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

doi:10.13481/j.1671-587X.20250616

摘要/Abstract

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160 μmol·L^-1 BBR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20 μmol·L^-1 BBR处理24 h）、M-BBR组（感染HSV-1后，用40 μmol·L^-1 BBR处理24 h）、H-BBR组（感染HSV-1后，用80 μmol·L^-1 BBR处理24 h）和740 Y-P组（感染HSV-1后，用80 μmol·L^-1 BBR和10 μmol·L^-1 740 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0 μmol·L^-1 BBR组比较，120和160 μmol·L^-1 BBR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80 μmol·L^-1 BBR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。

关键词: 盐酸小檗碱, 磷脂酰肌醇3-激酶, 蛋白激酶B, 哺乳动物雷帕霉素靶蛋白通路, 1型单纯疱疹病毒, 自噬

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.

Key words: Berberine hydrochloride, Phosphatidylinositol 3-kinase, Protein kinase B, Mammalian target of rapamycin pathway, Herpes simplex virus type 1, Autophagy

中图分类号:

R373.9

朱海东,吕长坤,师玮. 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[J]. 吉林大学学报(医学版), 2025, 51(6): 1607-1617.

Haidong ZHU,Changkun LYU,Wei SHI. Effect of berberine hydrochloride on autophagy of HeLa cells infected with herpes simplex virus type 1 by regulating PI3K/AKT/mTOR signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2025, 51(6): 1607-1617.

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Primer	Forward （5'-3'）	Reverse （5'-3'）
ICP0	TGTGCACGGATGAGATCG	TCGTTCACGATCGGGATG
ICP22	ATGGACAAGGTAACCATCGG	TTGAAAAACGGAAGGGGGTA
ICP8	TGAAGTTCCTCCCCCTCCAC	TGAAGTTCCTCCCCCTCCACGGAGACCATCATCACCAACC
TK	CGATGACTTACTGGCGGGTGT	GCGTCGGTCACGGCATAA
gB	GCCTTCTTCGCCTTTCGC	CGCTCGTGCCCTTCTTCTT
gD	CCCGATCACGGTTTACTACG	AGCGATGGTCAGGTTGTAGG
GAPDH	CAGCCTCAAGATCATCAGCAA	CCATCACGCCACAGTTTCC

Group	ICP0	ICP22	ICP8	TK	gB	gD
HSV-1	1.01±0.02	0.98±0.10	1.06±0.13	1.01±0.09	1.05±0.12	0.99±0.09
L-BBR	0.85±0.09^*	0.79±0.06^*	0.80±0.09^*	0.83±0.08^*	0.79±0.07^*	0.71±0.08^*
M-BBR	0.61±0.08^*△	0.50±0.07^*△	0.58±0.07^*△	0.66±0.05^*△	0.59±0.08^*△	0.52±0.06^*△
H-BBR	0.33±0.05^*#	0.23±0.04^*#	0.22±0.05^*#	0.30±0.06^*#	0.20±0.05^*#	0.23±0.04^*#
740 Y-P	0.65±0.07^○	0.56±0.07^○	0.64±0.08^○	0.49±0.05^○	0.58±0.06^○	0.59±0.08^○
F	462.461	330.150	247.229	401.737	289.045	309.713
P	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

Group	Apoptotic rate
HSV-1	5.36±0.25
L-BBR	11.03±1.16^*
M-BBR	17.94±2.29^*△
H-BBR	30.23±4.91^*△#
740 Y-P	16.62±3.15^○
F	31.734
P	<0.01

Group	Beclin-1	BNIP	LC3-Ⅱ/LC3-Ⅰ
HSV-1	0.29±0.04	0.35±0.05	0.15±0.04
L-BBR	0.62±0.07^*	0.67±0.08^*	0.47±0.06^*
M-BBR	0.98±0.09^*△	0.93±0.10^*△	0.91±0.10^*△
H-BBR	1.33±0.14^*△#	1.41±0.15^*△#	1.32±0.13^*△#
740 Y-P	0.59±0.07^○	0.53±0.07^○	0.92±0.09^○
F	64.124	60.267	78.190
P	<0.01	<0.01	<0.01

Group	Caspase-1	Caspase-3	Bcl-2	Bax
HSV-1	0.23±0.04	0.35±0.05	1.41±0.12	0.29±0.04
L-BBR	0.45±0.06^*	0.61±0.07^*	1.12±0.08^*	0.47±0.05^*
M-BBR	0.71±0.07^*△	0.94±0.09^*△	0.89±0.09^*△	0.88±0.09^*△
H-BBR	1.06±0.11^*△#	1.35±0.12^*△#	0.33±0.04^*△#	1.13±0.12^*△#
740 Y-P	0.66±0.07^○	0.53±0.08^○	0.64±0.07^○	0.82±0.07^○
F	56.375	66.705	92.733	53.700
P	<0.01	<0.01	<0.01	<0.01