祛湿化瘀方对脂肪肝大鼠肝脏基因表达谱的调节作用

Regulatory Effect of Qushi Huayu Recipe on Gene Expression Profiles of Fatty Liver Rats

目的观察祛湿化瘀方对高脂饮食诱导脂肪肝大鼠肝脏基因表达谱的干预作用及其作用机制。方法20只SD雄性大鼠采用单纯高脂饮食(88%普通饲料+2%胆固醇+10%猪油)制备脂肪肝大鼠模型。造模4周后按随机数字表法分为祛湿化瘀方组(10只)和模型组(10只),继续造模同时分别给予祛湿化瘀方汤剂(0.93 g生药/100 g体重)和蒸馏水灌胃,每日1次。同时设10只SD雄性大鼠为正常组,给予等量蒸馏水灌胃,各组均于8周末取材。采用生化法检测肝组织甘油三酯(triglyceride,TG)、游离脂肪酸(free fatty acid,FFA)含量及血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)活性;采用HE及油红染色观察肝脏组织的病理变化;采用Affymetrix基因芯片检测肝组织基因表达,比较祛湿化瘀方组与模型组差异表达基因、分析差异基因的功能以及涉及的信号通路;并选取祛湿化瘀方组与模型组间10个差异倍数大于2的涉及糖脂代谢的差异基因进行RT-PCR验证。结果(1)与正常组比较,模型组大鼠肝组织TG、FFA含量和血清ALT、AST活性均明显升高(P<0.01);与模型组比较,祛湿化瘀方组大鼠TG、FFA含量和ALT、AST活性均明显降低(P<0.05,P<0.01)。祛湿化瘀方能降低高脂饮食诱导的大鼠脂肪肝模型的肝细胞脂肪变性程度,减少炎症,改善肝组织病理。(2)祛湿化瘀方组与模型组比较,P<0.05且差异倍数大于2的功能明确、有指定基因名称的差异基因共80个,其中上调基因44个,下调基因36个。80个差异基因涉及27条差异有统计学意义的信号通路(包括甘油酯类代谢、通路脂肪细胞信号通路、胰岛素信号通路及药物代谢信号通路等,P<0.05)。(3)对甘油激酶(Gk)、硬脂酰CoA去饱和酶-1(Scd1)、甘油-3-磷酸转移酶(Gpat2)、葡萄糖-6-磷酸酶(G6pc)、Irs1等10个调节糖脂代谢基因的RT-PCR验证实验结果显示:所有基因RT-PCR与基因芯片结果上调或下调趋势完全一致,80%基因差异倍数非常接近。结论祛湿化瘀方可调节高脂饮食诱导的脂肪肝大鼠脂肪代谢、糖类代谢、抗脂质过氧化及药物代谢等相关基因表达,表现出中药复方的综合药理作用。

Objective To observe the intervention and mechanism of Qushi Huayu Recipe（QHR） on gene expression profiles in high lipid diet induced fatty liver rats.Methods Fatty liver model was prepared in 20 male SD rats using single high fat diet（88%common forage ＋2%cholesterol ＋10%lard）.Four weeks after modeling they were divided into the model group and the QHR group according to random digit table,10 in each group.QHR（at 0.93 g crude drug/100 g body weight） and distilled water was respectively to rats in the QHR group and the model group by gastrogavage while modeling,.once per day.Meanwhile,10 SD male rats were recruited in a normal group,administered with equal volume of distilled water by gastrogavage.At the end of week 8 all rats were sacrificed,and blood and livers were collected for subsequent analysis.Contents of liver triglyceride（TG） and free fatty acid（FFA）,activities of serum alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were detected using biochemical assay.Pathological changes of liver tissue were observed using H＆E and oil red O stain.Liver gene expressions were detected by Affymetrix gene expression profiles.Differentially expressed genes were compared between the QHR group and the model group,functions of differentially expressed genes and signal pathways involved analyzed.Ten differentially expressed genes involved in glycolipid metabolism with fold change more than 2 were selected for verification by real-time PCR.Results（1） Compared with the normal group,contents of liver TG and FFA,and serum activities of ALT and AST obviously increased in the model group（P 〈0.01）.Compared with the model group,contents of liver TG and FFA,and activities of ALT and AST obviously decreased in the QHR group（P 〈0.05,P 〈0.01）.QHR could reduce high fat induced fatty degeneration of liver cells,alleviate inflammation,and improve pathological changes of liver tissue.（2） Compared with the model group,there were 80 differentially expressed genes（with fold change 〉2,P 〈0.05） with clear functions and appointed gene names,including44 up-regulated and 36 down-regulated genes.Eighty genes were involved in 27 signal pathways with statistical difference,including glycerolipid metabolism,adipocytokine signaling pathway,insulin signal pathway,drug metabolism signal pathway,etc（P〈0.05）.（3） RT-PCR results of 10 glycolipids metabolism regulating genes such as Gk,Scd1,Gpat2,G6 pc,Irs1,and so on showed that all RT-PCR genes were completely coincide with up-regulated or down-regulated tendency in results of gene chips.80%genes had approximate fold change.Conclusion QHR could regulate gene expressions related to fat metabolism,carbohydrate metabolism,anti-lipid peroxidation,and drug metabolism in high fat diet induced fatty liver rats,and its comprehensive pharmacological actions could be manifested.