Effect and mechanism of high concentration glucose on cholesterol absorption of human colon cancer epithelial Caco-2 cells / 中华消化外科杂志

ABSTRACT

Objective To investigate the effect and mechanism of high concentration glucose on cholesterol absorption of human colon cancer epithelial Caco-2 cells.Methods The experimental study was used.(1) CCK-8 detected cell proliferationthe proliferation rate changes of Caco-2 cells were detected by CCK-8 when different concentrations (12.5,100.0,300.0,700.0,1 000.0,1 388.0 mmol/L) of glucose solution effects on Caco-2 cells in order to ensure the half hindering concentration of glucose concentration on Caco-2 cells.(2)Cholesterol absorption of Caco-2 cells was detectedCaco-2 cells were divided into the cholesterol group,cholesterol plus ezetimibe (cholesterol inhibitor) group and blank control group.Cholesterol group100 μmol/L cholesterol solution and different concentrations (5.0,25.0,50.0 mmol/L) of glucose solution were added.Cholesterol plus ezetimibe group100 μmol/L ezetimibe,100 μmol/L cholesterol solution and different concentrations (5.0,25.0,50.0 mmol/L) of glucose solution were added.Blank control groupDMEM culture medium and corresponding concentrations of DMSO were added.The cholesterol absorption amounts of Caco-2 cells were measured.(3) The relative expressions of ATP binding cassette G8 (ABCG8),ATP binding cassette G5 (ABCG5),Nickman-Pick CI Like 1 (NPC1L1) and scavenger receptor class B type Ⅰ (SR-B Ⅰ) were examined by Western blot in the different groups.Caco cells were divided into the glucose group,glucose plus ezetimibe group and control group.The different concentrations (5.0,25.0,50.0 mmol/L) of glucose solution were added into the glucose group,different concentrations (5.0,25.0,50.0 mmol/L) of glucose solution and 100 μmol/L ezetimibe were added into the glucose plus ezetimibe group,and 100 μmol/L ezetimibe were added into the control group.The relative expressions of ABCG8,ABCG5,NPC1L1 and SR-B Ⅰ were detected by Western blot.Measurement data were presented as (x) ±s,repeated measure variance analysis was used to perform variation trend test,and t test was utilized to conduct comparisons among groups.Results (1) CCK-8 results showedproliferation rates of Caco-2 cells with the glucose solution concentration of 12.5,100.0,300.0,700.0,1 000.0 and 1 388.0 mmol/L were 1.380 ±0.043,1.238 ±0.072,0.736 ±0.035,0.336 ±0.021,0.316 ±0.020 and 0.288 ±0.010,respectively,with a statistically significant difference in the proliferation rates (F =11.019,P ＜ 0.05).The half hindering concentration of glucose solution on Caco-2 cells was 283.54 mmol/L.(2)Cholesterol absorption of Caco-2 cells① the cholesterol absorption amounts of Caco-2 cells with the glucose solution concentration of 5.0,25.0 and 50.0 mmol/L were 0.282 ± 0.042,0.380 ± 0.063,0.390 ± 0.060 in the cholesterol group and 0.042 ± 0.012,0.197 ± 0.015,0.277 ± 0.029 in the cholesterol plus ezetimibe group,respectively,showing a statistically significant difference between the 2 groups (F =55.566,P ＜ 0.05).②There was a statistically significant difference in cholesterol absorption amounts of Caco-2 cells with different glucose solution concentration in the cholesterol group (F =79.117,P ＜ 0.05).The cholesterol absorption amounts of Caco-2 cells with the glucose solution concentrations of 5.0 mmol/L was lower than that with the glucose solution concentrations of 25.0 mmol/L and 50.0 mmol/L,respectively (t =11.207,11.532,P ＜0.05).There was no statistically significant difference in the cholesterol absorption amounts of Caco-2 cells between the glucose solution concentrations of 25.0 mmol/L and 50.0 mmol/L (t =12.389,P ＞ 0.05).③There were statistically significant differences in cholesterol absorption amounts of Caco-2 cells with the glucose concentration of 5.0 mmol/L and 25.0 mmol/L between cholesterol group and cholesterol plus ezetimibe group (t =10.908,10.644,P ＜ 0.05).(3) The results of Western blot showed① the relative expression of NPC1L1 protein in Caco-2 cells with the glucose solution concentrations of 5.0,25.0 and 50.0 mmol/L were respectively 0.277 ±0.019,0.558 ±0.015,0.576 ±0.003 in the glucose group and 0.057 ±0.002,0.054 ±0.005,0.077 ±0.005 in the glucose plus ezetimibe group,showing a statistically significant difference (F =482.207,P ＜0.05).② The relative expression of NPC1L1 protein of Caco-2 cells with the different concentration of glucose solution in the glucose group were compared,with a statistically significant difference (F =8.112,P ＜ 0.05).There was a statistically significant difference in the relative expression of NPC1L1 protein in Caco-2 cells with the different concentration of glucose solution in the glucose plus ezetimibe group (F =11.708,P ＜ 0.05).③ The relative expression of NPC1L1 protein in Caco-2 cells with the glucose solution concentrations of 5.0,25.0 and 50.0 mmol/L in the glucose group was statistically different from that in the glucose plus ezetimibe group (t =8.112,11.708,13.920,P ＜ 0.05).Conclusion High concentration glucose solution could promote the reabsorption of cholesterol through increasing NPC1L1 protein expression in Caco-2 cells,and increase the risk of suffering from cholelithiasis in diabetes patients.