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目的:观察6-姜酚对Caco-2细胞肽转运载体PepT1转运二肽的能力及其蛋白、mRNA表达的影响。方法:Caco-2细胞融合后连续培养28 d后给予6-姜酚处理,并设立正常对照组及cAMP抑制剂对照组,用放射性同位素示踪技术比较Caco-2细胞转运二肽化合物Glycyl-Sarcosine的能力,采用Western blot方法测定Caco-2细胞膜上PepT1蛋白的表达,荧光定量PCR方法测定PepT1 mRNA(SLC15A1)的表达水平。结果:6-姜酚处理组Caco-2细胞60,120 min时点Glycyl-Sarcosine吸收转运累计量分别为(6.84±0.46),(8.61±0.54)μmol/孔,高于正常对照组(P<0.05),后者分别为(5.62±0.20)、(6.54±0.54)μmol/孔;6-姜酚与Rp-8-Br-cAMP合用组120 min时点为(6.92±0.67)μmol/孔,明显低于相应时点单用6-姜酚组(P<0.05);6-姜酚处理后Caco-2细胞膜PepT1蛋白表达增加,PepT1表达与内参GAPDH表达灰度比值6-姜酚组为(0.317±0.022),高于空白对照组(0.220±0.019)(P<0.01);荧光定量PCR结果,6-姜酚能上调Caco-2细胞SLC15A1 mRNA表达,但与cAMP抑制剂合用后该抑制作用被阻断。结论:6-姜酚具有促进正常培养Caco-2细胞转运二肽化合物Glycyl-Sarcosine的作用,该作用可能与上调PepT1蛋白及mRNA表达有关,该过程调控与胞内第二信使cAMP有一定的关系。
OBJECTIVE: To observe the effect of 6-gingerol on the ability of PepT1 to transport dipeptide and its protein and mRNA expression in Caco-2 cell. Methods: Caco-2 cells were fused and continuously treated with 6-gingerol for 28 days. A normal control group and a cAMP inhibitor control group were established. Caco-2 cells were used to transport the dipeptide compound Glycyl-Sarcosine The expression of PepT1 protein in Caco-2 cell membrane was determined by Western blot and the expression level of PepT1 mRNA (SLC15A1) by fluorescence quantitative PCR. Results: The cumulative amount of Glycyl-Sarcosine uptake and transport in Caco-2 cells treated with Gingerol treatment for 6 and 120 min were (6.84 ± 0.46) and (8.61 ± 0.54) μmol / well, respectively, higher than that of the normal control group (P <0.05) , And the latter were (5.62 ± 0.20) and (6.54 ± 0.54) μmol / well, respectively. The time point of combination of 6-gingerol and Rp-8-Br-cAMP was (6.92 ± 0.67) μmol / (P <0.05). The expression of PepT1 protein in Caco-2 cell membrane increased with 6-Gingerol treatment, and the ratio of PepT1 expression to GAPDH expression level in 6-Gingerol group was (0.317 ± 0.022), which was higher than that of the blank control group (0.220 ± 0.019) (P <0.01); 6-gingerol could up-regulate the SLC15A1 mRNA expression in Caco-2 cells by fluorescence quantitative PCR, but this inhibition was blocked with cAMP inhibitor Off. CONCLUSION: 6-Gingerol can promote the transport of Glycyl-Sarcosine, a dipeptide compound, in normal cultured Caco-2 cells. This effect may be related to the up-regulation of PepT1 protein and mRNA expression. The regulation of this process may be related to the intracellular second messenger cAMP .