Molecular analyses of antifibrotic effect of BK channel activatior on rat livers in fibrosis

Liver fibrosis is a high-morbidity and high-mortality chronic disease throughout the world without any satisfying treatment Large-conductance Ca2+- and voltage-activated K+ (Slo1, BK) channels are widely expressed in human body and important for numerous physiological processes. Previous studies have shown that BK channels are expressed in HSCs of patients with liver fibrosis and they are involved in contraction/relaxation of the HSCs To investigate the molecular mechanism of antifibrotic effect of BK channel opener rottlerin using in vivo fibrosis models. transcriptomic analyses of differential expression genes in livers from rats of vehicle, CCl4 and CCl4 combined with rottlerin treatment were for discrimination. The RNA samples were extracted from three samples of each group for microarray profiling and performed in Affymetrix Rats Genome 230 2.0 arrays for gene expression profiling analysis, which contained 31 042 probe sets. The biotinylated cRNA targets were hybridized with the microarray. After hybridization, arrays were stained in the Fluidics Station 450 and scanned on the Affymetrix Scanner 3000. The microarray experiments were performed by following the protocol of Affymetrix Inc at Shanghai Biotechnology Corporation. Under the criteria fold change > 1.5 or < 0.67, we obtained 10672 differential expressed genes (DEGs) between CCl4 and CCl4 combined with rottlerin treatment group, and the data was applied to further analysis.肝纤维化（Liver fibrosis）是全球范围内高发病率与高死亡率并存的慢性疾病，目前尚无令人满意的治疗方案。大电导钙激活电压依赖性钾通道（Large-conductance Ca²⁺- and voltage-activated K⁺ channels，简称Slo1、BK通道）广泛表达于人体各组织，对诸多生理过程具有关键调控作用。既往研究表明，BK通道在肝纤维化患者的肝星状细胞（Hepatic Stellate Cells, HSCs）中有所表达，并参与肝星状细胞的收缩与舒张过程。为探究BK通道开放剂罗特林（rottlerin）发挥抗纤维化作用的分子机制，本研究采用体内纤维化模型开展相关实验，对赋形剂对照组、四氯化碳（Carbon Tetrachloride, CCl4）模型组以及CCl4联合罗特林治疗组大鼠的肝脏组织进行差异表达基因转录组分析以区分不同处理组。每组各取3份RNA样本进行基因芯片检测，采用Affymetrix大鼠基因组230 2.0芯片完成基因表达谱分析，该芯片共包含31042个探针集。将生物素标记的cRNA靶标与基因芯片进行杂交，杂交完成后，在Fluidics Station 450工作站中对芯片进行染色，并通过Affymetrix Scanner 3000扫描仪完成图像扫描。本基因芯片实验严格遵循Affymetrix公司的官方实验流程，由上海生物技术公司（Shanghai Biotechnology Corporation）完成。以折叠变化（fold change）>1.5或<0.67作为筛选标准，我们在CCl4模型组与CCl4联合罗特林治疗组间共筛选得到10672个差异表达基因（Differentially Expressed Genes, DEGs），所得数据将用于后续深入分析。