Expression-based In Silico Screening Of Candidate Therapeutic Drugs For Liver Fibrosis And Related Experiment

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 fibrosisObjective: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 databankRaw 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 fibrosisThe 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 fibrosisWe 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 ToppgeneThe 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 ToppgeneThe 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 fibrosisThe 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 fibrosisFinally,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 fibrosisObjective: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 genesWe 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 fibrosisThe 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 vennyThe 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 genesThe 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 ratsObjective: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 CCl4The 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 levelsSince 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 manner3. Pathological examination and determination of hepatic hydroxyproline concentrationAfter 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 substancesGlutathione (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.