基于转录组学分析复方胆草颗粒对非酒精性脂肪性肝炎小鼠肝细胞凋亡的影响  

Transcriptomics-based analysis of the effect of compound Dancao granules on hepatocyte apoptosis in mice with non-alcoholic steatohepatitis

作　　者：李旭涛 王四园 阮天音 张昊[2,3] 彭渊 刘成海[1,4] 陶艳艳[1] LI Xutao;WANG Siyuan;RUAN Tianyin;ZHANG Hao;PENG Yuan;LIU Chenghai;TAO Yanyan(Institute of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Xinxiang Medical College,Xinxiang 453003,China;Shanghai Hongwen Biotechnology Technology Co.,Ltd.,Shanghai 201403,China;Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院·肝病研究所,上海201203 [2]新乡医学院,河南新乡453003 [3]上海泓文生物科技有限公司,上海201403 [4]上海市中医临床重点实验室,上海201203

出　　处：《中国实验动物学报》2024年第12期1543-1555,共13页Acta Laboratorium Animalis Scientia Sinica

基　　金：上海市科委创新项目(19401901500)。

摘　　要：目的通过转录组测序技术(RNA sequencing,RNA-seq)分析复方胆草颗粒干预高脂饲料复合四氯化碳(carbon tetrachloride,CCl4)诱导的非酒精性脂肪性肝炎的作用机制。方法45只雄性C57BL/6J小鼠随机分为正常对照组、模型对照组、奥贝胆酸组(10 mg/(kg·d))、复方胆草颗粒低、高剂量组(3.74 g/(kg·d)、7.48 g/(kg·d)),每组9只。正常对照组喂养普通饲料,其余各组小鼠给予高脂饲料并复合CC1_(4)皮下注射,首次注射100%CC1_(4)溶液(4 mL/kg),之后40%CC1_(4)-橄榄油溶液(2 mL/kg),每周2次,共6周。奥贝胆酸组、复方胆草颗粒低、高剂量组从第3周开始给药,共给药4周。末次给药12 h后,取各组小鼠血清和肝组织,生化试剂盒检测小鼠血清肝功能;苏木素-伊红(HE)、天狼猩红及油红O染色观察小鼠肝组织病理学变化;酶联免疫吸附实验(ELISA)检测小鼠肝组织白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和转化生长因子-β(transforming growth factor beta,TGF-β)水平;免疫组化观察小鼠肝组织α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达;应用RNA-seq分析差异基因并进行功能富集分析、采用荧光定量逆转录PCR(quantitative reverse transcription PCR,qRT-PCR)验证差异基因mRNA的表达,再以脱氧核苷酸末端转移酶介导的dUTP缺口末端标记(TdT-mediated dUTP nick-end labeling,TUNEL)染色检测凋亡情况。结果与正常对照组比较,模型对照组小鼠血清谷丙转氨酶(alanine aminotransferase,ALT)、总胆固醇(total cholesterol,TC)、谷草转氨酶(aspartate transaminase,AST)、甘油三酯(triglyceride,TG)水平均明显升高(P<0.01),肝组织炎性细胞浸润明显,汇管区及小叶间胶原沉积,油红O染色显示红色脂滴面积显著增加(P<0.01),肝组织IL-6和TNF-α水平明显升高(P<0.01),IL-10和TGF-β水平下降(P<0.01),α-SMA的表达明显升高(P<0.01);与模型对照�ObjectiveTranscriptome sequencing technology(RNA-seq)was used to analyze the mechanism of compound Dancao granules as an intervention for high-fat feed combined with carbon tetrachloride(CCl4)-induced non-alcoholic steatohepatitis.Methods45 male C57BL/6J mice were split into two groups at random:normal control group,model control group,obeccholic acid group 10 mg/(kg·d),and compound Dancao granules low-and high-dose groups 3.74g/(kg·d)and 7.48g/(kg·d),with 9 mice in each group.Normal diet was made available to the control group,and the mice in the model group were given a high-fat diet combined with the subcutaneous injection of CC1_(4),with 100%CC1_(4)solution(4 mL/kg)in the first application,and 40%CC1_(4)-olive oil solution(2 mL/kg)in the second application,twice a week for a total of 6 weeks.Each drug group was administered the respective drug from week 3 for a total of 4 weeks.12 h after the last administration,the serum and liver tissues of mice in each group were collected,and a biochemical kit was used to detect serum liver function.Hematoxylin-eosin(HE),sirius scarlet,and oil red O staining were used to examine histopathological changes to the liver.The levels of IL-6,IL-10,TNF-αand TGF-βin mice liver were detected via ELISA,and the expression ofα-SMA was observed by immunohistochemistry.Differential gene expression was analyzed by RNA-seq and functional enrichment analysis.To verify the differential expression of mRNA,quantitative reverse transcription PCR(qRT-PCR)was used.TDT-mediated dUTP nick-end labeling(TUNEL)staining was employed to identify apoptosis.ResultsThe model control groups had significantly higher levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),and triglycerides(TG)than normal control group(P<0.01).Additionally,there was obvious inflammatory cell infiltration in the liver tissue,collagen deposition in the sink and interlobule areas,and a significant increase in lipid droplet area(P<0.01).The levels of IL-6 and TNF-αin liver tissu

关 键 词：复方胆草颗粒 非酒精性脂肪性肝炎 转录组测序 凋亡 

分 类 号：Q95-33[生物学—动物学]