Fuzhenghuayu Rat Serum Containing Hsc-t6 Cells Mrna And Mirm Of Expression Profiles Regulation

BACKGROUNDLiver fibrosis, an early pathologic stage of cirrhosis, is the outcome of many chronic liver diseases. Hepatic stellate cell (HSC) plays a central role in this process. In the recent 3 decades, Chinese medicine was committed lot of researches in this field and considered effective on the regulation of gene expression, intracellular signaling pathways, cell proliferation and cell apopatosis. The regulation feature of Chinese medicine is multi-target and multi-level, which is difficult to be reflected by the regular research.The genome-wide gene chip and high-throughput sequencing technology are the lastest technologies in the gene research field, which can give a global view of the total mRNA and miRNA gene expression profiles at a special time point. The develepment of bioinfomatics provides the powerful tools for the analysis of huge amounts of data. The above progresses make an unprecedented good chance for the development of Chinese medicine research.FuZheng HuaYu recipe (capsule) is a commonly used and effective anti-hepatic fibrosis drug. On the while, it is one of the drugs with systematic studies. This study is designed to explore the anti-hepatic fibrosis mechanism of Chines medicine by reseaching the mechanism of FuZheng HuaYu recipe with the technologies of the genome-wide gene chip and high-throughput sequencing.AIM:This study is designed to explore the mRNA and miRNA expression profiles features of HSC-T6 cells treated by FuZheng HuaYu (FZHY) rat drug serum and elucidate the anti-hepatic fibrosis mechanism in the level of gene.METHOD:1. The FZHY rat drug serum and its control serum were prepared from the rats fed with FZHY solution by gavage, according to the serum pharmacological method. The HSC-T6 cells were cultured in the normal culture solution containing 90% DMEM high glucose medium and 10% fetal bovine serum for the first 24 hour and then treated by the FZHY culture solution (90% DMEM high glucose medium and 10% FZHY rat serum) or the control culture solution (90% DMEM high glucose medium and 10% control rat serum) for the next 24 hours. Then the total RNA of the treated HSC-T6 cells was abstracted to commit the real-time PCR experiment. We detected the mRNA levels ofα-SMA, TGF-β1, collagen I and collagen III, which relating to the liver fibrosis closely, to confirm the stability of our research system.2. The total RNA was detected by the Affymetrix Rat 2302.0 gene chip to achieve the mRNA gene expression profile. Then the differentially expressed genes with more than 2-fold changing were screened out and annotated by the gene ontology and KEGG signaling pathway with the tools and databases integrated by the website DAVID. By the above analysis, the main functions and signaling pathways enriched with differentially expressed genes were found out.3. The total RNA was detected by the method of Solexa miRNA High-throughput sequencing to achieve the mRNA gene expression profile. The differentially expressed miRNA with more than 2-fold changing were screened out and used to achieve the target genes based on the prediction database of website TARGETSCAN. Then the target genes were annotated by the gene ontology and KEGG signaling pathway with the tools and databases integrated by the website DAVID. The analysis results of mRNA and target genes were compared to find the clues for the follow-up research.4. The MAPK signaling pathway screened out by the comparison of the annotation results of both mRNA and miRNA gene expression profiles was further confirmed by the western-blot experiment on the key proteins and their Phosphorylated types including ERK, JNK and p38 to elucidate the regulating details.5. The apoptosis was strongly suggested by the gene ontology analysis of mRNA gene expression profiles and miRNA target gene profile, the regulation features of MAPK pathway and HSC-T6 culture. We made a hypothesis that the FZHY rat serum can induce the apoptosis of HSC-T6 and made a further observation by the annexin V/7aad double staining with the flow cytometry on the apoptosis.RESULT:1. In the study of mRNA gene expression profiles, we found 637 differentially expressed genes with more than 2-fold changing and 334 genes within them researched before. The gene ontology analysis suggested that these genes enriched in the process of developmental process, biological regulation, cellular process and biological adhesion. The KEGG pathway analysis suggested the RIG-I-like receptor pathway and MAPK pathway were significantly regulated (P<0.05). The MAPK pathway had a closely relationship with the liver fibrosis and there were 11 genes regulated in this signaling pathway including Prkcg, TNF, Faslg, Rasa2, Gna12, Mapk8, Illr2, Fgf23, Fgf12, Map3k3 and Rasgrp2. These genes had key role in the ERK, JNK and p38 pathway.2. In the study of miRNA gene expression profiles, we found 75 differentially expressed genes with more than 2-fold changing. Based on the database of website TARGETSCAN, we achieved 1962 target genes. In the following GO analysis, we found these genes enriched in the biological processes:developmental process, biological regulation, cellular process, cellular component organization, growth and et al. In the KEGG signaling pathway analysis, we found that 29 pathway were regulated. Excepting the pathway relating to tumor, there were 16 signaling pathway left, most of which were proved to relating to liver fibrosis. In the 16 pathways, the MAPK signaling pathway was significantly regulated by the miRNA. After comparing the result with mRNA gene expression profiles, we confirm the MAPK signaling pathway for the following up research.3. In the experiment of real-time PCR, we found that all the 4 kinds of gene expression levels had been down regulated. The mRNA of collagen I, collagen III,α-SMA and TGF-β1 decreased 27%,36%,43% and 23% separately.4. In the western-blot study about the MAPK key proteins and its Phosphorylated types, we found that the treatment of FZHY rat serum had an important regulation on the MAPK pathway. The ERK pathway was unregulated (P<0.05), the JNK pathway was downregulated (P>0.05) and p38 pathway was upregulated (P<0.05). Considering that the HSC-T6 is the spontaneously activated cell strain, we suggested the above changing may induce the apoptosis of the cells.5. In the study of apoptosis by annexinV/7aad double staining with flow cytometry, we found the treatment of FZHY rat serum could induce the cell apoptosis comparing to the control serum. Whether the early stage apoptosis or the later stage apoptosis and the total apoptosis, the differences were significant.CONCLUSION:By the above 5 experiments, we proved that:1. FZHY rat serum had a broad regulation on the mRNA and miRNA expression profiles of HSC-T6 cells.2. The Gene Ontology analysis on the two profiles suggested that the differentially expressed genes mainly enriched in the cellular adhesion, development and apoptosis.3. The KEGG pathway analysis suggested that the MAPK pathway was the most important regulation target.4. The negative regulation of miRNA played an important role in the effection of the drug.5. The FZHY rat serum could induce the apoptosis of HSC-T6 cells.6. The regulation on the MAPK pathway embodied on the upregulation of p38 and downregulation of JNK, which was probably the most important way to induce the apoptosis of HSC-T6 cells.7. FZHY rat serum had a stable effect on decreasing the mRNA expression of TGF-β1, which is the mainly mechanism to inhibit the persistent activation of HSC and decrease the collagen I and collagen III...