小檗碱对肝癌细胞活性与增殖能力的影响及其机制  

Effect and mechanism of berberine on the activity and proliferation of hepatoma cells

作　　者：吴文龙 王权成 孟强 张玄 窦科峰 Wu Wen-Long;Wang Quan-Cheng;Meng Qiang;Zhang Xuan;Dou Ke-Feng(Department of Hepatobiliary Surgery,Xijing Hospital,Air Force Military Medical University,Xi’an,Shaanxi 710032,China)

机构地区：[1]空军军医大学西京医院肝胆外科,陕西西安710032

出　　处：《解放军医学杂志》2023年第6期653-662,共10页Medical Journal of Chinese People's Liberation Army

基　　金：国家重点研发计划(2016YFC0905902);陕西省院士工作站(陕科协[2018]函字5号);西京医院学科助推计划(XJZT21CM34)。

摘　　要：目的探讨小檗碱(BBR)对肝癌细胞的活性与增殖能力的影响及其机制。方法取正常人肝细胞MIHA和人肝癌细胞Hep3B、HepG2,设置对照组与0、12.5、25、50μmol/L BBR组,采用CCK-8实验、克隆形成实验探究无糖环境下BBR对MIHA、Hep3B、HepG2细胞活性及克隆形成的影响;SYTOX荧光染色检测细胞死亡情况;流式细胞术检测BBR孵育后肝癌细胞活性氧(ROS)水平的变化;Western blotting检测BBR孵育后Nrf2及其相关蛋白的变化;将蛋白酶体抑制剂MG132及蛋白合成抑制剂环己酰胺(CHX)与BBR共同孵育,研究其对Nrf2蛋白的影响;实时荧光定量PCR(qRT-PCR)检测BBR孵育后Hep3B细胞的Nrf2及HO-1在基因水平的变化;于裸鼠皮下注射Hep3B细胞,构建裸鼠成瘤模型,在BBR组及对照组裸鼠腹腔分别注射BBR或等量生理盐水,观察BBR在体内对肿瘤生长的影响。HE及免疫组化染色研究在体内环境BBR对Nrf2及相关蛋白的影响。结果无糖环境下BBR孵育后,肝癌细胞Hep3B、HepG2的活性逐渐下降(P<0.05),正常肝细胞MIHA的活性无明显变化。BBR可以抑制肝癌细胞克隆形成,促进肝癌细胞死亡,并且呈浓度依赖性(P<0.05)。BBR孵育后,肝癌细胞的ROS水平增高,Nrf2、HO-1、c-Myc蛋白水平降低,而Keap1蛋白无明显变化;GSK3β基本不变,P-GSK3β(Ser9)降低,P-GSK3β(Tyr216)增高。MG132可以明显逆转BBR引起的肝癌细胞Nrf2蛋白的降低;CHX则可以增强BBR引起的肝癌细胞Nrf2蛋白的降低。BBR孵育后,Nrf2 mRNA水平增高,HO-1 mRNA水平降低(P<0.05)。BBR在裸鼠体内可以明显抑制Hep3B细胞生长。注射BBR后,肿瘤组织坏死增多,肿瘤内Nrf2及c-Myc降低,而Keap1基本不变。结论BBR可以抑制肝癌细胞的活性及增殖能力,促进其死亡;体内注射BBR后可以显著抑制肿瘤细胞生长,导致肿瘤细胞坏死增多,其作用机制可能是BBR通过非Keap1依赖的Nrf2/GSK3β通路导致Nrf2蛋白通过泛素-蛋白酶体系统降解,从而降低肿瘤细胞的抗�Objective To investigate the effect and mechanism of berberine(BBR)on the activity and proliferation of hepatoma cells.Methods Normal human hepatocytes(MIHA)and human hepatoma cells(Hep3B,HepG2)were respectively divided into control group(high glucose)and different concentration(0,12.5,25,50μmol/L)of BBR groups(glucose-free).CCK-8 and clone formation experiment were used to explore the effect of BBR on the viability and clone formation.SYTOX Green nucleic acid stain was used to determine the cell death and flow cytometry to detect the changes in reactive oxygen species(ROS)levels in hepatoma cells.The changes of Nrf2 and its related proteins after BBR incubation were measured by Western blotting.Weincubated the proteasome inhibitor MG132 and the protein synthesis inhibitor cycloheximide(CHX)with BBR to study its effect on the Nrf2 protein.We quantified the expressions of Nrf2 and HO-1 in Hep3B cells cultured with BBR using Quantitative realtime PCR(qRT-PCR).We established a tumor model by subcutaneous injection of Hep3B cells into nude mice.To observe the effect of BBR on tumor growth,we injected tumor-bearing nude mice in the BBR groups and the control group with BBR or the same amount of normal saline,respectively.We evaluated the effect of BBR on Nrf2 and its related proteins in vivo with HE and immunohistochemical staining.Results The viability of Hep3B and HepG2 cells decreased after BBR incubation in a glucosefree environment(P<0.05).No appreciable changes were observed in MIHA cell viability under the same experiment condition.BBR could inhibit clone formation and promote the death of hepatoma cells in a concentration-dependent manner(P<0.05).We observed an increase in the ROS and a decrease in Nrf2,HO-1,and c-Myc with no significant change in Keap1 in hepatoma cells after BBR treatment.While total GSK3βremained unchanged,the treatment-induced decrease in P-GSK3β(Ser9)decreased but increase in P-GSK3β(Tyr216).MG132 co-treatment reversed the BBR-induced reduction of Nrf2 protein while CHX enhanced the B

关 键 词：肝细胞癌 小檗碱 NRF2 GSK3Β 

分 类 号：R773.4[医药卫生—眼科]