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 proliferation: the 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 detected: Caco-2 cells were divided into the cholesterol group, cholesterol plus ezetimibe (cholesterol inhibitor) group and blank control group. Cholesterol group: 100 μmol/L cholesterol solution and different concentrations (5.0, 25.0, 50.0 mmol/L) of glucose solution were added. Cholesterol plus ezetimibe group: 100 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 group：DMEM 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) , NickmanPick CI Like 1 (NPC1L1) and scavenger receptor class B type Ⅰ (SRBⅠ) 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 SRBⅠ were detected by Western blot. Measurement data were presented as
±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 showed: proliferation 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 cells: ① 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). ② There 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). ③ There 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 showed: ① 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.
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