冰片对血肿瘤屏障通透性的影响及机制

Effect of Borneol on the Permeability of Blood Tumor Barrier Model and Its Mechanism Study

目的观察天然冰片对体外血肿瘤屏障（blood tumor barrier,BTB）模型通透性和丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPKs）信号转导通路相关激酶表达与激活的影响。方法大鼠C6脑胶质瘤细胞与人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）共培养建立BTB模型后,设空白组,冰片低、中、高剂量组（25、50、100μg/m L）,每组7个时点（0、10、30、60、120、180、240 min）,每个时点3个样本。空白组在冰片组给药同时更换空白培养基,冰片组给不同剂量冰片,给药后不同时间点收集细胞。辣根过氧化物酶（horseradish peroxidase,HRP）流量测定BTB通透性,Western blot检测细胞外信号调节激酶（extracellular signal regulated protein kinase,ERK）,磷酸化ERK（phosphorylation extracellular signal regulated protein kinase,P-ERK）,P38MAPK,磷酸化P38MAPK（phosphor-P38,P-P38MAPK）,氨基末端激酶（c-Jun N-terminal kinase,JNK）和磷酸化JNK（phosphorylation c-Jun N-terminal kinase,P-JNK）的表达。结果与本组0 min比较,低、中、高剂量组各时间点通透率升高（P〈0.01）,P-ERK表达先升高,在30 min达极高值,逐渐恢复初始水平（P〉0.05）。与空白组比较,低剂量组10~240 min HRP通透率升高（P〈0.01）,30、60 min PERK蛋白表达升高（P〈0.05）,低、中、高剂量组180 min P-JNK表达升高（P〈0.05）,240 min P-JNK表达降低（P〈0.05）。与低剂量组比较,中剂量组10~180 min P-ERK表达升高（P〈0.05）,30~180 min通透率升高（P〈0.05）,180 min和240 min P-JNK表达降低（P〈0.05）。与中剂量组比较,高剂量组10~180 min通透率升高（P〈0.05）,10~180 min P-ERK表达升高（P〈0.05）,180 min和240 min P-JNK表达降低（P〈0.05）。结论冰片通过激活MAPKs信号通路的ERK磷酸化进而可逆性下调相关蛋白的表达,达到调节可逆性BTB开放的效果。

Objective To observe the effect of natural borneol on the permeability of blood tumor barrier（BTB） model and the expression and activation of mitogen-activated protein kinase（MAPKs） signal transduction pathway related protein kinase in vitro.Methods C6 rat glioma cells and human umbilical vein endothelial cells（HUVECs） were co-cultured to establish BTB model.Then 4 groups were set up,the blank control group,low,middle,and high dose borneol groups（25,50,100 μg/mL）,3 samples collected at 7 time points（0,10,30,60,120,180,240 min,respectively）.Blank culture medium was exchanged in the blank control group while medication.Different doses of natural borneol were administered to the 3 borneol groups.Cells were collected at different time points.BTB permeability was determined using horseradish peroxidase（HRP）.Expression levels of extracellular signal regulated protein kinase（ERK）,phosphorylation extracellular signal regulated protein kinase（P-ERK）,P38 MAPK,phosphor-P38 MAPK,c-Jun N-terminal kinase（JNK）,and phosphorylation c-Jun N-terminal kinase（PJNK） were detected using Western blot.Results Compared with the same group at min 0,the permeation rate obviously increased（P 〈0.0 1） in the 3 borneol groups at the rest time points.P-ERK expression was elevated first,reached the peak at 3 0 min,and gradually recovered to the initial level（P 〈0.05）.Compared with the blank control group,HRP permeation rate increased from 10 min to 240 min（P 〈0.01）,and expression of P-ERK protein increased at 30 min and 60 min（P0.05） in the low dose borneol group;expression of P-JNK protein decreased in the 3 borneol groups at 1 80 min and 240 min（P0.05）.Compared with the low dose borneol group,expression of P-ERK protein increased from 10 min to 180 m in（P0.05）,HRP permeation rate increased from 30 min to 180 min（P0.05）,expression of P-JNK protein decreased at 1 80 and 240 min（P 〈0.05） in the middle dose borneol group.Compared with the middle dose borneol group,HRP permeation rate increased from 1 0 min to 1 80 min（P 〈0.05）,expression ofP-ERK protein increased from 10 min to 180 min（P0.05）,expression of PJNK protein increased at 1 80 min and decreased at 240 min（both P0.05） in the high dose borneol group.Conclusion Natural borneol arrived at the effect of regulating reversible BTB patency possibly through activating phosphorylation of ERK in MAPKs signal transduction pathway,and further reversibly down-regulating expression of associated proteins.