Identification and Verification of Potential Therapeutic Target Genes in Berberine-Treated Zucker Diabetic Fatty Rats through Bioinformatics Analysis.

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. CONCLUSION: 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.

[Protective effect of ß-asarone on PC12 cells injury induced by Aß1₋42 astrocytic activation].

This study was aimed to investigate the protective effect and mechanism of ß-asarone on PC12 cells injury induced byAß1₋42 activated astrocytes, and provide experimental basis for ß-asarone application in the prevention and control of Alzheimer's disease (AD). Firstly, RA-h and PC12 cells were co-cultured in the special transwell chamber, and the Real time cell analysis (RTCA) system was used to real-time observe its effect on PC12 cells survival rate in the co-culture system after astrocytes injury induced by Aß1₋42. The best intervention time of ß-asarone was selected according to the survival curve and parameters generated automatically. ß-asarone with different concentrations was used for intervention on astrocytes, then the changes of PC12 cells survival rate in the co-culture system were observed. Secondly, MTT assay was used to detect the effect of Aß1₋42 on PC12 cells survival rate as well as the intervention effect of ß-asarone, and verify the testing results of RTCA. The levels of IL-1ß, TNF-α and BDNF in culture media of the lower chamber were detected by ELISA. The NF-κB activity and phosphorylation levels of ERK, p38 and JNK were detected by Western blot. Results showed that ß-asarone (55.5 mg•L⁻¹) could significantly slowdown the decline of PC12 cells survival rate caused by Aß1₋42-induced RA-h activation (P<0.01), significantly reduce the levels of IL-1ß, TNF-α and the phosphorylation levels of ERK, p38 and JNK in culture media of the lower chamber (P<0.01). ß-asarone(166.7 mg•L⁻¹) could promote the release of BDNF in culture media of the lower chamber(P<0.05). These results indicated that Aß1₋42 could induce RA-h activation and its release of IL-1ß, TNF-α and other inflammatory factors to aggravate the PC12 cells injury; ß-asarone could reduce the levels of IL-1ß, TNF-α, promote the release of BDNF, and inhibit the NF-κB activity as well as phosphorylation levels of ERK, p38 and JNK protein in PC12 cells.