基于基因表达谱的肝纤维化治疗药物筛选及相关实验研究
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摘要
肝纤维化(hepatic/liver fibrosis,HF)是一种常见的病症,是指由各种致病因素引起的肝脏损伤和炎症,在修复过程中导致肝脏细胞外基质异常增多和过度沉积的病理过程。引起肝纤维化的因素很多,如病毒感染、乙醇、寄生虫、化学毒物等,有的也可能是多种致病因素共同作用的结果。肝纤维化具有地区差异性,在欧美国家以酒精性肝纤维化为主,在我国主要与病毒性肝炎有关,其中乙型肝炎病毒感染与肝纤维化有着非常密切的关系。肝纤维化治疗效果差,重则演变为肝硬化和肝癌,预后极不良。
目前,治疗肝纤维化的药物种类很多,主要是通过作用于肝纤维化发生的不同环节,达到抗肝纤维化的目的,包括针对肝星状细胞(hepatic stellate cell,HSC)的药物、针对细胞因子的药物、影响胶原合成及代谢的药物、保护和阻止肝损伤的药物以及抗肝纤维化的中医药等,其中中医药治疗肝纤维化有很多的优势。肝纤维化和肝硬化在中医理论中属正虚血瘀症的范畴,治疗以活血化瘀、益气养阴为主,兼以养血柔肝和滋补肝肾；中药治疗肝纤维化的作用是多环节和多靶点的：减轻肝细胞炎症坏死、改善肝脏血液循环、促进肝细胞再生、直接抑制HSC活化和增殖、减少胶原分泌、促进胶原降解和再吸收等。近年来,国内外大量实验和临床研究发现,中药特别是复方中药治疗肝纤维化的作用为多环节、多靶点的,与化学合成药往往仅作用在单一环节或靶点相比,有独特的优势,值得深入研究。
基因芯片是20世纪生命科学领域发展起来的一项重要技术平台,具有高通量和快速测量等优点。随着基因芯片技术的广泛应用以及生物信息学的飞速发展,肝纤维化发生和发展的许多分子事件得到更全面的揭示。实验研究及临床试验证实,针对单一靶基因的药物治疗疗效有限,而针对多种靶基因或靶蛋白的药物治疗可能会更有效。
本研究将利用基于基因表达谱的药物发现模式,通过Toppgene等生物信息学分析方法筛选肝纤维化治疗候选化合物或药物,并进行相关实验验证,为肝纤维化的综合治疗及提高疗效提供理论及实验依据,同时也为加速肝纤维化中药治疗药物发现进程、为中药“老药新用”及中药现代化提供依据。
本研究内容主要分为三个部分：
第一部分基于基因表达谱的肝纤维化生物信息学分析
目的：本研究利用基因芯片数据分析软件BRB-ArrayTools 3.7.1,对来自基因芯片公共数据库(Gene Expression Omnibus, GEO)的肝纤维化基因表达谱芯片数据进行挖掘,并进行生物信息学分析,探讨基于基因表达谱的途径筛选和发现新的肝纤维化治疗药物。
方法与结果：
1.从GEO获取肝纤维化基因表达谱数据集
从NCBI的GEO数据库获得肝纤维化基因表达谱数据集,即GSE11536。该数据集来源于法国ROUEN医药系,采用GPL5215芯片平台,共48个样本,包括8个轻度肝纤维化样本和8个重度肝纤维化样本,每个样本三个重复。
2.肝纤维化差异表达基因分析
为了获得肝纤维化差异基因表达谱,我们采用BRB软件对GSE11536数据集进行差异表达基因分析,获得差异表达基因297个,其中上调基因115个,下调基因182个(Fold change> 1.5)。
3.文献挖掘获得已知肝纤维化疾病基因
采用Genecards和FABLE在线工具分析,共获得已知肝纤维化相关基因663个。
4. Toppgene筛选肝纤维化相关基因
首先建立已知肝纤维化相关基因集文件,然后通过toppgene在线分析工具进行肝纤维化候选基因筛选,结果得到7个肝纤维化候选新基因,分别是：SERPING1、ETS2、TSG101、CDH2、LMO2、PAWR、MST1。
5.肝纤维化信号通路富集分析
为从生物学意义水平上发现一些与肝纤维化密切相关的信号通路,采用Toppgene对所获取的差异基因表达谱进行基因集富集分析,共富集到44条通路(P<0.05)。对44条富集通路进行分析,结果显示,28条富集通路与凝血有关,3条与血小板生成有关,4条与心肌梗塞有关,6条与脂代谢有关,2条涉及MAPK途径。
6.肝纤维化差异基因富集相关疾病分析
为了解与肝纤维化相关联的疾病,采用Toppgene对所获取的肝纤维化差异基因进行富集相关疾病分析,共富集到4种疾病(P<0.05),分别是肝炎、先天性纤维蛋白原缺乏血症、后天纤维蛋白原缺乏血症和高密度脂蛋白胆固醇血症,这提示肝纤维化可能与凝血障碍和血脂代谢异常有关。
7.肝纤维化候选药物分析
Toppgene还建立了疾病、基因与药物之间的联系,为了获得一些可以逆转或是改善肝纤维化基因表达谱的治疗药物,进行Toppgene分析,结果得到69种药物(P<0.001)。结合文献,对69种药物进行分析,结果显示,17种药物是性激素类药物,12种是凝血相关药物,11种是降血脂药,6种是降血糖药,9种是消炎止痛药,这提示性激素类、凝血相关药物和降血脂药物可能可以成为肝纤维化治疗药物或辅助治疗药物。
结论：基于基因表达谱的途径筛选和发现肝纤维化治疗相关药物是可行的,并筛选到一些激素类药物、凝血相关药物和降血脂药物可能可以成为肝纤维化候选治疗药物或辅助治疗药物。
第二部分肝纤维化的治疗药物Dioscorea panthaica筛选
目的：黄山药(Dioscorea panthaica Prain et Burkill)是一种十分重要的食药两用植物资源,中医常用于治疗炭疽、胃痛、风湿性心脏病和风湿性关节炎；黄山药水提取物还可以用于治疗鼠高胆固醇血症。以黄山药根茎提取物为主要原料制成的地奥心血康,是国家二类新药,年销售收入达六亿元,临床主要用于心血管疾病、降血脂、降血糖,其主要化学成分是薯蓣皂甙元,还可作为性激素等多种药物的原料药等。结合第一部分结果：激素类药物、凝血相关药物和降血脂药物可能可以成为肝纤维化候选治疗药物或辅助治疗药物,因此本部分采用生物信息学方法寻找Dioscorea panthaica抗肝纤维化的可能作用靶点,并进行深入分析确认Dioscorea panthaica具有抑制肝纤维化的作用,为下一步进行相关实验验证提供依据。
方法与结果：
1.文献挖掘Dioscorea panthaica相关靶基因
采用FABLE在线工具,结合FACTA文献分析,共获得Dioscorea panthaica相关靶基因1142个。
2. Dioscorea panthaica抗肝纤维化的可能作用靶点
采用venny分析工具,分析已知肝纤维化相关基因和Dioscorea panthaica相关靶基因的共同表达基因,结果表明,在663个已知肝纤维化相关基因中,存在Dioscorea panthaica的可能作用靶基因322个(占48.6%),这进一步提示Dioscorea panthaica有抑制肝纤维化的作用。
3.获得共同表达基因
为了解Dioscorea panthaica抑制肝纤维化的可能作用机制,结合肝纤维化差异基因集,采用venny分析工具进行分析,获得肝纤维化差异基因、已知肝纤维化相关基因和Dioscorea panthaica相关靶基因的共同表达基因14个,分别是：APOA1、APOB、F2、GC、NR3Cl、TF、VTN、C3、C5、KNG1、NCF1、PAH、STAT3、TGFBR1。
4.共同表达基因集通路富集分析
将14个共同表达基因进行通路富集分析,结果显示有11条生物信号通路可能参与Dioscorea panthaica抑制肝纤维化的分子机制(P<0.05),这些信号通路主要涉及局部急性炎症应答反应、补体凝血过程、活性肽诱导通路.FOXA2/FOXA3转录因子调控网络、脂蛋白代谢等信号通路。
结论：通过生物信息学分析预测,进一步提示Dioscorea panthaica有抑制肝纤维化的作用。
第三部分Dioscorea panthaica抑制肝纤维化的相关实验验证
目的：为验证前两部分的分析结果,本部分采用CCl4法造大鼠肝纤维化模型,探讨Dioscorea panthaica提取物抑制肝纤维化的效果。
方法与结果：
1.CCl4诱导大鼠肝纤维化模型的建立、分组及给药方法
健康雄性SD大鼠,36只,随机分为6组,每组6只：①正常对照组(n=6),给予橄榄油1mL/kg体重(2次/周)腹腔内注射,共8周；②模型诱导对照CCl4组(n=6),给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；③阳性对照秋水仙碱组(n=6),按0.1 mg/kg体重腹腔注射秋水仙碱,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；④DPE-50mg/kg组(n=6),按50 mg/kg体重灌胃Dioscorea panthaica提取物(DPE),每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；⑤DPE-100mg/kg组(n=6),按100mg/kg体重灌胃DPE,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；⑥DPE-200mg/kg组(n=6),按200mg/kg体重灌胃DPE,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8次。各组大鼠均于8周后处死,处死前禁食24h。处死时,大鼠用水合氯醛麻醉,迅速从心脏穿刺取血,分离血清,-80℃保存。平卧状态下剖腹,去除肝脏筋膜,取右叶相同的部分置于冰生理盐水中充分洗涤后,一部分用10%中性甲醛固定,3天内石蜡包埋,切片,以备组织病理检测；其余肝组织迅速置于液氮中保存。
2.DPE对血清ALT、AST含量的影响
血清AST和ALT是评价肝脏损伤常用的生化指标。取大鼠血液约3mL,置干燥玻璃管内,37℃温浴30min,4℃冰箱静置2h,3000g离心10min,取血清,-80℃保存。通过全自动生化分析仪测定各组大鼠血清ALT、AST含量。与正常对照组相比,肝纤维化模型对照组的血清ALT含量(P=0.000)和AST含量(P=0.000)显著提高,DPE处理组的血清ALT、AST含量呈剂量依赖性下降。
3.肝组织病理学改变及检测肝组织中羟脯氨酸水平
运用HE染色和Masson'染色对各组大鼠肝组织纤维化程度进行分析,结果显示,正常对照组大鼠的肝组织肝索结构清晰,肝细胞以中央静脉为中心呈放射状,肝组织内无脂肪空泡,仅汇管区和中央静脉有少许胶原纤维存在。模型对照组肝小叶结构破坏,纤维组织增生明显,将肝小叶分隔成大小不等的肝细胞团,肝细胞广泛变性坏死,汇管区扩大、胶原大量沉积。与模型对照组相比,治疗组肝细胞的变性坏死程度及肝小叶结构破坏程度降低,肝脏胶原纤维增生亦明显减轻。
在检测肝脏组织病理学改变的同时,通过检测肝组织中羟脯氨酸水平,对肝损伤纤维化程度进行定量分析。与正常对照组相比,模型对照组的羟脯氨酸含量显著提高,差异有统计学意义(P=0.000),DPE处理组的羟脯氨酸含量呈剂量依赖性下降。
4.DPE对肝组织抗氧化能力的影响
通过检测大鼠肝组织匀浆中的GSH和TBARS浓度,探讨DPE治疗对肝组织氧化状态的影响。GSH构成了拮抗自由基的第一道防线。与正常对照组GSH含量相比,肝纤维化模型对照组GSH含量显著降低(P=0.000)；与肝纤维化模型对照组GSH含量相比,阳性对照秋水仙碱组(P=0.000)和DPE处理组(P=0.000)肝组织GSH含量差异均有统计学意义。为检测肝组织氧化性能,结果显示,与正常对照组比较,肝纤维化模型对照组TBARS含量增加约3.2倍；DPE (200mg/Kg)处理组肝脏TBARS含量降至1.03±0.12μmol/g,表明肝组织抗氧化能力增强。
结论：经动物实验证实,Dioscorea panthaica提取物能显著降低CCl4诱导的大鼠肝纤维化。
Liver fibrosis (hepatic fibrosis, HF) is a disease of reversible deposition of extracellular matrix(ECM) induced by chronic liver injuried. Many things could cause liver fibrosis, such as viral infection, ethanol, parasites, chemical poisons, and so on. Some fibrosis patients may be the result from a variety of pathogenic factors. Liver fibrosis is regional differences. Alcoholic liver fibrosis is common in Europe and America, while liver fibrosis with hepatitis B virus infection is common in China. With a high incidence of liver diseases, Chinese government takes heavy burden in the treatment of liver fibrosis and cirrhosis. Liver fibrosis can eventually lead to hepatic cirrhosis or liver cancer with poor prognosis.
The gene-chip (bio-chip) is major technique in bioscience field in the 21 century, which has the outstanding of high-throughput and rapid detecting, etc. The analysis of expression profiling chip is useful for the reveal of the mechanism of liver fibrosis, the diagnosis and therapy of liver fibrosis. Bioinformatics is a newly rising subject which elucidates and reveals the biological meaning hided in much data set. Bioinformatics provids a new approach for the analysis of DNA microarray data,also become a new tool for the discovery of the drug and speed up the process of drug discovery.In the study, based on the mode of gene expression profiles-based drug discovery approach, we set out to discover agents for liver fibrosis with computational and system biology tool "Toppgene",which provided the theory and experiment proof for the combined therapy of liver fibrosis, and also accelerate the discovery of drugs for liver fibrosis.
The study includes the following three parts:
Part I gene expression profiling based approach identifies candidate therapeutic compounds for liver fibrosis
Objective:Bioinformatics provids a new approach for the analysis of DNA microarray data, also become a new tool for the discovery of the drug and speed up the process of drug discovery. In the study, we explore gene expression profiles-based approach for the screening candidate therapeutic compounds against liver fibrosis, and screen the candidate compounds for liver fibrosis.
Methods and Results:
1. Acquisition liver fibrosis public microarray data from GEO databank
Raw data (.Cel files) of a published microarray data (GSE11536) used in the study was obtained from Gene Expression Omnibus(GEO) website, Amonge gene datasets GSE 11536, containing 48 samples, from Faculty of Medicine and Pharmacy, France. The dataset were based on GPL5215 platform.
2. BRB analysis of liver fibrosis
The aim of this study is to compare mild and advanced liver fibrosis gene expression profiling and to identify novel markers of fibrosis progression. By transcriptome analysis with a cDNA array virtually covering every transcript in liver, we compared transcript levels in mild (F1 Metavir score) and advanced (F4 Metavir score) fibrosis. A stringent selection identified a list of 16 transcripts which completely separated the 2 groups of patients (8 F1 and 8 F4). Mild fibrosis (F1) vs advanced fibrosis (F4) were alone performed with the software packages BRB Array Tools. Finally, we obtained 297 differentially expression genes, amonge 115 up-regulated genes and 182 down-regulated genes (Fold change> 1.5)
3. Data mining of known gene expression profile in liver fibrosis
We obtained 663 known genes correlated with liver fibrosis by data mining tools genecards and FABLE.
4. The screen of candidate genes for liver fibrosis with Toppgene
The 297 differential expression genes were defined as"test gene set", and 663 genes correlated with liver fibrosis were regarded as"train gene set".7 candidate genes were screened out by Toppgene, such as SERPING1、ETS2、TSG101、CDH2、LMO2、PAWR、MST1, which had nearly no report in liver fibrosis.
5. The pathway enrichment analysis of liver fibrosis with Toppgene
The pathway enrichment analysis showed that there were 44 biological pathways related with liver fibrosis(P<0.05). We found out that blood coagulation signal pathway(28), platelet signal pathway(3), myocardial infarction pathway(4), lipid metabolic pathway(6), MAPK signal pathway(2) were very significance in the progression of liver fibrosis.
6. The related deseases enrichment analysis of liver fibrosis
The related deseases enrichment analysis showed that there were 4 deseases related with liver fibrosis (P<0.05), such as hepatitis, afibrinogenemia congenital, afibrinogenemia, HDL cholesterol.
7. The candidate therapeutic drugs enrichment analysis of liver fibrosis
Finally,69 candidate therapeutic compounds for liver fibrosis were screened by Toppgene analysis (P<0.001). We found out some candidate therapeutic compounds, such as sexual hormone drugs (17), blood clotting drugs(12), descendens blood fat drugs(11), hypoglycemic agent(6), acesodyne (9),which were very significance in treatment of liver fibrosis.
Conclusion:Our results demonstrated that gene expression profiles-based approach is perspective for screening the candidate therapeutic compounds for liver fibrosis, which accelerates the introduction of compounds into the clinic.
Part II Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis
Objective:Dioscorea panthaica is a herb commonly used in traditional Chinese medicine to treat anthrax, gastropathy, rheumatic heart disease, and rheumarthritis. The anti-hypercholesterolemia activity of an aqueous extract of saponins from Dioscorea panthaica was also demonstrated in rats. It is well known that the chemical composition of herbal medicines is very complex, and detailed information on the bioactive compounds of Dioscorea panthaica remains largely incomplete despite several phytochemical studies on this medicinal plant. Currently, the effects of DPE on liver fibrosis have not been reported. In the study, the target genes which involved Dioscorea panthaica inhibits liver fibrosis were screened out by data mining tools, that further prompted Dioscorea panthaica could inhibit liver fibrosis.
Methods and Results:
1. Data minining of Dioscorea panthaica correlated genes
We obtained 1142 known genes correlated with Dioscorea panthaica by data mining tools genecards and FABLE.
2. Data mining of the target genes which involved Dioscorea panthaica inhibits liver fibrosis
The target genes which involved Dioscorea panthaica inhibits liver fibrosis were screened out by data mining tools. Among the 663 known liver fibrosis related genes, there were 322 potential target genes (48.6%) of Dioscorea panthaica, which further prompted Dioscorea panthaica could inhibit liver fibrosis.
3. co-expression genes of three gene sets were screened out by data mining tools venny
The 14 co-expression genes of three gene sets, differentially expression genes(297) of liver fibrosis, the known genes(663) correlated with liver fibrosis and Dioscorea panthaica correlated genes(1142), were screened out by data mining tools venny, which are APOAl、APOB、F2、GC、NR3Cl、TF、VTN、C3、C5、KNG1、NCF1、PAH、STAT3、TGFBR1。
4. The pathway enrichment analysis of co-expression genes
The pathway enrichment analysis showed that there were 11 biological pathways involved Dioscorea panthaica inhibits liver fibrosis(P<0.05), such as local acute inflammatory response, complement and coagulation cascades, lipoprotein metabolic pathway, and so on.
Conclusion:Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis by bioinformatics analysis.
PartⅢDioscorea panthaica inhibits CCl4-induced liver fibrosis in rats
Objective:Through bioinformatics analysis, Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis, which can be further validated throuth experiment. Therefore, the present study was undertaken to evaluate the protective effects of Dioscorea panthaica extract(DPE) on CCl4-induced liver fibrosis in rat model.
Materials and methods:
1. Liver fibrosis was established by hypodermics injection of CCl4
The male SD rats (190±10 g) were housed in conventional cages (21±1℃,12h light dark cycle) with free access to water and rodent chow. All animal experiments were performed under approved protocols of the institutional animal use and care committee of Sun Yat-Sen University. Rats were randomly divided into six groups. Groups 1 (normal control) and 2 (induction control) received water for eight weeks. Group 3 received colchicine (0.1 mg/kg, p.o. daily) for 8 weeks. Groups 4-6 received DPE (50,100,200 mg/kg per day, respectively) for 8 weeks. All animals except Group 1 were administrated with CCl4 (3 ml/kg body weight,4:6 in olive oil, twice a week). At 24 h after the final injection of CCl4, a laparotomy was performed and the blood was drawn from the abdominal aorta under ether anesthesia. The serum was stored at-80℃after separation until assayed as described below. Liver samples were collected and frozen in liquid nitrogen.
2. Measurement of serum aminotransferase levels
Since the serum levels of AST and ALT are regarded as biochemical markers for the liver injury, as the first step to evaluate the effects of DPE on liver injury we examined AST and ATL levels. A 3ml sample of blood was treated and centrifuged at 3000×g for 10 min at 4℃to obtain plasma, which was then stored at-80℃until assayed. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined. Compared to the normal control group, the serum AST and ALT levels were significantly increased in the liver fibrosis groups. However, in DPE-treated groups DPE treatment led to the decreases of AST and ALT levels in a dose-dependent manner
3. Pathological examination and determination of hepatic hydroxyproline concentration
After formalin fixation, tissue samples were sliced, embedded in a standard manner. Liver fibrosis of the rats was evaluated by two histological methods, H&E staining and Masson's trichrome staining, and both methods showed similar results. The histological analysis of the livers from normal control rats indicated normal architecture. In contrast, both qualitative and quantitative histopathological examinations demonstrated that CCl4-induced liver fibrosis was evidenced by the disruption of tissue architecture, extension of fibers, large fibrous septa formation, pseudolobe separation, and collagen accumulation. These alterations were remarkably reduced in the liver sections of the rats that received both DPE and CCl4 treatment for 8 weeks. It was verified that the scoring of the DPE-treated group had obviously been improved compared to the liver fibrosis group.
In parallel to the observed improvement of liver histology, liver fibrosis was also quantified by measurement of hepatic hydroxyproline level. There was a significant increase in hepatic hydroxyproline level in rats with CCl4-induced liver fibrosis compared to normal controls and the administration of DPE prevented the increase in hepatic hydroxyproline content in CCl4-treated rats.
4. Measurement of hepatic glutathione and thiobarbituric acid-reactive substances
Glutathione (GSH) constitutes the first line of defense against free radicals. A significant reduction in the GSH content (p<0.05) was observed in the liver of the CCl4-treated group rats, compared to that of the normal controls. The treatment of DPE at the dosage of 100 mg/kg significantly recovered the GSH depletion caused by CCl4 (p<0.01).
Compared with the normal controls, the concentration of TBARS was higher in the chronic liver injury group. DPE treatment at the dosage of 100 mg/kg was able to alleviate oxidative damage of lipids significantly, and continual DPE administration decreased the hepatic level of TBARS in a dose-dependent manner
conclusion:Oral administration of DPE significantly reduces CCl4-induced liver fibrosis in rats.
目前,治疗肝纤维化的药物种类很多,主要是通过作用于肝纤维化发生的不同环节,达到抗肝纤维化的目的,包括针对肝星状细胞(hepatic stellate cell,HSC)的药物、针对细胞因子的药物、影响胶原合成及代谢的药物、保护和阻止肝损伤的药物以及抗肝纤维化的中医药等,其中中医药治疗肝纤维化有很多的优势。肝纤维化和肝硬化在中医理论中属正虚血瘀症的范畴,治疗以活血化瘀、益气养阴为主,兼以养血柔肝和滋补肝肾；中药治疗肝纤维化的作用是多环节和多靶点的：减轻肝细胞炎症坏死、改善肝脏血液循环、促进肝细胞再生、直接抑制HSC活化和增殖、减少胶原分泌、促进胶原降解和再吸收等。近年来,国内外大量实验和临床研究发现,中药特别是复方中药治疗肝纤维化的作用为多环节、多靶点的,与化学合成药往往仅作用在单一环节或靶点相比,有独特的优势,值得深入研究。
基因芯片是20世纪生命科学领域发展起来的一项重要技术平台,具有高通量和快速测量等优点。随着基因芯片技术的广泛应用以及生物信息学的飞速发展,肝纤维化发生和发展的许多分子事件得到更全面的揭示。实验研究及临床试验证实,针对单一靶基因的药物治疗疗效有限,而针对多种靶基因或靶蛋白的药物治疗可能会更有效。
本研究将利用基于基因表达谱的药物发现模式,通过Toppgene等生物信息学分析方法筛选肝纤维化治疗候选化合物或药物,并进行相关实验验证,为肝纤维化的综合治疗及提高疗效提供理论及实验依据,同时也为加速肝纤维化中药治疗药物发现进程、为中药“老药新用”及中药现代化提供依据。
本研究内容主要分为三个部分：
第一部分基于基因表达谱的肝纤维化生物信息学分析
目的：本研究利用基因芯片数据分析软件BRB-ArrayTools 3.7.1,对来自基因芯片公共数据库(Gene Expression Omnibus, GEO)的肝纤维化基因表达谱芯片数据进行挖掘,并进行生物信息学分析,探讨基于基因表达谱的途径筛选和发现新的肝纤维化治疗药物。
方法与结果：
1.从GEO获取肝纤维化基因表达谱数据集
从NCBI的GEO数据库获得肝纤维化基因表达谱数据集,即GSE11536。该数据集来源于法国ROUEN医药系,采用GPL5215芯片平台,共48个样本,包括8个轻度肝纤维化样本和8个重度肝纤维化样本,每个样本三个重复。
2.肝纤维化差异表达基因分析
为了获得肝纤维化差异基因表达谱,我们采用BRB软件对GSE11536数据集进行差异表达基因分析,获得差异表达基因297个,其中上调基因115个,下调基因182个(Fold change> 1.5)。
3.文献挖掘获得已知肝纤维化疾病基因
采用Genecards和FABLE在线工具分析,共获得已知肝纤维化相关基因663个。
4. Toppgene筛选肝纤维化相关基因
首先建立已知肝纤维化相关基因集文件,然后通过toppgene在线分析工具进行肝纤维化候选基因筛选,结果得到7个肝纤维化候选新基因,分别是：SERPING1、ETS2、TSG101、CDH2、LMO2、PAWR、MST1。
5.肝纤维化信号通路富集分析
为从生物学意义水平上发现一些与肝纤维化密切相关的信号通路,采用Toppgene对所获取的差异基因表达谱进行基因集富集分析,共富集到44条通路(P<0.05)。对44条富集通路进行分析,结果显示,28条富集通路与凝血有关,3条与血小板生成有关,4条与心肌梗塞有关,6条与脂代谢有关,2条涉及MAPK途径。
6.肝纤维化差异基因富集相关疾病分析
为了解与肝纤维化相关联的疾病,采用Toppgene对所获取的肝纤维化差异基因进行富集相关疾病分析,共富集到4种疾病(P<0.05),分别是肝炎、先天性纤维蛋白原缺乏血症、后天纤维蛋白原缺乏血症和高密度脂蛋白胆固醇血症,这提示肝纤维化可能与凝血障碍和血脂代谢异常有关。
7.肝纤维化候选药物分析
Toppgene还建立了疾病、基因与药物之间的联系,为了获得一些可以逆转或是改善肝纤维化基因表达谱的治疗药物,进行Toppgene分析,结果得到69种药物(P<0.001)。结合文献,对69种药物进行分析,结果显示,17种药物是性激素类药物,12种是凝血相关药物,11种是降血脂药,6种是降血糖药,9种是消炎止痛药,这提示性激素类、凝血相关药物和降血脂药物可能可以成为肝纤维化治疗药物或辅助治疗药物。
结论：基于基因表达谱的途径筛选和发现肝纤维化治疗相关药物是可行的,并筛选到一些激素类药物、凝血相关药物和降血脂药物可能可以成为肝纤维化候选治疗药物或辅助治疗药物。
第二部分肝纤维化的治疗药物Dioscorea panthaica筛选
目的：黄山药(Dioscorea panthaica Prain et Burkill)是一种十分重要的食药两用植物资源,中医常用于治疗炭疽、胃痛、风湿性心脏病和风湿性关节炎；黄山药水提取物还可以用于治疗鼠高胆固醇血症。以黄山药根茎提取物为主要原料制成的地奥心血康,是国家二类新药,年销售收入达六亿元,临床主要用于心血管疾病、降血脂、降血糖,其主要化学成分是薯蓣皂甙元,还可作为性激素等多种药物的原料药等。结合第一部分结果：激素类药物、凝血相关药物和降血脂药物可能可以成为肝纤维化候选治疗药物或辅助治疗药物,因此本部分采用生物信息学方法寻找Dioscorea panthaica抗肝纤维化的可能作用靶点,并进行深入分析确认Dioscorea panthaica具有抑制肝纤维化的作用,为下一步进行相关实验验证提供依据。
方法与结果：
1.文献挖掘Dioscorea panthaica相关靶基因
采用FABLE在线工具,结合FACTA文献分析,共获得Dioscorea panthaica相关靶基因1142个。
2. Dioscorea panthaica抗肝纤维化的可能作用靶点
采用venny分析工具,分析已知肝纤维化相关基因和Dioscorea panthaica相关靶基因的共同表达基因,结果表明,在663个已知肝纤维化相关基因中,存在Dioscorea panthaica的可能作用靶基因322个(占48.6%),这进一步提示Dioscorea panthaica有抑制肝纤维化的作用。
3.获得共同表达基因
为了解Dioscorea panthaica抑制肝纤维化的可能作用机制,结合肝纤维化差异基因集,采用venny分析工具进行分析,获得肝纤维化差异基因、已知肝纤维化相关基因和Dioscorea panthaica相关靶基因的共同表达基因14个,分别是：APOA1、APOB、F2、GC、NR3Cl、TF、VTN、C3、C5、KNG1、NCF1、PAH、STAT3、TGFBR1。
4.共同表达基因集通路富集分析
将14个共同表达基因进行通路富集分析,结果显示有11条生物信号通路可能参与Dioscorea panthaica抑制肝纤维化的分子机制(P<0.05),这些信号通路主要涉及局部急性炎症应答反应、补体凝血过程、活性肽诱导通路.FOXA2/FOXA3转录因子调控网络、脂蛋白代谢等信号通路。
结论：通过生物信息学分析预测,进一步提示Dioscorea panthaica有抑制肝纤维化的作用。
第三部分Dioscorea panthaica抑制肝纤维化的相关实验验证
目的：为验证前两部分的分析结果,本部分采用CCl4法造大鼠肝纤维化模型,探讨Dioscorea panthaica提取物抑制肝纤维化的效果。
方法与结果：
1.CCl4诱导大鼠肝纤维化模型的建立、分组及给药方法
健康雄性SD大鼠,36只,随机分为6组,每组6只：①正常对照组(n=6),给予橄榄油1mL/kg体重(2次/周)腹腔内注射,共8周；②模型诱导对照CCl4组(n=6),给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；③阳性对照秋水仙碱组(n=6),按0.1 mg/kg体重腹腔注射秋水仙碱,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；④DPE-50mg/kg组(n=6),按50 mg/kg体重灌胃Dioscorea panthaica提取物(DPE),每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；⑤DPE-100mg/kg组(n=6),按100mg/kg体重灌胃DPE,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8周；⑥DPE-200mg/kg组(n=6),按200mg/kg体重灌胃DPE,每天1次,同时给予CCl4(CCl4橄榄油=4：6)3mL/kg体重,每周两次,共8次。各组大鼠均于8周后处死,处死前禁食24h。处死时,大鼠用水合氯醛麻醉,迅速从心脏穿刺取血,分离血清,-80℃保存。平卧状态下剖腹,去除肝脏筋膜,取右叶相同的部分置于冰生理盐水中充分洗涤后,一部分用10%中性甲醛固定,3天内石蜡包埋,切片,以备组织病理检测；其余肝组织迅速置于液氮中保存。
2.DPE对血清ALT、AST含量的影响
血清AST和ALT是评价肝脏损伤常用的生化指标。取大鼠血液约3mL,置干燥玻璃管内,37℃温浴30min,4℃冰箱静置2h,3000g离心10min,取血清,-80℃保存。通过全自动生化分析仪测定各组大鼠血清ALT、AST含量。与正常对照组相比,肝纤维化模型对照组的血清ALT含量(P=0.000)和AST含量(P=0.000)显著提高,DPE处理组的血清ALT、AST含量呈剂量依赖性下降。
3.肝组织病理学改变及检测肝组织中羟脯氨酸水平
运用HE染色和Masson'染色对各组大鼠肝组织纤维化程度进行分析,结果显示,正常对照组大鼠的肝组织肝索结构清晰,肝细胞以中央静脉为中心呈放射状,肝组织内无脂肪空泡,仅汇管区和中央静脉有少许胶原纤维存在。模型对照组肝小叶结构破坏,纤维组织增生明显,将肝小叶分隔成大小不等的肝细胞团,肝细胞广泛变性坏死,汇管区扩大、胶原大量沉积。与模型对照组相比,治疗组肝细胞的变性坏死程度及肝小叶结构破坏程度降低,肝脏胶原纤维增生亦明显减轻。
在检测肝脏组织病理学改变的同时,通过检测肝组织中羟脯氨酸水平,对肝损伤纤维化程度进行定量分析。与正常对照组相比,模型对照组的羟脯氨酸含量显著提高,差异有统计学意义(P=0.000),DPE处理组的羟脯氨酸含量呈剂量依赖性下降。
4.DPE对肝组织抗氧化能力的影响
通过检测大鼠肝组织匀浆中的GSH和TBARS浓度,探讨DPE治疗对肝组织氧化状态的影响。GSH构成了拮抗自由基的第一道防线。与正常对照组GSH含量相比,肝纤维化模型对照组GSH含量显著降低(P=0.000)；与肝纤维化模型对照组GSH含量相比,阳性对照秋水仙碱组(P=0.000)和DPE处理组(P=0.000)肝组织GSH含量差异均有统计学意义。为检测肝组织氧化性能,结果显示,与正常对照组比较,肝纤维化模型对照组TBARS含量增加约3.2倍；DPE (200mg/Kg)处理组肝脏TBARS含量降至1.03±0.12μmol/g,表明肝组织抗氧化能力增强。
结论：经动物实验证实,Dioscorea panthaica提取物能显著降低CCl4诱导的大鼠肝纤维化。
Liver fibrosis (hepatic fibrosis, HF) is a disease of reversible deposition of extracellular matrix(ECM) induced by chronic liver injuried. Many things could cause liver fibrosis, such as viral infection, ethanol, parasites, chemical poisons, and so on. Some fibrosis patients may be the result from a variety of pathogenic factors. Liver fibrosis is regional differences. Alcoholic liver fibrosis is common in Europe and America, while liver fibrosis with hepatitis B virus infection is common in China. With a high incidence of liver diseases, Chinese government takes heavy burden in the treatment of liver fibrosis and cirrhosis. Liver fibrosis can eventually lead to hepatic cirrhosis or liver cancer with poor prognosis.
The gene-chip (bio-chip) is major technique in bioscience field in the 21 century, which has the outstanding of high-throughput and rapid detecting, etc. The analysis of expression profiling chip is useful for the reveal of the mechanism of liver fibrosis, the diagnosis and therapy of liver fibrosis. Bioinformatics is a newly rising subject which elucidates and reveals the biological meaning hided in much data set. Bioinformatics provids a new approach for the analysis of DNA microarray data,also become a new tool for the discovery of the drug and speed up the process of drug discovery.In the study, based on the mode of gene expression profiles-based drug discovery approach, we set out to discover agents for liver fibrosis with computational and system biology tool "Toppgene",which provided the theory and experiment proof for the combined therapy of liver fibrosis, and also accelerate the discovery of drugs for liver fibrosis.
The study includes the following three parts:
Part I gene expression profiling based approach identifies candidate therapeutic compounds for liver fibrosis
Objective:Bioinformatics provids a new approach for the analysis of DNA microarray data, also become a new tool for the discovery of the drug and speed up the process of drug discovery. In the study, we explore gene expression profiles-based approach for the screening candidate therapeutic compounds against liver fibrosis, and screen the candidate compounds for liver fibrosis.
Methods and Results:
1. Acquisition liver fibrosis public microarray data from GEO databank
Raw data (.Cel files) of a published microarray data (GSE11536) used in the study was obtained from Gene Expression Omnibus(GEO) website, Amonge gene datasets GSE 11536, containing 48 samples, from Faculty of Medicine and Pharmacy, France. The dataset were based on GPL5215 platform.
2. BRB analysis of liver fibrosis
The aim of this study is to compare mild and advanced liver fibrosis gene expression profiling and to identify novel markers of fibrosis progression. By transcriptome analysis with a cDNA array virtually covering every transcript in liver, we compared transcript levels in mild (F1 Metavir score) and advanced (F4 Metavir score) fibrosis. A stringent selection identified a list of 16 transcripts which completely separated the 2 groups of patients (8 F1 and 8 F4). Mild fibrosis (F1) vs advanced fibrosis (F4) were alone performed with the software packages BRB Array Tools. Finally, we obtained 297 differentially expression genes, amonge 115 up-regulated genes and 182 down-regulated genes (Fold change> 1.5)
3. Data mining of known gene expression profile in liver fibrosis
We obtained 663 known genes correlated with liver fibrosis by data mining tools genecards and FABLE.
4. The screen of candidate genes for liver fibrosis with Toppgene
The 297 differential expression genes were defined as"test gene set", and 663 genes correlated with liver fibrosis were regarded as"train gene set".7 candidate genes were screened out by Toppgene, such as SERPING1、ETS2、TSG101、CDH2、LMO2、PAWR、MST1, which had nearly no report in liver fibrosis.
5. The pathway enrichment analysis of liver fibrosis with Toppgene
The pathway enrichment analysis showed that there were 44 biological pathways related with liver fibrosis(P<0.05). We found out that blood coagulation signal pathway(28), platelet signal pathway(3), myocardial infarction pathway(4), lipid metabolic pathway(6), MAPK signal pathway(2) were very significance in the progression of liver fibrosis.
6. The related deseases enrichment analysis of liver fibrosis
The related deseases enrichment analysis showed that there were 4 deseases related with liver fibrosis (P<0.05), such as hepatitis, afibrinogenemia congenital, afibrinogenemia, HDL cholesterol.
7. The candidate therapeutic drugs enrichment analysis of liver fibrosis
Finally,69 candidate therapeutic compounds for liver fibrosis were screened by Toppgene analysis (P<0.001). We found out some candidate therapeutic compounds, such as sexual hormone drugs (17), blood clotting drugs(12), descendens blood fat drugs(11), hypoglycemic agent(6), acesodyne (9),which were very significance in treatment of liver fibrosis.
Conclusion:Our results demonstrated that gene expression profiles-based approach is perspective for screening the candidate therapeutic compounds for liver fibrosis, which accelerates the introduction of compounds into the clinic.
Part II Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis
Objective:Dioscorea panthaica is a herb commonly used in traditional Chinese medicine to treat anthrax, gastropathy, rheumatic heart disease, and rheumarthritis. The anti-hypercholesterolemia activity of an aqueous extract of saponins from Dioscorea panthaica was also demonstrated in rats. It is well known that the chemical composition of herbal medicines is very complex, and detailed information on the bioactive compounds of Dioscorea panthaica remains largely incomplete despite several phytochemical studies on this medicinal plant. Currently, the effects of DPE on liver fibrosis have not been reported. In the study, the target genes which involved Dioscorea panthaica inhibits liver fibrosis were screened out by data mining tools, that further prompted Dioscorea panthaica could inhibit liver fibrosis.
Methods and Results:
1. Data minining of Dioscorea panthaica correlated genes
We obtained 1142 known genes correlated with Dioscorea panthaica by data mining tools genecards and FABLE.
2. Data mining of the target genes which involved Dioscorea panthaica inhibits liver fibrosis
The target genes which involved Dioscorea panthaica inhibits liver fibrosis were screened out by data mining tools. Among the 663 known liver fibrosis related genes, there were 322 potential target genes (48.6%) of Dioscorea panthaica, which further prompted Dioscorea panthaica could inhibit liver fibrosis.
3. co-expression genes of three gene sets were screened out by data mining tools venny
The 14 co-expression genes of three gene sets, differentially expression genes(297) of liver fibrosis, the known genes(663) correlated with liver fibrosis and Dioscorea panthaica correlated genes(1142), were screened out by data mining tools venny, which are APOAl、APOB、F2、GC、NR3Cl、TF、VTN、C3、C5、KNG1、NCF1、PAH、STAT3、TGFBR1。
4. The pathway enrichment analysis of co-expression genes
The pathway enrichment analysis showed that there were 11 biological pathways involved Dioscorea panthaica inhibits liver fibrosis(P<0.05), such as local acute inflammatory response, complement and coagulation cascades, lipoprotein metabolic pathway, and so on.
Conclusion:Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis by bioinformatics analysis.
PartⅢDioscorea panthaica inhibits CCl4-induced liver fibrosis in rats
Objective:Through bioinformatics analysis, Dioscorea panthaica were identified as candidate therapeutic compounds for liver fibrosis, which can be further validated throuth experiment. Therefore, the present study was undertaken to evaluate the protective effects of Dioscorea panthaica extract(DPE) on CCl4-induced liver fibrosis in rat model.
Materials and methods:
1. Liver fibrosis was established by hypodermics injection of CCl4
The male SD rats (190±10 g) were housed in conventional cages (21±1℃,12h light dark cycle) with free access to water and rodent chow. All animal experiments were performed under approved protocols of the institutional animal use and care committee of Sun Yat-Sen University. Rats were randomly divided into six groups. Groups 1 (normal control) and 2 (induction control) received water for eight weeks. Group 3 received colchicine (0.1 mg/kg, p.o. daily) for 8 weeks. Groups 4-6 received DPE (50,100,200 mg/kg per day, respectively) for 8 weeks. All animals except Group 1 were administrated with CCl4 (3 ml/kg body weight,4:6 in olive oil, twice a week). At 24 h after the final injection of CCl4, a laparotomy was performed and the blood was drawn from the abdominal aorta under ether anesthesia. The serum was stored at-80℃after separation until assayed as described below. Liver samples were collected and frozen in liquid nitrogen.
2. Measurement of serum aminotransferase levels
Since the serum levels of AST and ALT are regarded as biochemical markers for the liver injury, as the first step to evaluate the effects of DPE on liver injury we examined AST and ATL levels. A 3ml sample of blood was treated and centrifuged at 3000×g for 10 min at 4℃to obtain plasma, which was then stored at-80℃until assayed. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined. Compared to the normal control group, the serum AST and ALT levels were significantly increased in the liver fibrosis groups. However, in DPE-treated groups DPE treatment led to the decreases of AST and ALT levels in a dose-dependent manner
3. Pathological examination and determination of hepatic hydroxyproline concentration
After formalin fixation, tissue samples were sliced, embedded in a standard manner. Liver fibrosis of the rats was evaluated by two histological methods, H&E staining and Masson's trichrome staining, and both methods showed similar results. The histological analysis of the livers from normal control rats indicated normal architecture. In contrast, both qualitative and quantitative histopathological examinations demonstrated that CCl4-induced liver fibrosis was evidenced by the disruption of tissue architecture, extension of fibers, large fibrous septa formation, pseudolobe separation, and collagen accumulation. These alterations were remarkably reduced in the liver sections of the rats that received both DPE and CCl4 treatment for 8 weeks. It was verified that the scoring of the DPE-treated group had obviously been improved compared to the liver fibrosis group.
In parallel to the observed improvement of liver histology, liver fibrosis was also quantified by measurement of hepatic hydroxyproline level. There was a significant increase in hepatic hydroxyproline level in rats with CCl4-induced liver fibrosis compared to normal controls and the administration of DPE prevented the increase in hepatic hydroxyproline content in CCl4-treated rats.
4. Measurement of hepatic glutathione and thiobarbituric acid-reactive substances
Glutathione (GSH) constitutes the first line of defense against free radicals. A significant reduction in the GSH content (p<0.05) was observed in the liver of the CCl4-treated group rats, compared to that of the normal controls. The treatment of DPE at the dosage of 100 mg/kg significantly recovered the GSH depletion caused by CCl4 (p<0.01).
Compared with the normal controls, the concentration of TBARS was higher in the chronic liver injury group. DPE treatment at the dosage of 100 mg/kg was able to alleviate oxidative damage of lipids significantly, and continual DPE administration decreased the hepatic level of TBARS in a dose-dependent manner
conclusion:Oral administration of DPE significantly reduces CCl4-induced liver fibrosis in rats.
引文
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