左金丸对IEC6、HCT116细胞共培养模型肠上皮屏障功能的保护及机制研究  

Protection and Mechanism of Zuojin Pills on Intestinal Epithelial Barrier Function in A Co-culture Model of IEC6 and HCT116

作　　者：余彩雁 靳淑颖 周四桂 王桂香 YU Caiyan;JIN Shuying;ZHOU Sigui;WANG Guixiang(School of Pharmacy,Guangdong Pharmaceutical University,Guangzhou 510006 Guangdong,China)

机构地区：[1]广东药科大学药学院,广东广州510006

出　　处：《中药新药与临床药理》2023年第1期1-7,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81873225)。

摘　　要：目的 研究左金丸对IEC6、HCT116细胞共培养模型肠上皮屏障功能的影响,并初步探讨其作用机制。方法 采用左金丸醇提物低、中、高剂量(5、10、20μg·mL^(-1))处理HCT116、IEC6细胞,采用MTT法检测细胞增殖情况。通过体外构建IEC6、HCT116细胞共培养体系模拟结肠癌微环境,以左金丸(5、10、20μg·mL^(-1))进行干预;检测IEC6细胞跨膜电阻(TEER)值及细胞通透性;采用高效液相色谱(HPLC)法检测HCT116、IEC6细胞多胺含量;微量法检测HCT116细胞的葡萄糖消耗量、乳酸及ATP含量;Western Blot法检测IEC6细胞Zonula occluden 1(ZO-1)、Occludin蛋白和HCT116细胞鸟氨酸脱羧酶(ODC)、精眯/精胺N1-乙酰基转移酶(SSAT)、鸟氨酸脱羧酶抗酶1(OAZ1)、SLC22A1、缺氧诱导因子1(HIF1α)、C-Myc、己糖激酶2(HK2)、丙酮酸激酶M2亚型(PKM2)、丙酮酸脱氢酶激酶1(PDK1)、葡萄糖转运体1(GLUT1)、乳酸脱氢酶(LDHA)蛋白表达情况。结果 (1)与对照组比较,左金丸低、中、高剂量(5、10、20μg·mL^(-1))处理HCT116细胞24、48、72 h后,细胞增殖抑制率显著升高(P<0.01);左金丸低、中、高剂量(5、10、20μg·mL^(-1))处理IEC6细胞24 h后,细胞增殖抑制率无明显变化(P>0.05)。(2)与IEC6细胞单培养组比较,共培养模型组细胞的TEER及FD4荧光强度显著降低(P<0.01);紧密连接蛋白ZO-1、Occludin表达量显著降低(P<0.01);IEC6细胞的多胺含量显著降低(P<0.01)。与HCT116细胞单培养组比较,共培养模型组HCT116细胞的多胺含量显著增加(P<0.01)。与共培养模型组比较,左金丸中、高剂量组细胞的TEER及FD4荧光强度显著升高(P<0.01);左金丸高剂量组IEC6细胞ZO-1、Occludin蛋白表达明显上调(P<0.05,P<0.01);左金丸低、中、高剂量组的IEC6细胞多胺含量显著增加(P<0.01),而左金丸中、高剂量组HCT116细胞的多胺含量显著降低(P<0.01)。(3)与对照组比较,左金丸能明显下调HCT116细胞多胺合成酶ODC、多胺转运体SLC22A1蛋�Objective To study the effect of Zuojin Pills on the function of intestinal epithelial barrier in a co-culture model of IEC6 and HCT116 cells and to preliminarily investigate its mechanism of action.Methods HCT116 and IEC6 cells were treated with low-,medium-and high-doses(5,10 and 20μg·mL^(-1))of the ethanol extraction of Zuojin Pills,and cell proliferation was detected by MTT assay.In vitro co-culture system of IEC6 and HCT116 cells was constructed to stimulate the microenvironment of colon cancer,and the intervention was performed with Zuojin Pills(5,10 and 20μg·mL^(-1));the trans epithellal electric resistance(TEER)values and cell permeability of IEC6 cells were measured;the polyamine content of HCT116 and IEC6 cells was measured by high performance liquid chromatography(HPLC);glucose consumption,lactate concentration and ATP content of HCT116 cells were measured by microvolume method;protein expressions of Zonula occluden 1(ZO-1),Occludin protein and ornithine decarboxylase(ODC),spermidine/spermine N1-acetyltransferase(SSAT),ornithine OAZ1,SLC22A1,hypoxia-inducible factor 1(HIF1α),C-Myc,hexokinase 2(HK2),pyruvate kinase M2 isoform(PKM2),pyruvate dehydrogenase kinase 1(PDK1),glucose trans porter 1(GLUT1),and lactic dehydrogenase(LDHA)were detected by Western Blot.Results (1)Compared with the control group,the proliferation inhibition rate of HCT116 cells treated with low-,medium-and high-doses(5,10 and 20μg·mL^(-1))of Zuojin Pills for 24,48 and 72 hours was significantly increased(P<0.01);the proliferation inhibition rate of IEC6 cells treated with low-,medium-and high-doses(5,10 and 20μg·mL^(-1))of Zuojin Pills for 24 hours did not change significantly(P>0.05).(2)Compared with the IEC6 cells monoculture group,the fluorescence intensity of TEER and FD4 was significantly reduced in the co-culture model group(P<0.01);the expressions of tight junction protein ZO-1 and Occludin were significantly reduced(P<0.01);the polyamine content of IEC6 cells was significantly reduced(P<0.01).Compared with the HCT11

关 键 词：左金丸 结肠癌HCT116细胞 肠上皮IEC6细胞 细胞共培养 多胺 糖酵解 

分 类 号：R285.5[医药卫生—中药学]