可视化软件对PPI网络进行分析，如图8B所示为排名前10位的节点基因。为了避免高通量测序Ⅰ类错误假阳性的出现，我们对排名前5位的节点进行验证，如图9，通过独立的RT-qPCR分析，我们发现LX-2细胞IL-6、CXCL8、 MMP9、VCAM1、CXCL1基因表达水平的变化趋势与RNA测序结果相一致。如图10A-B 所示，小鼠肝脏Mmp9、Vcam1基因表达水平的变化趋势与高通量RNA测序结果一致。
为了寻找IRI的作用靶点，我们通过SwissTargetPrediction和Bindingdb网站(http://www.swisstargetprediction.ch/；http://www.bindingdb.org/)，对IRI可能的靶点进行了预测，并对预测结果进行VENN聚类分析，得到13个相关蛋白(图11A-B)。如图12A所示，将13个蛋白与RNA测序结果的差异基因进行PPI分析，得到EGFR处于互作前 20位的节点，与差异基因具有强相关性。接下来对已经有研究报道的IRI的靶点FN1与 RNA测序结果的差异基因进行PPI分析(图12B)，提示了IRI处理HSCs后，FN1与IRI 给药调控的过程同样具有强相关性。因此，EGFR和FN1可能是IRI调控HSCs活化的潜在作用靶点。
Claims (6)
1.野鸢尾黄素在制备治疗肝纤维化或肝纤维化相关疾病药物中的应用。
2.野鸢尾黄素在制备抑制肝星状细胞的活化的抑制剂中的应用。
3.野鸢尾黄素在制备抑制细胞外基质生成的抑制剂中的应用。
4.根据权利要求1所述的应用，其特征在于，所述野鸢尾黄素在调控肝星状细胞活化中的分子靶点预测方面的应用。
5.根据权利要求2所述的应用，其特征在于，所述肝纤维化相关疾病包括病毒性肝炎、酒精性肝病、非酒精性脂肪性肝病、自身免疫性肝病、胆汁淤积性肝病、中毒和药物性肝病。
6.根据权利要求1所述的应用，其特征在于，所述野鸢尾黄素的给药剂型包括口服制剂和注射制剂。
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| XIAONAN SU 等: "FN-EDA mediates angiogenesis of hepatic fibrosis via integrin-VEGFR2 in a CD63 synergetic manner", 《CELL DEATH DISCOVERY》 * |
| 苏骁男: "纤维连接蛋白额外结构域A促进肝纤维化及血管异常增生的作用与机制研究", 《中国优秀博硕士学位论文全文数据库(博士) 医药卫生科技辑》 * |
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