藤茶提取物对卡波西肉瘤相关疱疹病毒裂解复制及相关淋巴肿瘤细胞生长活性的影响  

Effect of Extract of Tengcha(Ampelopsis Grossedentata)on Lytic Replication of Kaposi’s Sarcoma Associated Herpes Virus and Growth Activity of Associated Lymphoma Cell

作　　者：张宏赐 何嘉伟 何吉甜 肖岚泽 李晓娟[1] ZHANG Hongci;HE Jiawei;HE Jitian;XIAO Lanze;LI Xiaojuan(Hubei University of Chinese Medicine,Wuhan 430060,China)

机构地区：[1]湖北中医药大学,湖北武汉430060

出　　处：《山东中医药大学学报》2024年第6期699-707,共9页Journal of Shandong University of Traditional Chinese Medicine

基　　金：湖北省自然科学基金项目(编号:2023AFD109)。

摘　　要：目的:观察藤茶水提取物(AGE)对卡波西肉瘤相关疱疹病毒(KSHV)裂解复制的影响,检测AGE抗KSHV相关淋巴瘤的安全性和细胞特异性,探索其可能的细胞通路机制。方法:采用组织型纤溶酶原激活剂(TPA)诱导人卡波式肉瘤相关疱疹病毒感染细胞系(BCBL-1)中的KSHV裂解复制,用AGE制备的低、中、高浓度溶液处理48~72 h,随后收集细胞样品,逆转录PCR(RT-PCR)检测KSHV潜伏基因LANA、即时早期基因RTA和ORF45以及晚期基因K8.1的转录水平;实时荧光定量PCR检测KSHV子代病毒含量;蛋白质免疫印迹法(WB)检测KSHV潜伏蛋白LANA、裂解蛋白RTA、ORF45和K8.1的表达及KSHV裂解复制中相关信号通路蛋白激酶B(Akt)、细胞外调节蛋白激酶(ERK)、丝裂原活化蛋白激酶p38抗体(p38 MAPK)和哺乳动物雷帕霉素靶蛋白(mTOR)的活化水平;噻唑蓝比色法(MTT)检测AGE对BCBL-1和正常外周血单个核细胞(PBMC)的细胞毒性。结果:与对照组比较,低浓度AGE对潜伏基因LANA、即时早期基因RTA和ORF45的转录无明显抑制作用,对晚期基因K8.1的转录具有轻微的抑制作用(P<0.05);中浓度AGE对LANA、RTA和ORF45的转录具有轻微的抑制作用,并显著抑制K8.1的转录(P<0.05,P<0.01);高浓度AGE对LANA、RTA的转录无明显抑制作用,但显著抑制即时早期基因ORF45和晚期基因K8.1的转录(P<0.01)。20μg/mL浓度AGE能抑制73%子代KSHV颗粒的产生。与对照组比较,低、中、高浓度AGE对潜伏蛋白LANA的表达抑制均不明显,但对裂解蛋白RTA、ORF45和K8.1的表达均有不同程度的抑制作用(P<0.05,P<0.01,P<0.001),且呈剂量依赖性抑制。低、中、高浓度的AGE对Akt的磷酸化均没有明显的抑制作用,但对mTOR(2448)、ERK1/2和p38 MAPK(180/182)的磷酸化均有明显抑制作用(P<0.01,P<0.001)。100μg/mL的AGE显著抑制BCBL-1细胞的生长,最大细胞抑制率为67.7%,AGE对PBMC最大细胞抑制率为37.0%。BCBL-1细胞在潜伏期和裂解期的半数毒性浓度(CC50)Objective:To observe the effect of aqueous extract of Tengcha(Ampelopsis Grossedentata)(AGE)on lytic replication of Kaposi’s sarcoma associated herpes virus(KSHV),detect the safety and cell specificity of AGE against KSHV associated lymphoma,and explore its possible cellular pathway mechanisms.Methods:Tissue plasminogen activator(TPA)was used to induce KSHV lysis and replication in BCBL1 lymphoma cells.Low,medium,and high dose of solutions prepared with AGE were treated for 48~72 hours.Subsequently,cell samples were collected and RT-PCR was used to detect the transcription levels of KSHV latent antigen LANA,real-time early genes RTA and ORF45,as well as late gene K8.1.Quantitative real-time PCR was used to detect the copy number of viral DNA.Western blot was applied to detect the expression of latent protein LANA,lytic protein RTA,ORF45,and K8.1 in KSHV,as well as the activation levels of related signaling pathways Akt,ERK,p38 MAPK,and mTOR in KSHV lytic replication.The cytotoxicity of AGE on lymphoma cells and normal PBMC was determined by using MTT.Results:Compared with the control group,low concentration of AGE had no significant inhibitory effect on the transcription of latent gene LANA,immediate early genes RTA and ORF45,and only a slight inhibitory effect on the transcription of late gene K8.1(P<0.05);medium concentration of AGE had a slight inhibitory effect on the transcription of LANA,RTA and ORF45,and significantly inhibited the transcription of late gene K8.1(P<0.05,P<0.01);high concentration of AGE had no significant inhibitory effect on the transcription of LANA and RTA,but significantly suppressed the transcription of immediate early gene ORF45 and late gene K8.1(P<0.01).AGE of 20μg/mL inhibited the generation of KSHV particles in 73%of progeny.Compared with the control group,low,medium and high concentrations of AGE had no significant inhibitory effect on the expression of latent protein LANA,but had different degrees of inhibitory effect on the expression of lytic proteins of RTA,ORF45 and K8.1

关 键 词：藤茶 卡波西肉瘤相关疱疹病毒 裂解复制 淋巴瘤 ERK/MAPK/mTOR通路 

分 类 号：R285[医药卫生—中药学]