Effect of berberine hydrochloride on autophagy of HeLa cells infected with herpes simplex virus type 1 by regulating PI3K/AKT/m TOR signaling pathway
10.13481/j.1671-587X.20250616
- VernacularTitle:盐酸小檗碱调控PI3K/AKT/mTOR信号通路对感染1型单纯疱疹病毒HeLa细胞自噬的影响
- Author:
Haidong ZHU
1
;
Changkun LYU
;
Wei SHI
Author Information
1. 商丘医学高等专科学校基础医学部,河南 商丘 476100
- Keywords:
Berberine hydrochloride;
Phosphatidylinositol 3-kinase;
Protein kinase B;
Mammalian target of rapamycin pathway;
Herpes simplex virus type 1;
Autophagy
- From:
Journal of Jilin University(Medicine Edition)
2025;51(6):1607-1617
- CountryChina
- Language:Chinese
-
Abstract:
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 160 μmol·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 20 μmol·L-1 BBR for 24 h),M-BBR group(were infected with HSV-1 and then treated with 40 μmol·L-1 BBR for 24 h),H-BBR group(were infected with HSV-1 and then treated with 80 μmol·L-1 BBR for 24 h),and 740 Y-P group(were infected with HSV-1 and then treated with 80 μmol·L-1 BBR and 10 μmol·L-1 740 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 0 μmol·L-1 BBR group,the activities of the HeLa cells in 120 and 160 μmol·L-1 BBR groups were significantly decreased(P<0.05),which had certain toxicity to the cells;20,40,and 80 μmol·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-Ⅰ in 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-Ⅰ ratio 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 dose-dependent 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.
