CN115487238A - 一种中药组合物在制备预防或治疗肝纤维化药物中的应用 - Google Patents
一种中药组合物在制备预防或治疗肝纤维化药物中的应用 Download PDFInfo
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Abstract
本发明属于中药医药技术领域,具体涉及一种中药组合物在制备预防和/或治疗肝纤维化药物中的医药用途。所述中药组合物主要由羌活、独活、茯苓、防风、荆芥、川芎、桔梗、柴胡、前胡、枳壳、甘草制备而成。本发明中药组合物能够对肝组织中TGF‑β、Smad4及α‑SMA的蛋白表达水平较模型组显著降低,表明荆防败毒散可能干预TGF‑β/Smad4信号通路,从而有效减轻肝纤维化的程度,对于肝纤维化具有显著的治疗作用。
Description
技术领域
本发明属于中药医药技术领域,具体涉及一种中药组合物在制备预防和/或治疗肝纤维化药物中的医药用途。
背景技术
肝纤维化是由于各种因素导致的慢性肝损伤引起的反应,例如饮酒、非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)、病毒性肝炎、自身免疫性肝炎、非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)和胆汁淤积性肝病。这些因素对肝脏的共同作用是产生慢性炎症,导致异常的伤口愈合反应,它会产生和沉积包括胶原纤维在内的细胞外基质(extracellular matrix,ECM)蛋白,从而导致组织瘢痕形成。肝脏通常在肝损伤后再生,然而,当肝损伤和炎症持续和进展时,肝脏不能正常再生并导致纤维化。进行性肝纤维化会导致肝硬化,最终导致肝细胞癌(hepatocellular carcinoma,HCC)。然而,肝纤维化是一个可逆的过程,延缓、减轻或逆转肝纤维化已成为预防肝硬化、肝癌的重要方法。
目前,西医尚无有效治疗肝纤维化的药物获批上市,现有治疗肝纤维化的药物如干扰素、皮质类固醇等存在药物长期服用后毒性大、不良反应多等问题,因此,越来越的研究者将目光投向中医药的开发和利用上,以期寻找安全、有效抗肝纤维化的药物。
荆防败毒散是由人参败毒散去人参加荆芥、防风而成。人参败毒散最早出自宋代官方成药医籍《太平惠民和剂局方》,药物组成为柴胡、甘草、桔梗、人参、川芎、茯苓、枳壳、前胡、羌活、独活,长于治疗疫病。1550年,明代张时彻《摄生众妙方》亦载有荆防败毒散,即人参败毒散去人参,加荆芥、防风,即荆防败毒散组方为荆芥、防风、羌活、独活、柴胡、前胡、川芎、枳壳、茯苓、桔梗、甘草,后世所用的荆防败毒散多遵《摄生众妙方》而不用人参。
荆防颗粒是荆防败毒散的中成药制剂。荆防(冲剂)颗粒是《卫生部药品标准》中药成方制剂第二册收载的品种,标准编号为WS3-B-0328-90。王乖娟等(王乖娟,刘卫兵,荆鲁华.荆防颗粒治疗血虚风燥型皮肤瘙瘁症临床观察[J].中国中西医结合皮肤性病学杂志,2009,8(05):315)自拟荆防颗粒治疗血虚风燥型皮肤瘙痒症,实验发现荆防颗粒治疗血虚风燥型皮肤瘙痒症远期临床疗效满意。谢舜辉等(谢舜辉,郑慕雄,陈昌鹏,陈玉兴.荆防颗粒对大鼠I型超敏反应及肥大细胞释放组胺的影响[J].皮肤性病诊疗学杂,2010,17(01):21-23)研究发现荆防颗粒能明显抑制大鼠I型超敏反应和肥大细胞释放组胺。专利CN202011009484.8公开了荆防制剂在制备胃溃疡药物中的应用。专利CN201410174688.5公开了一种荆防颗粒的检测方法。
迄今为止,未见有关荆防制剂治疗肝纤维化生物活性或临床应用方面的研究。
发明内容
本发明提供了一种预防或治疗肝纤维化作用的中药组合物,所述的中药组合物主要由羌活、独活、茯苓、防风、荆芥、川芎、桔梗、柴胡、前胡、枳壳、甘草制备而成。
荆芥、防风辛温解表,祛风散寒为君药羌活、独活祛风除湿止痛,且助荆芥、防风解表之力;川芎祛血中之风而宣痹止痛,共为臣药。柴胡辛凉透表,可疏散肌表郁热,并与前胡、桔梗、枳壳共用有升有降,共起宣肺化痰、开胸利气之功效;茯苓甘淡而平,可健脾渗湿,此4味共为佐药。甘草为使,调和诸药。诸药合用,共奏发汗解表、祛风散寒除湿之功。它具有发汗解表,散风祛湿的作用。临床上用于治疗感冒风寒,头痛身痛,恶寒无汗,鼻塞流涕,咳嗽。是目前市场上用于治疗感冒风寒,头痛身痛,恶寒无汗,鼻塞流涕,咳嗽的常用药品。
本发明目的之一在于,提供上述中药组合物的组成,即所述中药组合物主要由以下原料制备而成:
进一步地,所述中药组合物主要由以下原料制备而成:
进一步地,所述中药组合物主要由以下原料制备而成:
本发明目的之二在于提供上述中药组合物在制备预防或治疗肝纤维化药物中的用途。本发明实施例显示本发明中药组合物能够对肝组织中TGF-β、Smad4及α-SMA的蛋白表达水平较模型组显著降低,表明荆防败毒散可能干预TGF-β/Smad4信号通路,从而有效减轻肝纤维化的程度,对于肝纤维化具有显著的治疗作用。
本发明中药组合物在制备预防或治疗肝纤维化药物中的用途。
进一步地,所述肝纤维化为炎症导致的肝纤维化。
进一步地,所述炎症导致的肝纤维化包括但不限于自身免疫性疾病,代谢紊乱,药物或毒素诱导的疾病,慢性胆汁淤积性疾病和遗传性疾病引起的肝纤维化。
本发明目的之三在于提供上述中药组合物的制备方法,所述制备方法主要包括以下步骤:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成10-30%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮,煎煮液浓缩后备用;
步骤E:将步骤C所得渗漉液、步骤D所得煎煮液混合,浓缩,加入步骤A所得挥发油,即得。
优选的,所述制备方法主要包括以下步骤:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成20%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮,煎煮液浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,浓缩成清膏,加入步骤A所得挥发油,即得。
本发明目的之四在于提供含有上述中药组合物的中药制剂,所述的中药制剂为临床上可接受的口服制剂;
优选的,所述临床上可接受的口服制剂为丸剂、胶囊剂、片剂、颗粒剂或液体口服制剂中的一种或多种;
进一步更优选的,所述口服制剂为颗粒剂。
与现有技术相比,本发明的优势在于:
本发明中,CCL4诱导的肝纤维化模型小鼠血清中ALT、AST、TBA和TG的含量显著升高,表明小鼠肝脏严重受损,给予荆防败毒散干预后,小鼠血清中ALT、AST、TBA和TG的含量显著降低,表明荆防败毒散能明显改善肝脏受损程度,对CCL4诱导的肝纤维化有显著的治疗作用,此结果与肝脏组织病理学结果观察一致。
本发明中,荆防败毒散对小鼠肝组织中TGF-β、Smad4及α-SMA的蛋白表达水平较模型组显著降低,表明荆防败毒散可能干预TGF-β/Smad4信号通路,从而有效减轻肝纤维化的程度。
附图说明
图1为各组小鼠肝纤维化肝脏形态及病理学组织切片图(×200)
图2为各组小鼠血清中肝功指标水平的比较(x±s,n=7)
图3为各组小鼠血清中TNF-α、IL-6、IL-1β含量水平的比较(x±s,n=7)
图4为各组小鼠肝组织匀浆中SOD、MDA、GSH含量水平的比较(x±s,n=7)
图5为各组小鼠肝脏中TGF-β、α-SMA、Smad4蛋白表达(x±s,n=3)
图6为各组小鼠肝脏中TGF-β、α-SMA、Smad4蛋白表达的电泳图
具体实施方式
实施例
实施例1颗粒剂制备
处方:
制备方法:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成20%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,制成颗粒,干燥,加入步骤A所得挥发油,混匀,即得。
实施例2颗粒剂制备
处方:
制备方法同实施例1。
实施例3颗粒剂制备
处方:
制备方法同实施例1。
实施例4糖浆剂制备
处方:
制备方法:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成30%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,加入步骤A所得挥发油和单糖浆500ml,混匀,静置,滤过,加水至1000ml,即得。
实施例5口服液制备
处方:
制备方法:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成10%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,加入步骤A所得挥发油,混匀,加水至1000ml,即得。
实施例6胶囊剂制备
处方:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成30%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,制成颗粒,干燥,加入步骤A所得挥发油,混匀,制成颗粒,干燥,粉碎,装入胶囊,即得。
实施例7丸剂制备
处方:
制备方法:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成20%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,制成颗粒,干燥,加入步骤A所得挥发油,混匀,干燥,粉碎,过筛,加炼蜜30-50g,适量的水泛丸,干燥,即得。
实施例8片剂制备
处方:
制备方法:
步骤A:将荆芥、防风、羌活、独活、前胡、川芎、枳壳,分别蒸馏提取挥发油备用,蒸馏后的药渣、蒸馏后的川芎、枳壳水溶液备用;
步骤B:将步骤A所得蒸馏后的川芎、枳壳水溶液配制成15%的乙醇溶液,备用;
步骤C:将茯苓、步骤A所得蒸馏后的川芎、枳壳药渣混合,以步骤B所得乙醇溶液渗漉提取,渗漉液备用;
步骤D:将柴胡、桔梗、甘草、步骤A所得蒸馏后的荆芥、防风、羌活、独活、前胡药渣,加水煎煮两次,每次2小时,合并两次的煎煮液,过滤后浓缩成稠膏,备用;
步骤E:将步骤C所得渗漉液、步骤D所得稠膏混合,静置,过滤后浓缩成清膏,加蔗糖适量,混匀,制成颗粒,干燥,加入步骤A所得挥发油,混匀,制成颗粒,加入辅料适量,混匀,压片,即得。
实施例9本发明中药组合物对小鼠肝纤维化的治疗作用及其机制的探究
1材料
1.1仪器AU480全自动生化分析仪(美国贝克曼库尔特有限公司);Leica RM 2016石蜡切片机(美国莱卡公司);Nikon Eclipse Ti-SR倒置荧光显微镜(日本尼康);HI650离心机(湖南湘仪实验室仪器开发有限公司);DYY-7C电泳仪电源(北京六一仪器厂);DYCZ-24DN垂直电泳槽(北京六一仪器厂);DYCZ-40电转仪(北京六一仪器厂);TS-1水平摇床(江苏海门其林贝尔仪器制造有限公司);CPA电子天平(北京赛多利斯仪器系统有限公司);mμlISKANMK3酶标仪(美国Thermo公司)。
1.2药品与试剂由实施例1制备得到的荆防败毒散由荆芥、防风、羌活、独活、柴胡、前胡、枳壳、茯苓、桔梗、川芎各4.5g,炙甘草1.5g,制备成颗粒剂,1g颗粒剂相当于原药材1.08234g。水飞蓟宾胶囊购于天津天士力圣特制药有限公司,规格为35mg,批号文号:国药准字H20040299;四氯化碳(CCL4)溶液购于北京伊诺凯科技有限公司;超氧化物歧化酶(superoxide dismutase,SOD)测定试剂盒(批号:20211104)、丙二醛(malondialdehyde,MDA)测定试剂盒(批号:20211028)、谷胱甘肽(glutathione,GSH)测定试剂盒(批号:20211029)均购于南京建成生物工程研究所;肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)试剂盒(批号:L211005197)、白介素-6(interleukin-6,IL-6)试剂盒(批号:L211001917)、白介素-1β(interleukin-1β,IL-1β)试剂盒(批号:L210910073)均购于武汉云克隆科技股份有限公司;RIPA裂解液购于上海碧云天生物技术有限公司;二喹啉甲酸(bicinchoninic acid,BCA)蛋白浓度测定试剂盒购于上海碧云天生物技术有限公司;鼠单抗α-平滑肌肌动蛋白(α-smooth muscle actin,a-SMA)购于美国Cell SignalingTehnology公司;兔多抗Smad4购于武汉三鹰生物技术有限公司;鼠单抗转化生长因子-β(transforming growth factor-β,TGF-β)购于美国Santa Cruz公司;甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)购于杭州贤至生物有限公司;辣根过氧化物酶(horseradish peroxidase,HRP)标记羊抗小鼠、兔二抗均购于武汉博士德生物工程有限公司;增强化学发光法(enhanced chemiluminescence,ECL)底物液购于北京普利莱基因技术有限公司;显影定影试剂盒购于天津市汉中摄影材料厂;4%多聚甲醛溶液(批号:CR2106123)购于武汉赛维尔生物科技有限公司;生理盐水(批号:2107262011)购于石家庄四药有限公司,去离子水由北京中医药大学科研综合楼提供。
1.3动物健康SPF级8周龄雄性C57BL/6J小鼠49只,体质量(20±2)g,质量合格证编号为No.110324211103471337,实验动物均购于斯贝福(北京)生物技术有限公司,许可证号为SCXK(京)2019-0010。实验期间动物均饲养于同一环境下,保持室温(24.0±1.0)℃,空气湿度55%~65%,环境安静,动物随意进食进水,明、暗12h循环饲养,适应性饲养1周。本实验相关动物实验遵循北京中医药大学有关实验动物管理和使用的规定,整个实验过程均遵循3R原则,符合动物伦理学的规定。
2方法
2.1分组、造模与给药将49只8周龄雄性C57BL/6J小鼠随机分为7组,每组7只,分别为空白对照组、模型组(CCL4)、水飞蓟宾组(阳性对照,100mg/kg)[13]、荆防高剂量(16g/kg)组、荆防高剂量(16g/kg)+CCL4组、荆防中剂量(8g/kg)+CCL4组、荆防低剂量(4g/kg)+CCL4组。实验方法如下[14-16]:除空白对照组和荆防高剂量组外,其余各组小鼠腹腔注射10%CCL4橄榄油溶液(5mL/kg)诱导肝纤维化,每周2次,间隔3d,连续8周,空白对照组小鼠腹腔注射等体积的橄榄油溶液。同时,除空白对照组和模型组给予等量的生理盐水外,其余各组小鼠灌胃给予相应剂量的药物,每天1次,连续8周,灌胃体积为0.1mL/10g。
2.2样本的取材及处理末次给药后,禁食不禁水12h后摘取眼球取血,常温静置4h后置离心机中,3000r/min,离心10min,取上层血清,置于-80℃冰箱中保存以备用。将采血后的小鼠麻醉后颈椎脱臼,取出肝组织和脾组织,分别称重后,取肝大叶相同部位,用体积分数为4%的多聚甲醛溶液固定。剩余肝组织和脾组织分别装入冻存管后迅速投入液氮中放-80℃冰箱保存,备用。
2.3体质量、肝质量和脏器指数分析实验期间,每天记录小鼠体质量,分析小鼠体质量的变化情况。小鼠处死后,取出肝组织和脾组织,分别称取质量后,计算脏器指数(脏器指数=脏器质量/体质量×100%)。
2.4肝组织病理学观察取上述经4%多聚甲醛溶液固定的肝组织后,常规石蜡包埋,切片,厚5μm,分别进行苏木精一伊红(hematoxylin-eosin,HE)染色、Masson染色、天狼猩红染色,通过光学显微镜观察各组肝组织的结构变化和胶原纤维沉积情况,并拍照分析。
2.5血清肝功能指标的检测取上述冻存后的血清,解冻后用AU480全自动生化分析仪检测血清肝功能指标,包括丙氨酸转氨酶(alanine transaminase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、总胆汁酸(total bile acid,TBA)、甘油三酯(triglyceride,TG)。
2.6血清中TNF-α、IL-6、IL-1β含量检测取上述冻存后的血清,解冻后参照试剂盒操作说明测定小鼠血清中TNF-α、IL-6、IL-1β的含量。
2.7肝组织匀浆中SOD、MDA、GSH含量检测取上述冻存后的剩余肝组织,室温解冻后,取肝组织约0.5g,剪碎,在冰水浴中研磨制成10%肝组织匀浆液,离心后取上清液,检测各组小鼠肝组织中SOD、MDA、GSH的含量。具体操作按照相应试剂盒说明书进行。
2.8蛋白印迹法检测相关蛋白表达将上述冻存后的剩余肝组织取出,剪取少量肝组织并剪碎后置于2mL EP管中,加入清洗干净的钢珠。每管加入200μL RIPA裂解液(含2μL苯甲基磺酰氟、2μL磷酸酶抑制剂),置于自动匀浆机中匀浆。匀浆完成后将EP管置于冰上30min使其充分裂解。随后用移液器将裂解液移至1.5mL离心管中,利用离心机将其离心(12000rpm,5min)。离心完成后取上清分装于0.5mL离心管中并置于-20℃冰箱保存。采用BCA蛋白浓度测定试剂盒对肝组织蛋白样品进行定量分析。将上述获取的蛋白上清与5×蛋白上样缓冲液按体积比4∶1混合后,将其放入沸水浴中10min使蛋白变性。依次行十二烷基磺酸钠-聚丙烯酰胺(SDS-PAGE)凝胶电泳进行蛋白分离,转膜。用含5%脱脂奶粉的封闭液(TBST)浸泡PVDF膜,室温摇床封闭2h。TGF-β、α-SMA及Smad4蛋白一抗稀释后4℃孵育过夜,随后用TBST充分漂洗5次,每次5min,之后用TBST稀释相应的HRP标记二抗(1∶10000),将PVDF膜浸泡其中,室温摇床孵育2h。取出PVDF膜后用TBST充分漂洗5次,每次5min。采用GAPDH作为内参,按ECL试剂盒说明进行曝光显影,用BandScan分析胶片灰度值。
2.9统计学方法采用SPSS 25.0软件统计分析所有的实验数据结果。所有实验数据以均值±标准差()表示。多组间数据的比较当方差齐时,用单因素方差分析法,方差不齐时用非参数检验法进行分析。组间比较用LSD法处理。P<0.05为实验数据间差异有统计学意义。
3结果
3.1荆防败毒散对肝纤维化小鼠体质量的影响结果由表1所示,在第三周时,模型组小鼠体质量明显低于空白对照组、荆防中剂量+CCL4组,差异具有统计学意义(P<0.05或P<0.01)。随实验的进行,在第六周时,除上述两组外,模型组小鼠体质量显著低于水飞蓟宾组、荆防低剂量+CCL4组,差异具有统计学意义(P<0.05)。在第七周时,除上述各组外,模型组小鼠体质量显著低于荆防高剂量+CCL4组,差异具有统计学意义(P<0.05或P<0.01)。此外,一到八周小鼠体质量统计结果表明,荆防高剂量组小鼠体质量均显著低于空白对照组(P<0.05或P<0.01),表明荆防败毒散对正常小鼠体质量有一定的影响。
3.2荆防败毒散对肝纤维化小鼠肝质量和脏器指数的影响结果由表2所示,与空白对照组相比,模型组小鼠肝质量及肝指数和脾指数均显著升高(P<0.05或P<0.01)。与模型组相比,水飞蓟宾组、荆防高、中、低剂量+CCL4组小鼠肝质量及肝指数和脾指数均显著降低(P<0.05或P<0.01)。此外,与空白对照组相比,荆防高剂量组小鼠肝质量、肝指数及脾指数均无明显差异,表明荆防败毒散对正常小鼠肝质量、肝指数及脾指数均无明显影响。
注:与空白对照组相比,#P<0.05,##P<0.01;与模型组相比,*P<0.05,**P<0.01。
3.3荆防败毒散对肝纤维化小鼠肝脏形态及病理学组织的影响图1为各组小鼠肝脏形态及并病理学组织切片观察结果(×200)。空白对照组小鼠肝脏外观表面光滑,色泽红润(图1A)。模型组小鼠肝脏颜色暗沉,欠光泽,表面有明显的粗颗粒状物质,边缘钝化。水飞蓟宾组和荆防高、中、低+CCl4组表面虽仍有细小颗粒状物质但较模型组比均有不同程度的改善。HE切片结果显示(图1B),空白对照组小鼠肝组织结构正常,肝细胞无坏死、排列整齐,大小均匀,以中央静脉为中心呈放射状排列,细胞核位于细胞中央,圆整、边界清晰,肝小叶结构完整。模型组小鼠肝细胞排列紊乱,肝组织损伤严重,出现大量的脂肪变性及肝细胞坏死,明显的炎症细胞浸润。水飞蓟宾组和荆防高、中、低+CCl4组肝细胞变性、坏死及炎症细胞浸润的现象较模型组显著改善。Masson染色(图1C)、天狼猩红染色结果显示(图1D),空白对照组小鼠肝组织切片细胞结构完整,未见明显的胶原纤维产生。模型组小鼠肝组织切片中可明显观察到大量增生的胶原纤维,并有纤维架桥的形成。水飞蓟宾组和荆防高、中、低+CCl4组中虽仍有一定量的胶原纤维形成,但与模型组比较轻。此外,与空白对照组相比,荆防高剂量组小鼠肝组织外观形态及病理学组织切片观察结果未见明显异常,提示荆防败毒散对正常小鼠肝脏形态无明显影响。
注:(A)肝组织的外观形态;(B)H&E染色结果;(C)Masson染色结果;(D)天狼猩红染色结果。NC:空白对照组;Model:模型组;Silybin:水飞蓟宾组;JFH:荆防高剂量组;JFH+CCL4:荆防高剂量+CCl4组;JFM+CCL4:荆防中剂量+CCL4组;JFL+CCL4:荆防低剂量+CCL4组
3.4荆防败毒散对肝纤维化小鼠血清中肝功指标的影响结果如图2所示,与空白对照组相比,模型组小鼠肝功指标ALT、AST、TBA、TG含量水平均极显著性升高(P<0.01)。与模型组相比,水飞蓟宾组及荆防高、中、低+CCL4组均能不同程度的降低上述肝功指标的含量水平(P<0.05或P<0.01)。此外,与空白对照组相比,荆防高剂量组小鼠血清中ALT、AST、TBA、TG的含量水平未见显著变化,提示荆防败毒散对正常小鼠肝脏功能无明显影响。
注:(A)各组小鼠血清中ALT的水平;(B)各组小鼠血清中AST的水平;(c)各组小鼠血清中TBA的水平;(D)各组小鼠血清中TG的水平。NC:空白对照组;Model:模型组;Silybin:水飞蓟宾组;JFH:荆防高剂量组;JFH+CCL4:荆防高剂量+CCl4组;JFM+CCL4:荆防中剂量+CCL4组;JFL+CCL4:荆防低剂量+CCL4组。与空白对照组相比,#P<0.05,##P<0.01;与模型组相比,*P<0.05,**P<0.01。
3.5荆防败毒散对肝纤维化小鼠血清中TNF-α、IL-6、IL-1β含量水平的影响结果如图3所示,图3为各组小鼠血清中TNF-α、IL-6、IL-1β含量水平的比较(,n=7),与空白对照组相比,模型组小鼠TNF-α、IL-6、IL-1β的含量水平均极显著性升高(P<0.01)。与模型组相比,水飞蓟宾组及荆防高、中、低+CCL4组均可显著性降低血清中TNF-α、IL-6、IL-1β的含量水平(P<0.05或P<0.01)。此外,与空白对照组相比,荆防高剂量组小鼠血清中TNF-α、IL-6、IL-1B的含量水平未见显著变化,提示荆防败毒散对正常小鼠血清中TNF-α、IL-6、IL-1β的含量水平无明显影响。
注:(A)各组小鼠血清中TNF-α的水平;(B)各组小鼠血清中IL-6的水平;(C)各组小鼠血清中IL-1β的水平。NC:空白对照组;Model:模型组;Silybin:水飞蓟宾组;JFH:荆防高剂量组;JFH+CCL4:荆防高剂量+CCl4组;JFM+CCL4:荆防中剂量+CCL4组;JFL+CCL4:荆防低剂量+CCL4组。
与空白对照组相比,#P<0.05,##P<0.01;与模型组相比,*P<0.05,**P<0.01。
3.6荆防败毒散对肝纤维化小鼠肝组织匀浆中SOD、MDA、GSH含量水平的影响结果如图4所示,图4为各组小鼠肝组织匀浆中SOD、MDA、GSH含量水平的比较(x±s,n=7),与空白对照组相比,模型组小鼠肝匀浆中SOD、GSH的含量水平极显著性降低,MDA的含量水平极显著性升高(P<0.01)。与模型组相比,水飞蓟宾组及荆防高、中、低+CCL4组小鼠肝匀浆中SOD、GSH的含量水平均不同程度的升高,MDA的含量水平均不同程度的降低(P<0.05或P<0.01)。此外,与空白对照组相比,荆防高剂量组小鼠肝匀浆中SOD、MDA、GSH的含量水平未见显著变化,提示荆防败毒散对正常小鼠肝匀浆中SOD、MDA、GSH的含量水平无明显影响。
注:(A)各组小鼠肝匀浆SOD的水平;(B)各组小鼠肝匀浆MDA的水平;(C)各组小鼠肝匀浆GSH的水平。NC:空白对照组;Model:模型组;Silybin:水飞蓟宾组;JFH:荆防高剂量组;JFH+CCL4:荆防高剂量+CCl4组;JFM+CCL4:荆防中剂量+CcL4组;JFL+CCL4:荆防低剂量+CcL4组。
与空白对照组相比,#P<0.05,##P<0.01;与模型组相比,*P<0.05,**P<0.01。
3.7荆防败毒散对肝纤维化小鼠肝脏TGF-β、α-SMA、Smad4蛋白表达的影响结果如图5、图6所示,图5为各组小鼠肝脏中TGF-β、α-SMA、Smad4蛋白表达(,n=3),图6为各组小鼠肝脏中TGF-β、α-SMA、Smad4蛋白表达的电泳图,与空白对照组相比,模型组小鼠肝脏TGF-B、α-SMA、Smad4蛋白表达明显上调(P<0.01)。与模型组相比,荆防中剂量+CCL4组小鼠肝脏中TGF-β、α-SMA、Smad4蛋白表达明显下调(P<0.01)。
图5注:(A)TGF-β/GAPDH分析;(B)α-SMA/GAPDH分析;(C)Smad4/GAPDH分析。NC:空白对照组;Model:模型组;JFM+CCL4:荆防中剂量+CCL4组。
与空白对照组相比,#P<0.05,##P<0.01;与模型组相比,*P<0.05,**P<0.01。
图6注:A:空白对照组;B:模型组;C:荆防中剂量+CCL4组
在本发明中,通过H&E染色、Masson染色及天狼猩红染色对各组小鼠肝脏的组织病理学切片进行观察,结果显示,与空白对照组相比,模型组小鼠肝组织损伤严重,出现大量的脂肪变性及肝细胞坏死,明显的炎症细胞浸润,汇管区静脉和中央静脉周围均可见大量胶原纤维产生,并有纤维架桥形成,表明肝纤维化模型成功建立。给予荆防败毒散干预后,小鼠肝组织损伤较模型组明显减轻,肝细胞变性、坏死及炎性细胞浸润的现象明显改善,纤维化现象减轻,提示荆防败毒散对CCL4诱导的肝纤维化有明显的治疗作用。
在本发明中,CCL4诱导的肝纤维化模型小鼠血清中ALT、AST、TBA和TG的含量显著升高,表明小鼠肝脏严重受损,给予荆防败毒散干预后,小鼠血清中ALT、AST、TBA和TG的含量显著降低,表明荆防败毒散能明显改善肝脏受损程度,对CCL4诱导的肝纤维化有显著的治疗作用。此结果与肝脏组织病理学结果观察一致。
在本发明中,荆防败毒散组小鼠血清中TNF-α、IL-6及IL-1β的含量水平较模型组比显著降低,表明荆防败毒散能减轻炎症反应,改善肝纤维化的程度。
在本发明中,荆防败毒散组小鼠肝组织中SOD和GSH的含量水平较模型组比显著升高,MDA的含量水平较模型组比显著降低,说明荆防败毒散能提高机体的抗氧化能力,减轻肝纤维化中的氧化应激损伤。
HSC的激活和ECM的沉积是肝纤维化的主要特征。研究表明,TGF-β/Smads信号通路与HSC的激活和ECM的合成密切相关。TGF-β是一种有效的促纤维化细胞因子,通过调节下游通路(如Smad蛋白)参与激活HSC并增加胶原蛋白和ECM蛋白的合成。Smad4是Smad蛋白家族中的一个关键调节因子,它在TGF-β信号通路中充当介质并在肝纤维化的发生中发挥重要作用。α-SMA是HSC活化的重要指标,抑制α-SMA表达可以逆转肝纤维化。因此,本发明选取上述HSC活化通路上的三个关键节点检测荆防败毒散对TGF-β/Smads信号通路的影响[33,34]。结果表明,荆防败毒散对小鼠肝组织中TGF-β、Smad4及α-SMA的蛋白表达水平较模型组显著降低,表明荆防败毒散可能干预TGF-β/Smad4信号通路,从而有效减轻肝纤维化的程度。
Claims (10)
1.一种中药组合物的用途,其特征在于,所述中药组合物用于制备治疗肝纤维素药物中的应用。
2.如权利要求1所述的用途,其特征在于,所述肝纤维化为炎症导致的肝纤维化。
3.如权利要求2所述的用途,其特征在于,所述炎症导致的肝纤维化包括但不限于自身免疫性疾病,代谢紊乱,药物或毒素诱导的疾病,慢性胆汁淤积性疾病和遗传性疾病引起的肝纤维化。
4.如权利要求1所述的用途,其特征在于,所述中药组合物主要由羌活、独活、茯苓、防风、荆芥、川芎、桔梗、柴胡、前胡、枳壳、甘草制备而成。
8.一种中药制剂,其特征在于,所述中药制剂含有如权利要求1-7任一项所述的中药组合物。
9.如权利要求8所述的中药制剂,其特征在于,所述中药制剂为临床上可接受的口服制剂;优选的,临床上可接受的口服制剂为丸剂、胶囊剂、片剂、颗粒剂或液体口服制剂中的一种或多种;更优选的,所述口服制剂为颗粒剂,更优选的,所述颗粒剂为荆防颗粒。
10.如权利要求1所述的用途,其特征在于,所述中药组合物能够降低肝组织中TGF-β、Smad4及α-SMA的蛋白表达水平,能干预TGF-β/Smad4信号通路。
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