大黄素对人胚胎肝细胞L02细胞株法尼醇X受体的调节作用

摘要

Objective To investigate the regulation effect of emodin on human embryonic liver L02 cells strain farnesoid X receptor (FXR) pathways.Methods By using Guggulsterones,FXR genes were intervened with in L02 cells as model group,in three different concentrations of emodin (50.0 μ mol/L,25.0 μmol/L,12.5 μmol/L) of emodin in the model group cells,FXR,small heterodimer parter (SHP),UDP-glucuronosyltransferase 2B4 (UGT2 B4),bile salt export pump(BSEP) mRNA and protein expressions were detected by real-time fluorescent quantitative PCR and Western blot test.Results (1) The relative expressions of FXR mRNA and protein in the model group (0.240 ± 0.021,0.385 ±0.119) decreased significantly than those of control group (1.000 ± 0.088,1.000 ± 0.223),the differences were statistically significant (t =14.62,4.21,all P ＜ 0.01).Compared with the model group,the relative expressions of FXR mRNA in the high,the middle and the low-dose emodin groups (0.755 ±0.083,0.817 ±0.097,0.547 ± 0.080) were significantly higher (t =10.42,10.03,6.39,all P ＜ 0.01).The relative expressions of FXR protein in the medium-dose group (0.865 ± 0.203) increased significantly (t =3.53,P ＜ 0.01).The relative expressions of FXR mRNA in the high and the medium-dose groups (0.755 ± 0.083,0.817 ± 0.097) were higher than those in the low-dose group (0.547 ± 0.080),the differences were statistically significant (t =3.11,3.70,all P ＜ 0.01).(2)The relative expressions of SHP,UGT2B4,BSEP mRNA and protein in the model group (0.148 ±0.025,0.205 ± 0.039,0.184 ± 0.020;0.458 ± 0.130,0.255 ± 0.170,0.303 ± 0.100) were significantly lower than those in the control group (1.000 ±.0.099,1.000 ±0.104,1.000 ±0.125;1.000 ±0.129,1.000 ±0.157,1.000 ±0.162),the differences were statistically significant (t =14.50,12.44,11.19,5.13,5.57,6.33,all P ＜ 0.01).The relative expressions of SHP,UGT2B4 and BSEP mRNA in the high,the middle and the low-dose groups (0.610 ± 0.058,0.514 ± 0.041,0.707 ± 0.062;0.755 ± 0.108,0.800 ± 0.086,0.727 ± 0.076;0.470 ± 0.070,0.582 ± 0.050,0.500±0.108) were significantly lower than those in the model group (0.148 ± 0.025,0.205 ± 0.039,0.184 ± 0.020),the differences were statistically significant (t =12.75,9.38,13.94,9.46,10.90,11.96,7.53,10.31,5.00,all P ＜0.01).The relative expressions of SHP,UGT2B4 and BSEP mRNA in the high and the middle-dose emodin group (0.658 ±0.091,0.624 ±0.113,0.607 ±0.097;0.868 ±0.194,0.883 ±0.099,0.913 ±0.131) were significantly higher than those in the low-dose group (0.458 ±0.130,0.255 ±0.170,0.303 ±0.100),the differences were statistically significant (t =2.18,3.13,3.78,3.05,5.53,6.41,all P ＜ 0.01).The relative expression of SHP and BSEP protein in the low-dose group (0.645 ±0.135,0.572 ±0.076) increased,the differences were statistically significant (t =1.73,P ＜ 0.05,t =3.72,P ＜ 0.01).The relative expression of BSEP protein in the high and the medium-dose groups (0.607 ±0.097,0.913 ± 0.131) was significantly higher than those in the low-dose group (0.572 ± 0.076),the differences were statistically significant (t =1.99,3.90,all P ＜ 0.01).The relative expressions of SHP and UGT2B4 mRNA in the high and the low-dose group (0.610 ±0.058,0.470 ±0.070;0.514 ± 0.041,0.582 ± 0.050) were significantly lower than those in the medium-dose group (0.800 ± 0.086),the differences were statistically significant (t =3.75,6.47,3.83,3.42,all P ＜ 0.01).The expression levels of UGT2B4 and BSEP in the high and the low-dose groups (0.624 ± 0.113,0.644 ± 0.097;0.607 ± 0.097,0.572 ± 0.076) were significantly lower than those in the medium-dose group (0.883 ± 0.099,0.913 ± 0.131),the differences were statistically significant (t =4.27,2.98,6.30,3.90,all P ＜ 0.01).Conclusions Guggulsterones can inhibit FXR and downstream genes SHP,UGT2B4,BSEP expressions in L02,and emodin can enhance FXR gene expression,promote SHP,UGT2B4,BSEP gene expression,inhibit cholestasis pathway,protection of liver cell,which shows a dosage discreapancy.%目的 研究大黄素对人胚胎肝细胞L02细胞株法尼醇X受体(FXR)通路的调节作用.方法 模型组采用孕二烯二酮(Guggulsterones)对L02细胞株的FXR基因干预,根据大黄素剂量分为高剂量、中剂量、低剂量组(50.0 μmol/L、25.0 μmol/L、12.5 μmol/L),另设对照组.采用实时荧光定量PCR与Western blot检测FXR及下游小异源二聚体伴侣受体(SHP)、尿苷二磷酸葡萄糖苷酸转移酶2B4(UGT2B4)、胆盐输出泵(BSEP)mRNA及蛋白表达.结果 1.模型组FXR mRNA及蛋白相对表达量(0.240±0.021、0.385 ±0.119)较对照组(1.000±0.088、1.000±0.223)显著降低,差异均有统计学意义(t=14.62、4.21,均P＜0.01);与模型组比较,大黄素高、中、低剂量组FXR mRNA相对表达量(0.755±0.083、0.817±0.097、0.547±0.080)显著升高,差异均有统计学意义(t=10.42、10.03、6.39,均P＜0.01);中剂量组FXR蛋白相对表达量(0.865±0.203)显著升高,差异有统计学意义(t =3.53,P＜0.01);高、中剂量组FXR mRNA相对表达量(0.755±0.083、0.817±0.097)均高于低剂量组(0.547±0.080),差异均有统计学意义(t=3.11、3.70,均P＜0.01).2.模型组SHP、UGT2B4、BSEP mRNA及蛋白相对表达量(0.148 ±0.025、0.205±0.039、0.184 ±0.020,0.458±0.130、0.255±0.170、0.303 ±0.100)较对照组(1.000±0.099、1.000 ±0.104、1.000±0.125,1.000±0.129、1.000±0.157、1.000 ±0.162)显著降低,差异均有统计学意义(t=14.50、12.44、11.19、5.13、5.57、6.33,均P＜0.01).高、中、低剂量组SHP、UGT2B4、BSEP mRNA相对表达量(0.610±0.058、0.514±0.041、0.707±0.062,0.755±0.108、0.800±0.086、0.727±0.076,0.470 ±0.070、0.582 ±0.050、0.500 ±0.108)较模型组(0.148±0.025、0.205±0.039、0.184 ±0.020)显著升高,差异均有统计学意义(t=12.75、9.38、13.94、9.46、10.90、11.96、7.53、10.31、5.00,均P＜0.01);高、中剂量组SHP、UGT2B4、BSEP蛋白相对表达量(0.658 ±0.091、0.624±0.113、0.607 ±0.097,0.868 ±0.194、0.883±0.099、0.913 ±0.131)较模型组(0.458 ±0.130、0.255±0.170、0.303 ±0.100)显著升高,差异均有统计学意义(t=2.18、3.13、3.78、3.05、5.53、6.41,均P＜0.01),低剂量组SHP、BSEP蛋白相对表达量(0.645 ±0.135、0.572 ±0.076)不同程度升高,差异均有统计学意义(t=1.73,P＜0.05,t =3.72,P＜0.01).大黄素高、中剂量组BSEP蛋白相对表达量(0.607±0.097、0.913 ±0.131)均高于低剂量组(0.572 ±0.076),差异均有统计学意义(t=1.99、3.90,均P＜0.01);高、低剂量组SHP、UGT2B4 mRNA相对表达量(0.610 ±0.058、0.470 ±0.070,0.514 ±0.041、0.582 ±0.050)均低于中剂量组(0.800±0.086),差异均有统计学意义(t=3.75、6.47、3.83、3.42,均P＜0.01);高、低剂量组UGT2B4、BSEP蛋白表达量(0.624±0.113、0.644 ±0.097,0.607 ±0.097、0.572 ±0.076)均低于中剂量组(0.883±0.099、0.913 ±0.131),差异均有统计学意义(=4.27、2.98、6.30、3.90,均P＜0.01).结论 Guggulsterones能抑制L02细胞株FXR及下游SHP、UGT2B4、BSEP基因表达,大黄素可通过对FXR基因表达上调,促进SHP、UGT2B4、BSEP基因表达上调,抑制胆汁淤积通路来保护肝细胞,但有剂量差异.