健脾活血祛湿方经AQP9和线粒体细胞凋亡途径对H22肝癌荷瘤裸鼠的影响  

Effect of Jianpi Huoxue Qushi Decoction on nude mice bearing H22 hepatoma through AQP9 and mitochondrial apoptosis pathway

作　　者：李嘉 高玲 陈晓兰 杨孝芳 蒲翔 张雄 LI Jia;GAO Ling;CHEN Xiao-lan;YANG Xiao-fang;PU Xiang;ZHANG Xiong(Guizhou University of Traditional Chinese Medicine,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州中医药大学,贵州贵阳550025

出　　处：《中国临床研究》2020年第8期1014-1018,1049,共6页Chinese Journal of Clinical Research

基　　金：国家自然科学基金(81560771,81860887);黔苗医药项目(K字[2017]010号);贵州省中医药、民族医药科学技术专项课题项目(QZYY-2018-013);贵州中医药大学院内科研项目(200)。

摘　　要：目的观察健脾活血祛湿方对H22肝癌皮下移植瘤裸鼠的影响,探讨其是否通过干预水通道蛋白9(AQP9)蛋白靶点调控肝癌细胞线粒体凋亡途径,从而发挥抗肿瘤的可能机制。方法采用H22肝癌细胞成功建立裸鼠皮下移植瘤模型,选取成瘤裸鼠25只随机分为空白组(生理盐水灌胃,0.2 ml/10 g)、阳性对照组(环磷酰胺腹腔注射,30 mg/kg)和健脾活血祛湿方低、中、高剂量组(灌胃,25、50、100 mg/kg),每组5只,1次/d。计算各组H22肝癌移植瘤裸鼠的抑瘤率及脾脏指数,采用荧光探针JC-1和流式细胞术检测线粒体膜电位(ΔΨm)的变化;Western blot检测瘤组织B细胞淋巴瘤/白血病-2(Bcl-2)、B淋巴细胞瘤因子相关X蛋白(Bax)的表达变化;免疫组织化学检测AQP9表达。结果与空白组相比,阳性对照组裸鼠的生存状态较差,瘤体体积减小,抑瘤率、Bcl-2、Bax蛋白表达显著增高(P<0.05,P<0.01),脾指数、线粒体膜电位水平显著降低(P<0.01);免疫组织化学结果显示AQP9表达增多。与阳性对照组相比,健脾活血祛湿方高剂量组瘤体体积减小,瘤重和抑瘤率均无明显变化(P>0.05),脾指数升高(P<0.05),线粒体膜电位显著降低(P<0.05),Bcl-2、Bax表达和Bax/Bcl-2比值显著增高(P<0.05);免疫组织化学结果显示AQP9广泛表达,呈现AQP9棕褐色深染,部分细胞出现胞核的形态改变,细胞空泡显著增多。结论健脾活血祛湿方在一定剂量下对H22肝癌荷瘤裸鼠有明显的抑瘤作用,并可改善裸鼠免疫功能,其作用可能与调控AQP9干预肝癌细胞线粒体凋亡途径有关。Objective To observe the influence of Jianpi Huoxue Qushi Decoction on nude mice bearing H22 hepatoma,and to investigate the possible mechanism of Jianpi Huoxue Qushi Decoction by interfering with Aquaporin 9(AQP-9)protein target to regulate mitochondrial apoptosis pathway in hepatoma cells.Methods H22 hepatoma cells were used to establish subcutaneous xenograft models,25 nude mice were selected.All the nude mice bearing H22 were randomly divided into blank group(normal saline gavage,0.2 ml/10 g),positive control group(cyclophosphamide intraperitoneal injection,30 mg/kg)and low-,medium-and high-dose groups(Jianpi Huoxue Qushi Decoction intragastric administration,25,50,100 mg/kg,respectively),with 5 rats in each group.nude mice bearing H22 were intervened once a day.The inhibition rate and spleen index of nude mice bearing H22 in each group were calculated.Fluorescent probe JC-1 and flow cytometry were used to detect the expression of mitochondrial membrane potential(ΔΨm).Western blot was used to detect the expression of B-cell lymphoma/leukemia-2(Bcl-2)and B-lymphocyte tumor factor associated X protein(Bax).Immunohistochemistry was used to detect the expression of AQP-9.Results Compared with the blank group,the survival status of nude mice in the positive control group was worse,and the tumor volume,the spleen index andΔΨm were significantly reduced,the tumor inhibition rate,the expression of Bcl-2 and Bax protein were significantly increased in the positive control group(P<0.05,P<0.01).Immunohistochemical results showed that the expression of AQP9 was increased in the positive control group.Compared with the positive control group,the tumor volume in the high-dose group significantly reduced,without significant chang in the tumor weight and tumor inhibition rate(P>0.05),the spleen index increased(P<0.05),theΔΨm significantly decreased(P<0.05),the expression of Bcl-2 and Bax and the ratio of Bax/Bcl-2 significantly increased(P<0.05).The results of immunohistochemistry showed that AQP9 was widely expressed

关 键 词：健脾活血祛湿方 H22肝癌细胞 凋亡 水通道蛋白9 线粒体途径 

分 类 号：R273[医药卫生—中西医结合] R735.7[医药卫生—中医肿瘤科]