醋柴胡多糖联用奥沙利铂增效活性及机制研究

Synergistic activity and mechanism of vinegar baked radix bupleurum polysaccharides in combination with oxaliplatin

摘要

目的 探讨醋柴胡多糖联用奥沙利铂（oxaliplatin，OXA）增效活性及机制，为临床治疗原发肝细胞癌提供新的思路。 方法 噻唑蓝（methyl thiazolyl tetrazolium，MTT）法检测醋制柴胡多糖3-4（vinegar baked radix bupleurum polysaccharides 3-4，VBCP 3-4）、醋制柴胡多糖3-3（vinegar baked radix bupleurum polysaccharides 3-3，VBCP 3-3）联用OXA对Huh7细胞的细胞杀伤作用；电感耦合等离子体质谱（inductively coupled plasma mass spectrometry，ICP-MS）检测Huh7细胞对OXA的摄取率，评价醋柴胡多糖VBCP 3-4联用OXA的体外增效途径；蛋白印迹法测定Huh7相关转运蛋白的表达，探讨VBCP 3-4联用OXA的增效机制。 结果 VBCP 3-4、VBCP 3-3能够增加OXA对细胞的生长抑制作用。VBCP 3-4能够促进Huh7细胞对OXA的摄取，摄取率在4 h达到峰值，高、低剂量联用组增加摄取率为：分别43.43%（P=0.000）和29.02%（P=0.000）。OXA低、高剂量组单独作用Huh7细胞，能够促进细胞多药耐药相关蛋白（multidrug resistance-associated protein，MRP）2和MRP1蛋白表达，MRP2蛋白的上调幅度为18.11%、25.00%（P=0.008、P=0.001），MRP1蛋白的上调幅度为28.51%（P>0.05）、39.70%（P=0.015）。VBCP 3-4联用OXA后，MRP2、MRP1及乳腺癌耐药蛋白（breast cancer resistance protein，BCRP）蛋白表达受到抑制，联用高、低剂量组MRP2降低幅度分别为47.38%、15.18%（P=0.000、P=0.049），MRP1蛋白降低幅度分别为64.96%、34.63%（P=0.000、P=0.000），联用高剂量组BCRP蛋白表达降低幅度为29.00%（P=0.020）；VBCP 3-4单独作用Huh7细胞后，能够明显降低MRP2、MRP1及BCRP蛋白表达，VBCP 3-4高剂量组下调MRP2、MRP1幅度为24.91%、20.79%（P=0.004、P=0.005）。VBCP3-4下调BCRP蛋白的表达，高、中剂量下调幅度分别为15.02%、13.92%（P=0.003、P=0.030）。 结论 醋柴胡多糖VBCP3-4通过抑制外排型转运蛋白MRP1、MRP2、BCRP表达，促进Huh7细胞摄入OXA，提高胞内有效浓度实现增效作用。

Abstract

Objective To investigate the synergistic activity and mechanism of vinegar baked radix bupleurum polysaccharides（VBCP） in combination with oxaliplatin（OXA），and to provide new ideas for the clinical treatment of primary hepatocellular carcinoma. Methods MTT assay was used to detect the cytotoxic effect of VBCP 3-4 and VBCP 3-3 in combination with OXA on Huh7 cells；ICP-MS was used to measure the uptake rate of OXA by Huh7 cells and evaluate the in vitro synergistic pathway of VBCP 3-4 in combination with OXA； Western blotting was used to measure the expression levels of related transporter proteins in Huh7 cells and explore the synergistic mechanism of VBCP 3-4 in combination with OXA. Results VBCP 3-4 and VBCP 3-3 enhanced the inhibitory effect of OXA on the growth of cells. VBCP 3-4 promoted the uptake of OXA by Huh7 cells，with the peak uptake rate at 4 hours，and the uptake rate was increased to 43.43% in the high-dose combination group（P=0.000） and 29.02% in the low-dose combination group（P=0.000）. Acting on Huh7 cells，the low- and high-dose OXA alone could promote the protein expression levels of MRP2 and MRP1 in Huh7 cells，and the protein expression level of multidrug resistance-associated protein（MRP） 2 was upregulated to 18.11% and 25.00%，respectively（P=0.008，P=0.001），while that of MRP1 was upregulated to 28.51%（P>0.05） and 39.70%（P=0.015），respectively. After the combination of VBCP 3-4 and OXA，the protein expression of MRP2，MRP1，and breast cancer resistance protein（BCRP） was inhibited； MRP2 was reduced by 47.38% in the high-dose combination group（P=0.000） and 15.18% in the low-dose combination group（P=0.049）；MRP1 was reduced by 64.96% in the high-dose combination group（P=0.000） and 34.63% in the low-dose combination group（P=0.000）； BCRP was reduced by 29.00%（P=0.020） in the high-dose combination group. Acting on Huh7 cells alone，VBCP 3-4 significantly reduced the protein expression levels of MRP2，MRP1，and BCRP，and in the high-dose VBCP 3-4 group，MRP2 and MRP1 were reduced by 24.91% and 20.79%，respectively（P=0.004，P=0.005）. VBCP 3-4 downregulated the protein expression level of BCRP by 15.02% in the high-dose group（P=0.003） and 13.92% in the middle-dose group（P=0.030）. Conclusion VBCP 3-4 exerts a synergistic effect by inhibiting the expression of the efflux transporter proteins MRP1，MRP2，and BCRP，promoting the intake of OXA by Huh7 cells，and increasing the intracellular effective concentration.

关键词

醋柴胡多糖 / 奥沙利铂 / 联用增效 / 抗肿瘤

Key words

vinegar-processed Bupleurum chinense polysaccharides / OXA / synergistic effect / antitumor effect

基金资助

国家自然科学基金资助项目(82173981;82074086)

广东省科技计划资助项目(2023B1212060063;2021A1515110846)