摘要
该实验探讨真武汤对体外培养NRK-52E细胞AQP2相关的＂AVP-V2R-AQP2＂通路的调控作用,初步揭示真武汤调节水分转运平衡的部分分子机制。取SPF级雄性SD大鼠48只,随机分成8组,每组6只。分别给予蒸馏水或真武汤22.68 g·kg^-1·d^-1（按生药量计）灌胃,心脏采血,3 000 r·min^-1离心分离制备含药血清。然后体外培养大鼠肾小管上皮细胞NRK-52E细胞,给予上述不同给药天数和日给药次数制备的真武汤含药血清和血管加压素类似物1-去氨基-8-右旋-精氨酸加压素（1-desamino-8-D-arginine vasopressin,d DAVP）进行干预,采用实时无标记动态细胞分析技术（Real time cell analysis,RTCA）检测细胞增殖情况;免疫荧光法检测细胞中V2R,AQP2蛋白的分布情况;Western blot法检测V2R,PKA,AQP2的蛋白表达量;Real-time PCR法检测AQP2 mRNA水平。结果显示,与正常大鼠血清组比较,每天给药2次连续给药7 d制备的真武汤含药血清作用于NRK-52E细胞约24 h,可显著上调其AQP2的mRNA水平（P〈0.01）;显著增加V2R,PKA,AQP2的蛋白表达量（P〈0.05,P〈0.01,P〈0.05）;该方法制备的真武汤含药血清与d DAVP合用干预NRK-52E细胞后,可显著增加V2R,p-AQP2和AQP2的蛋白表达量（P〈0.01,P〈0.05,P〈0.01）。以上实验结果表明,真武汤含药血清能增加NRK-52E细胞＂AVP-V2R-AQP2＂通路中V2R,PKA及AQP2的蛋白表达量,与d DAVP合用存在协同效应,提示＂AVP-V2R-AQP2＂通路可能是真武汤调节水分转运平衡的分子机制之一。
This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52 E cells in vitro. Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22. 68 g·kg^-1·d^-1 Zhenwu Tang（ calculated by raw drug dosage meter） was given by gavage. Blood samples were collected by cardiac puncture,and the medicated serum was centrifuged from the blood by 3 000 r·min^-1. NRK-52 E cells were treated with different medicated serum or d DAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2R,PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA（ P〈0. 01） and protein expression of V2R,PKA and AQP2（ P〈0. 05,P〈0. 01,P〈0. 05） of Z7 d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2R,p-AQP2 and AQP2（ P〈0. 01,P〈0. 05,P〈0. 01） of Z7 d ＋d DAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2R,PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52 E,and there is synergistic effect between Zhenwu Tang medicated serum and d DAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.
作者
周小杰
包玉婷
陈红淑
宣玲
陈学明
张洁颖
杨元宵
李昌煜
ZHOU Xiao-jie;BAO Yu-ting;CHEN Hong-shu;XUAN Ling;CHEN Xue-ming;ZHANG Jie-ying;YANG Yuan-xiao;LI Chang-yu(Zhejiang Chinese Medicine University, Hangzhou 310053, China;Affiliated First Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China;Haagzhou Medical College, Hangzhou 310053, China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2018年第3期603-608,共6页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81373507,81673839)
浙江省自然科学基金项目(LQ16H280002)
浙江中医药大学校级科研基金项目(2017ZY23)
