摘要
目的:探讨盐酸小檗碱(BBR)对1型单纯疱疹病毒(HSV-1)感染HeLa细胞自噬的影响,并阐明其相关机制。方法:将处于对数生长期的HeLa细胞分别加入0、10、20、40、80、120和160μmol·L-1BBR中培养24 h。另将HeLa细胞分为HSV-1组(感染HSV-1)、 L-BBR组(感染HSV-1后,用20μmol·L-1BBR处理24 h)、M-BBR组(感染HSV-1后,用40μmol·L-1BBR处理24 h)、H-BBR组(感染HSV-1后,用80μmol·L-1BBR处理24 h)和740 Y-P组(感染HSV-1后,用80μmol·L-1BBR和10μmol·L-1740 Y-P处理24 h),进行病毒感染及相应的药物处理。四甲基偶氮唑盐(MTT)法检测检测不同浓度BBR处理HeLa细胞活性,空斑减数实验检测各组HeLa细胞中HSV-1增殖能力,实时荧光定量PCR(RT-qPCR)法检测各组Hela细胞中病毒复制相关基因mRNA表达水平,免疫荧光法检测各组Hela细胞中微管相关蛋白1轻链3(LC3)自噬小体形成情况,流式细胞术检测各组Hela细胞凋亡率,Western blotting法检测各组Hela细胞中自噬及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:MTT法检测,与0μmol·L-1BBR组比较,120和160μmol·L-1BBR组HeLa细胞活性明显降低(P<0.05),对细胞具有一定毒性;选择20、40和80μmol·L-1BBR进行后续实验。空斑减数实验检测,与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞中空斑形成单位(PFUs)明显减少(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞中PFUs明显增加(P<0.05)。RT-qPCR法检测,与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组感染细胞蛋白0(ICP0)、感染细胞蛋白22(ICP22)、感染细胞蛋白8(ICP8)、胸苷激酶(TK)、糖蛋白B(gB)和糖蛋白D(gD) mRNA表达水平均明显降低(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞中ICP0、ICP22、ICP8、TK、gB和gD mRNA表达水平均明显升高(P<0.05)。免疫荧光法检测,与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞中LC3自噬小体形成数量增加;与H-BBR组比较,740 Y-P组HeLa细胞中LC3自噬小体形成数量减少。流式细胞术检测,与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞凋亡率明显升高(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞凋亡率明显降低(P<0.05)。Western blotting法检测,与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞中苄氯素1(Beclin-1)及B细胞淋巴瘤2(Bcl-2)/腺病毒E1B 19kDa蛋白相互作用蛋白(BNIP)蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ比值均明显升高(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞中Beclin-1和BNIP蛋白表达水平及LC3-Ⅱ/LC3-Ⅰ比值均明显降低(P<0.05);与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞中含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)和Bcl-2相关X蛋白(Bax)蛋白表达水平均明显升高(P<0.05),Bcl-2蛋白表达水平明显降低(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞中Caspase-1、Caspase-3和Bax蛋白表达水平明显降低(P<0.05), Bcl-2蛋白表达水平明显升高(P<0.05);与HSV-1组比较,L-BBR组、M-BBR组和H-BBR组HeLa细胞中磷酸化PI3K(p-PI3K)/PI3K、磷酸化AKT(p-AKT)/AKT和磷酸化mTOR(p-mTOR)/mTOR比值均明显降低(P<0.05),并呈剂量依赖性;与H-BBR组比较,740 Y-P组HeLa细胞中p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR比值均明显升高(P<0.05)。结论:BBR能够促进HSV-1病毒感染HeLa细胞的自噬过程,诱导自噬依赖性凋亡,并显著抑制病毒的复制,其作用机制与调控PI3K/AKT/mTOR通路有关。
Objective:To discuss the effect of berberine hydrochloride(BBR)on autophagy in the HeLa cells infected with herpes simplex virus type 1(HSV-1),and to clarify its related mechanism.Methods:The HeLa cells at logarithmic growth phase were added with 0,10,20,40,80,120,and 160μmol·L-1 BBR,respectively,and cultured for 24 h.In addition,the HeLa cells were divided into HSV-1 group(the cells were infected with HSV-1),L-BBR group(were infected with HSV-1 and then treated with 20μmol·L-1 BBR for 24 h),M-BBR group(were infected with HSV-1 and then treated with 40μmol·L-1 BBR for 24 h),H-BBR group(were infected with HSV-1 and then treated with 80μmol·L-1 BBR for 24 h),and 740 Y-P group(were infected with HSV-1 and then treated with 80μmol·L-1 BBR and 10μmol·L-1740 Y-P for 24 h)for viral infection and corresponding drug treatment.MTT method was used to detect the activities of microtubule-associated protein 1 light chain 3(LC3)the HeLa cells after treated with different concentrations of BBR;plaque reduction assay was used to detect the proliferation ability of HSV-1 in the HeLa cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of virus replication-related genes in the HeLa cells in various groups;immunofluorescence method was used to detect the formation of autophagosomes in the HeLa cells in various groups;flow cytometry was used to detect the apoptotic rate of the HeLa cells in various groups;Western blotting method was used to detect the expression levels of autophagy and the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway pathway-related proteins in the HeLa cells in various groups.Results:The MTT method results showed that compared with 0μmol·L-1 BBR group,the activities of the HeLa cells in 120 and 160μmol·L-1 BBR groups were significantly decreased(P<0.05),which had certain toxicity to the cells;20,40,and 80μmol·L-1 BBR were selected for subsequent experiments.The plaque reduction assay results showed that compared with HSV-1 group,the plaque forming units(PFUs)in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the PFUs in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).The RT-qPCR results showed that compared with HSV-1 group,the mRNA expression levels of infected cell protein 0(ICP0),infected cell protein 22(ICP22),infected cell protein 8(ICP8),thymidine kinase(TK),glycoprotein B(gB),and glycoprotein D(gD)in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the expression levels of ICP0,ICP22,ICP8,TK,gB,and gD mRNA in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).The immunofluorescence method results showed that compared with HSV-1 group,the number of LC3 autophagosomes formed in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group was increased;compared with H-BBR group,the number of LC3 autophagosomes formed in the HeLa cells in 740 Y-P group was decreased.The flow cytometry results showed that compared with HSV-1 group,the apoptotic rates of the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the apoptotic rate of the HeLa cells in 740 Y-P group was significantly decreased(P<0.05).The Western blotting method results showed that compared with HSV-1 group,the expression levels of Beclin-1 and B-cell lymphoma 2(Bcl-2)/adenovirus E1B 19kDa protein interacting protein protein(BNIP)2 proteins and the ratios of LC3-Ⅱ/LC3-Ⅰin the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the expression levels of Beclin-1 and BNIP proteins and the LC3-Ⅱ/LC3-Ⅰratio in the HeLa cells in 740 Y-P group were significantly decreased(P<0.05);compared with HSV-1 group,the expression levels of cysteine-containing aspartate protein hydrolase 1(Caspase-1),cysteine-containing aspartate protein hydrolase 3(Caspase-3),and Bcl-2 associated X protein(Bax)proteins in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly increased(P<0.05),and the expression level of Bcl-2 protein was significantly decreased(P<0.05)in a dosedependent manner;compared with H-BBR group,the expression levels of Caspase-1,Caspase-3,and Bax proteins in the HeLa cells in 740 Y-P group were significantly decreased(P<0.05),and the expression level of Bcl-2 protein was significantly increased(P<0.05);compared with HSV-1 group,the ratios of phosphorylated PI3K(p-PI3K)/PI3K,phosphorylated AKT(p-AKT)/AKT,and phosphorylated mTOR(p-mTOR)/mTOR in the HeLa cells in L-BBR group,M-BBR group,and H-BBR group were significantly decreased(P<0.05)in a dose-dependent manner;compared with H-BBR group,the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR in the HeLa cells in 740 Y-P group were significantly increased(P<0.05).Conclusion:BBR can promote the autophagy process in the HeLa cells infected with HSV-1 by regulating the PI3K/AKT/mTOR pathway,induce autophagy-dependent apoptosis,and significantly inhibit the virus replication.
作者
朱海东
吕长坤
师玮
ZHU Haidong;LYU Changkun;SHI Wei(Department of Basic Medicine,Shangqiu Medical College,Shangqiu 476100,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第6期1607-1617,共11页
Journal of Jilin University:Medicine Edition
基金
河南省科技厅科技发展计划项目(242102310081)。
