丁酸钠通过G蛋白偶联受体43/109a-磷酸化蛋白激酶B-哺乳动物雷帕霉素靶蛋白通路影响脂肪变性HepG2细胞的增殖与凋亡  

Sodium butyrate affected the proliferation and apoptosis of steatosis HepG2 cells via G protein-coupled receptor 43/109a-phosphorylated protein kinase B-mammalian target of rapamycin signaling pathway

作　　者：李煜 李梦婷 刘韬韬 周达 Li Yu;Li Mengting;Liu Taotao;Zhou Da(Department of Gastroenterology and Hepatology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属中山医院消化科,上海200032 [2]宁波大学附属人民医院消化科,315000

出　　处：《中华消化杂志》2021年第7期471-477,共7页Chinese Journal of Digestion

基　　金：国家自然科学基金(青年基金)(81800510);上海市青年科技英才扬帆计划(18YF1415900)。

摘　　要：目的通过体外研究探索肠道菌群代谢产物丁酸钠对脂肪变性HepG2细胞增殖与凋亡的影响机制。方法采用人源性肝细胞系HepG2、游离脂肪酸(FFA;油酸与棕榈酸浓度比为2∶1)建立体外脂肪变性肝细胞模型。设置正常对照组、模型组、丁酸钠不同浓度(1、2、5、10、20、50 mmol/L)干预组,使用细胞计数试剂盒(CCK-8)检测丁酸钠对脂肪变性HepG2细胞增殖的抑制作用;设置正常对照组、模型组和丁酸钠5 mmol/L干预(丁酸钠干预)组,采用流式细胞术检测各组凋亡细胞比例;设置正常对照组、模型组和丁酸钠1、2、5、10 mmol/L干预组、空白小干扰RNA(siRNA)干扰组、G蛋白偶联受体(GPR)43干扰组、GPR109a干扰组、GPR43/GPR109a双敲组,采用蛋白质印迹法检测各组转染前后的磷酸化蛋白激酶B(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)蛋白质水平变化。统计学方法采用单因素方差分析、SNK-q检验和logistic回归分析。结果CCK-8检测结果提示丁酸钠对脂肪变性HepG2细胞增殖的抑制作用有剂量、时间依赖性。流式细胞术检测结果显示,丁酸钠干预组的细胞凋亡比例高于模型组和正常对照组[(3.400±0.100)%比(1.800±0.400)%、(1.067±0.451)%],差异均有统计学意义(t=6.721、8.705,P均<0.01);模型组凋亡细胞比例与正常对照组比较差异无统计学意义(P>0.05)。转染前,模型组的p-AKT、p-mTOR蛋白质水平均高于正常对照组(2.300±0.058比1.000±0.012、2.160±0.125比1.000±0.052),差异均有统计学意义(t=22.080、8.575,P均<0.05);丁酸钠1、2、5、10 mmol/L干预组的p-AKT和p-mTOR蛋白质水平均低于模型组(1.530±0.085、1.407±0.096、1.032±0.035、1.036±0.099比2.300±0.058,1.483±0.073、1.297±0.048、1.067±0.035、0.970±0.072比2.160±0.125),差异均有统计学意义(t=7.491、7.997、19.790、11.020,4.683、6.445、8.424、8.245,P均<0.05)。转染后,GPR43干扰组、GPR109a干扰组和GPR43/GPR109a双敲组的p-AKObjective To investigate the effects and mechanism of gut metabolite sodium butyrate on the proliferation and apoptosis of steatosis HepG2 cells in vitro.Methods The in vitro steatosis hepatocyte model was established with human liver cell line HepG2 and free fatty acid(FFA;the concentration ratio of oleic acid to palmitic acid was 2∶1).Normal control group,model group and intervention groups with different concentration(1,2,5,10,20 and 50 mmol/L)of sodium butyrate were set up.The inhibition of sodium butyrate on the proliferation of steatosis HepG2 cells was detected by cell counting kit(CCK-8).The proportion of apoptotic cells of normal control group,model group and sodium butyrate 5 mmol/L(sodium butyrate intervention)group was detected by flow cytometry.Normal control group,model group,intervention group with different concentration(1,2,5 and 10 mmol/L)of sodium butyrate,negative small interfering RNA(siRNA)control group,G protein-coupled receptor(GPR)43-siRNA group,GPR109a-siRNA group,GPR43+GPR109a double knockout group were set up.The change of the levels of phosphorylated protein kinase B(p-AKT)and phosphorylated mammalian target of rapamycin(p-mTOR)before and after transfection were detected by Western blotting.One-way analysis of varivance,SNK-q test and logistic regression analysis were used for statistical analysis.Results The results of CCK-8 test indicated that sodium butyrate inhibited the proliferation of steatosis HepG2 cells in a dose-dependent and time-dependent manner.The results of flow cytometry showed that the proportion of apoptotic cells of the sodium butyrate intervention group was higher than that of the model group and normal control group((3.400±0.100)%vs.(1.800±0.400)%and(1.067±0.451)%),and the differences were statistically significant(t=6.721 and 8.705,both P<0.01).There was no significant difference in the proportion of apoptotic cells between the model group and the normal control group(P>0.05).Before transfection,the expressions of p-AKT and p-mTOR at protein level of the mo

关 键 词：丁酸钠 脂肪变性 肝细胞 细胞增殖 细胞凋亡 

分 类 号：R575.5[医药卫生—消化系统]