title: Identification and Verification of Potential Therapeutic Target Genes in Berberine-Treated Zucker Diabetic Fatty Rats through Bioinformatics Analysis.
abstract: BACKGROUND
  Berberine is used to treat diabetes and dyslipidemia. However, the effect of berberine on specific diabetes treatment targets is unknown. In the current study, we investigated the effect of berberine on the random plasma glucose, glycated hemoglobin (HbA1C), AST, ALT, BUN and CREA levels of Zucker diabetic fatty (ZDF) rats, and we identified and verified the importance of potential therapeutic target genes to provide molecular information for further investigation of the mechanisms underlying the anti-diabetic effects of berberine.
  
  
  METHODS
  ZDF rats were randomly divided into control (Con), diabetic (DM) and berberine-treated (300 mg⋅kg-1, BBR) groups. After the ZDF rats were treated with BBR for 12 weeks, its effect on the random plasma glucose and HbA1C levels was evaluated. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), CREA and OGTT were measured from blood, respectively. The levels of gene expression in liver samples were analyzed using an Agilent rat gene expression 4x44K microarray. The differentially expressed genes (DEGs) were screened as those with log2 (Con vs DM) ≥ 1 and log2 (BBR vs DM) ≥ 1 expression levels, which were the genes with up-regulated expression, and those with log2 (Con vs DM) ≤ -1 and log2 (BBR vs DM) ≤ -1 expression levels, which were the genes with down-regulated expression; the changes in gene expression were considered significant at P<0.05. The functions of the DEGs were determined using gene ontology (GO) and pathway analysis. Furthermore, a protein-protein interaction (PPI) network was constructed using STRING and Cytoscape software. The expression levels of the key node genes in the livers of the ZDF rats were also analyzed using qRT-PCR.
  
  
  RESULTS
  We found that 12 weeks of berberine treatment significantly decreased the random plasma glucose, HbA1C levels and improved glucose tolerance. There was a tendency for berberine to reduce AST, ALT, BUN except increase CREA levels. In the livers of the BBR group, we found 154 DEGs, including 91 genes with up-regulated expression and 63 genes with down-regulated expression. In addition, GO enrichment analysis showed significant enrichment of the DEGs in the following categories: metabolic process, localization, cellular process, biological regulation and response to stimulus process. After the gene screening, KEGG pathway analysis showed that the target genes are involved in multiple pathways, including the lysine degradation, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and pyruvate metabolism pathways. By combining the results of PPI network and KEGG pathway analyses, we identified seven key node genes. The qRT-PCR results confirmed that the expression of the RHOA, MAPK4 and DLAT genes was significantly down-regulated compared with the levels in DM group, whereas the expression of the SgK494, DOT1L, SETD2 and ME3 genes was significantly up-regulated in the BBR group.
  
  
  CONCLUSIONS
  Berberine can significantly improve glucose metabolism and has a protective effects of liver and kidney function in ZDF rats. The qRT-PCR results for the crucial DEGs validated the microarray results. These results suggested that the RHOA, MAPK4, SGK494, DOT1L, SETD2, ME3 and DLAT genes are potential therapeutic target genes for the treatment of diabetes.
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Yang Sheng
authors.initials: YS
authors.lastname: Wu
authors.affiliation: College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Chen
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Bao
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Zhe-Ming
authors.initials: ZM
authors.lastname: Li
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Xiao-Jie
authors.initials: XJ
authors.lastname: Zhou
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Jia-Na
authors.initials: JN
authors.lastname: He
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Shi-Jie
authors.initials: SJ
authors.lastname: Dai
authors.affiliation: College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Chang Yu
authors.initials: CY
authors.lastname: Li
mesh_terms: D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D001599:Berberine / Q000008:administration & dosage*; D001786:Blood Glucose; D050260:Carbohydrate Metabolism / Q000235:genetics; D019295:Computational Biology; D003920:Diabetes Mellitus / Q000097:blood / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D006442:Glycated Hemoglobin / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism / Q000235:genetics; D053858:Metabolic Networks and Pathways / Q000187:drug effects* / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000235:genetics; D051381:Rats; D011924:Rats, Zucker
chemical_list: D000410:Alanine Transaminase; D001219:Aspartate Aminotransferases; D001599:Berberine; D001786:Blood Glucose; D006442:Glycated Hemoglobin