[關(guān)鍵詞]
[摘要]
目的 探索防己諾林堿在細(xì)胞水平抗H1N1病毒的作用,闡明防己諾林堿調(diào)控細(xì)胞自噬抑制H1N1病毒復(fù)制的分子機(jī)制。方法 CCK-8法檢測(cè)0.312 5、0.625 0、1.250 0、2.500 0、5.000 0、10.000 0、20.000 0、40.000 0、60.000 0 μmol·L-1的防己諾林堿對(duì)MDCK細(xì)胞活力的影響;MDCK細(xì)胞設(shè)置對(duì)照組、模型組和防己諾林堿(2.5、5.0、10.0 μmol·L-1)組,除對(duì)照組外,以感染復(fù)數(shù)(MOI)為0.1的H1N1病毒感染MDCK細(xì)胞,加入防己諾林堿共同孵育12 h,同時(shí)設(shè)置防己諾林堿(10.0 μmol·L-1)與病毒共同孵育4、8 h組,通過實(shí)時(shí)熒光定量PCR (qRT-PCR,檢測(cè)HIN1 mRNA表達(dá))和Western blotting [H1N1病毒核蛋白(NP)]檢測(cè)防己諾林堿對(duì)病毒復(fù)制的抑制作用;PI/Hoechst 33342染色檢測(cè)防己諾林堿(10.0 μmol·L-1)對(duì)MDCK細(xì)胞死亡的影響;qRT-PCR法檢測(cè)防己諾林堿檢測(cè)防己諾林堿預(yù)處理、病毒感染早期藥物處理、病毒感染晚期藥物處理以及吸附和進(jìn)入階段給藥對(duì)病毒復(fù)制的影響;MDCK細(xì)胞轉(zhuǎn)染EGFP-LC3或EGFP-mCherry-LC3雙熒光質(zhì)粒,觀察防己諾林堿對(duì)EGFP-LC3的熒光強(qiáng)度、EGFP-mCherry-LC3共定位的影響,設(shè)置自噬誘導(dǎo)劑雷帕霉素(0.5 μmol·L-1)、自噬晚期抑制劑氯喹(10 μmol·L-1)、巴佛洛霉素A1 (100 nmol·L-1)對(duì)照。轉(zhuǎn)染過EGFP-LC3質(zhì)粒的MDCK細(xì)胞感染H1N1病毒,免疫熒光檢測(cè)防己諾林堿對(duì)LC3與胞內(nèi)的病毒NP蛋白共定位的影響;體外培養(yǎng)A549細(xì)胞,以MOI為0.1的H1N1病毒感染,Western blotting檢測(cè)防己諾林堿對(duì)LC3II/LC3I蛋白表達(dá)的影響。結(jié)果 防己諾林堿對(duì)MDCK細(xì)胞的半數(shù)抑制濃度(IC50)為40.19 μmol·L-1 ;與模型組比較,防己諾林堿以濃度相關(guān)性的方式抑制HIN1 mRNA表達(dá),以濃度和時(shí)間相關(guān)性的方式抑制NP蛋白表達(dá)(P<0.01、0.001);顯著減少H1N1誘導(dǎo)的細(xì)胞死亡(P<0.001);抑制H1N1病毒進(jìn)入過程。與對(duì)照組比較,防己諾林堿處理誘導(dǎo)LC3熒光聚集,誘導(dǎo)EGFP-LC3和mCherry-LC3雙熒光共定位顯著增加(P<0.001)。與模型組比較,防己諾林堿處理明顯減少NP的熒光數(shù)量(P<0.001),同時(shí)造成LC3熒光積累并與胞內(nèi)的病毒NP蛋白共定位;引起LC3II積累(P<0.01、0.001)。結(jié)論 防己諾林堿同氯喹和巴佛洛霉素A1一致,阻斷自噬途徑,使病毒粒子在自噬體內(nèi)滯留,破壞病毒生命周期。
[Key word]
[Abstract]
Objective To investigate the antiviral effect of fangchinoline against the H1N1 virus and elucidate its molecular mechanism in regulating cellular autophagy. Methods CCK-8 method was used to detect the effect of tetrandrine of 0.312 5, 0.625 0, 1.250 0, 2.500 0, 5.000 0 0, 10.000 0 0, 20.000 0, 40.000 0, and 60.000 0 0 μmol·L?1 on viability of MDCK cells. MDCK cells were divided into control group, model group, and tetrandrine (2.5, 5.0, and 10.0 μmol·L?1) groups. Except for control group, MDCK cells were infected with H1N1 virus with a multiple infection index (MOI) of 0.1, and co-incubated with tetrandrine for 12 h. At the same time, tetrandrine (10.0 μmol·L?1) was set up incubated with the virus for 4 and 8 h, and detected the inhibitory effect of tetrandrine on virus replication through real-time fluorescence quantitative PCR (qRT-PCR, detecting HIN1 mRNA expression) and Western blotting [H1N1 virus nucleoprotein (NP)]. PI/Hoechst 33342 staining was used for detection of tetrandrine on MDCK cell death. QRT-PCR method was used to detect the effects of pre-treatment with tetrandrine, early drug treatment for viral infection, late drug treatment for viral infection, as well as adsorption and entry stage administration on virus replication. MDCK cells were transfected with EGFP-LC3 or EGFP-mCherry-LC3 dual fluorescent plasmids to observe the fluorescence intensity of EGFP-LC3 and EGFPmCherry-LC3 co-localization. MDCK cells transfected with EGFP-LC3 plasmid were infected with H1N1 virus, and immunofluorescence was used to detect the effect of tetrandrine on LC3 and co-localization with intracellular viral NP protein. A549 cells were cultured in vitro and infected with H1N1 virus with a MOI of 0.1. Western blotting was performed to detect the effect of tetrandrine on expression of LC3II/LC3I protein. Results The half inhibitory concentration (IC50) of tetrandrine on MDCK cells is 40.19 μmol·L?1. Compared with model group, tetrandrine inhibited HIN1 mRNA expression in a concentration dependent manner and NP protein expression in a concentration and time dependent manner (P < 0.01, 0.001), significantly reduced H1N1 induced cell death (P < 0.001), and inhibited the entry process of H1N1 virus. Compared with control group, treatment with tetrandrine induced LC3 fluorescence aggregation and significantly increased co-localization of EGFP-LC3 and mCherry-LC3 dual fluorescence (P < 0.001). Compared with model group, treatment with tetrandrine significantly reduced the fluorescence quantity of NP (P < 0.001), while causing the accumulation of LC3 fluorescence and co-localization with intracellular viral NP proteins, causing accumulation of LC3II (P < 0.01, 0.001). Conclusion Fangchinoline is consistent with bafilomycin A1, blocking the autophagy pathway, causing viral particles to remain in the autophagy, and destroying the life cycle of the virus.
[中圖分類號(hào)]
R285.5
[基金項(xiàng)目]
北京市科技新星計(jì)劃(2023107);中華中醫(yī)藥學(xué)會(huì)青年人才托舉工程項(xiàng)目(CACM-2023-QNRC2-A02);國家自然科學(xué)基金資助項(xiàng)目(82001663)