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Enterovi khuẩn 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to lớn treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European & Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, và viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive sầu oxyren species (ROS) generation; & translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus khổng lồ cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely lớn suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP.. A1 translocation và EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO & its constituent RA possess anti-EV71 activities, và may serve sầu as a candidate drug for therapeutic & prophylactic uses against EV71 infection.

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Hvà, foot, and mouth disease (HFMD) is a prevalent infectious childhood disease caused by several viral strains belonging lớn the genus Enterovi khuẩn within the family Picornaviridae. Enterovirut 71 (EV71) is one of the major pathogens causing HFMD in infants and children aged under 51,2. Patients with HFMD suffered from fever and vesicular exanthemas on hands, feet, buttocks & mouth3. A small proportion of EV71-infected patients develop severe central nervous system (CNS) infection, which can cause neurological cardiopulmonary failure, systemic complication, and even fatality4,5. Even those who survive sầu severe infection may have sầu long-term motor và cognitive sầu deficits6,7,8. EV71 was first isolated from patients with CNS disease in California, USA, during 1969 khổng lồ 19729, và caused several sporadic cases around the world in the 1970s và 1980s10. In recent two decades, severe EV71 outbreaks have affected more than millions of children, và claimed hundreds of lives in the Asia-Pacific region2,11,12,13,14,15. There has not been specific treatment for enteroviral CNS infection, và supportive sầu therapies have sầu been used to manage EV71-induced pulmonary heart failure16. Development of novel therapeutics is needed khổng lồ improve the clinical outcome of infection.

EV71 is a positive sầu single-stranded RNA vi khuẩn & its genome is approximately 7,400 nucleotides long. The RNA genome is enclosed in an icosahedral capsid shell. During viral infection, EV71 attaches to lớn cellular receptors và enter host cells through endocytosis17. Engagement of virus with scavenger receptor B2 (SCARB2) receptor results in release of viral RNA18. Viral RNA contains a highly structured type I internal ribosomal entry site (IRES) within 5′ untranslated region (UTR). The IRES interacts with host proteins, namely IRES trans-acting factors (ITAFs), to recruit ribosome và initiate translation19,20. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an RNA binding protein, acts as an ITAF lớn enhance EV71 IRES-mediated translation. Translocation of hnRNP A1 from nucleus to cytoplasm is required for viral translation and replication during EV71 infection21,22. Viral RNA is translated khổng lồ a single precursor polyprotein that is hydrolyzed by viral proteases, 2Apr° & 3Cpr°, to produce mature viral structural proteins (VP1, VP2, VP3 và VP4) & nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C & 3D)20. EV71 utilizes the translation apparatus of host cells to lớn produce viral proteins23, và switches the translation of capped mRNA khổng lồ viral RNA. Viral proteases, 2Apr° & 3Cpr°, cleave sầu eukaryotic initiation factor 4 G (eIF4G), eukaryotic initiation factor 4A (eIF4A) & poly(A)-binding protein (PABP), which are required for cap-dependent translation24. EV71 infection induces expression of miR-141 which targets eukaryotic initiation factor 4 G (eIF4G)25.

Several lines of evidence tư vấn the notion that the redox status of host affects viral pathogenesis26. We have sầu previously shown that EV71 infection induces oxidative găng, which in turn promotes viral replication27,28. Treatment with antioxidants, such as N-acetylcysteine (NAC) or mito-Tempo, suppresses EV71 replication27,28,29. Natural antioxidants, epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG), in green tea have sầu antiviral activity, which correlates well with their antioxidant capacity30. It is probable that the antiviral activities of natural products may be partly attributed khổng lồ their antioxidative activities.

Herbal plants are potential sources for development of antiviral drugs31. Melissa officinalis (MO), also known as lemon balm, is a perennial plant belonging to family Labiatae. In Southern Europe, Mediterranean region, Western Asia, và North Africa, fresh leaves of MO have sầu been used to lớn add flavor lớn dishes, herbal tea, vinegars, & oils for more than 2000 years. Dried or fresh leaves and stems of MO are used as medicine to lớn treat inflammatory, gastrointestinal, mental, neuralgic, and rheumatic disorders32. MO displays an antiviral activity against herpes simplex virut type 1, herpes simplex virus type 2, human immunodeficiency virus type 1, and influenza virus33,34,35,36,37,38. Choi et al. showed that the aqueous extract of MO prevents the viability loss in EV71-infected Vero cells39. However, the mechanism underlying the antiviral activity of MO is currently unknown.

In present study, we show that methanolic extract of MO (MOM) inhibits cytopathic effect, plaque formation, and viral protein synthesis in EV71-infected cells. Further characterization & identification reveals rosmarinic acid (RA) as an active antienteroviral constituent of MOM. Mechanistically, RA inhibits EV71-induced ROS accumulation, translocation of hnRNP A1, cleavage of eIF4G, và p38-mediated phosphorylation of epidermal growth factor receptor substrate 15 (EPS15). Our results suggest that RA acts on multiple pathways to exert its antiviral effect.

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Anti-EV71 activity of methanolic extract of MO (MOM)

The syrup of MOM was diluted in water, and tested for its antiviral activity. Using the plaque reduction assay, we examined the anti-EV71 activity of MOM. RD or Vero cell monolayers were infected with 100 PFU/well or 200 PFU/well of EV71 strains, BrCr, 1743, or 4643, for 1 h, & were overlaid with semisolid truyền thông containing 0.3% agarose and 0, 78, or 156 μg/ml of MOM. MOM diminished plaque formed by these virut strains in RD & Vero cells at concentrations up to 156 μg/ml (Fig. 1a). At these concentrations, MOM was non-cytotoxic to lớn RD cells (CC50 = 370.9 ± 1.07 μg/ml, Supplementary Fig. S1a) or Vero cells (CC50 = 555.4 ± 1.05 μg/ml, Supplementary Fig. S1b). Additionally, MOM inhibited virus-induced cytopathic effect (CPE) in RD & Vero cells (Supplementary Fig. S1c). Infected cells exhibited CPE characterized by cells rounding, and the presence of crescent-shaped nuclei with condensed chromatin due lớn CPE at 16 h post infection. The percentage of cells with crescent-shaped nuclei decreased after MOM treatment in a dose-dependent manner (Supplementary Fig. S1d,e). It was accompanied by diminished expression of EV71 proteins, including viral caspid (VP2), RNA dependent RNA polymerase (3Dp°l), và protease (3CDpr°), in MOM-treated infected cells. Protein levels of 3Dp°l were reduced by 98% và 99%, respectively, in infected RD cells treated with 156 và 312 μg/ml of MOM (Fig. 1b). Expression levels of VP2 were lowered by more than 99% in infected cells treated with 156 & 312 μg/ml (Fig. 1b). The 1/2 inhibitory concentration (IC50) of MOM for inhibitory effect on EV71 in RD cells was 45.92 ± 1.05 µg/ml (Fig. 1c). These findings indicate that MOM possesses an anti-EV71 activity.


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MOM inhibits EV71 infection. (a) RD and Vero cells were mock- or infected with BrCr, 1743 & 4643 strains for 1 h, and were overlaid with 0.3% agarose in DMEM/2% FBS supplemented with 0, 78, or 156 μg/ml MOM. RD cells were incubated for 3 days, và Vero cells were cultured for 5 days. The plates were fixed with 10% formalin, và stained with 1% crystal violet solution. Representative plates are shown here. (b) RD cells were infected with BrCr at an m. o. i. of 0.05 in the presence of indicated concentrations of MOM for 16 h. Cellular protein was harvested, & was subject to western blotting with antibodies to 3 chiều, VP2 & β-actin. The cropped images of the blots are shown. The full-length blots are presented in Supplementary Fig. S8. A representative sầu experiment out of three is shown. (c) RD cells were infected with BrCr at an m. o. i. of 10 in the presence of 9.75, 19.5, 39, 78, or 156 μg/ml of MOM for 24 h. The percentage of inhibition was determined. The results are expressed as the mean ± SD of three independent experiments.


Identification of antiviral constituents in MOM

To identify the antiviral constituents in MOM, we diluted the dark green syrup of MOM in water and partitioned with equal volume of ethyl acetate (EtOAc) (Fig. 2a). The aqueous & organic fractions were evaporated, và the resulting fractions MOMW & MOME were obtained. These fractions were tested for their antiviral activity. Both MOMW & MOME fractions reduced plaque formation for all viral strains tested (Fig. 2b). At these concentrations, MOMW (CC50 = 153.7 ± 1.07 μg/ml, Supplementary Fig. S2a) and MOME (CC50 = 21.61 ± 1.08 μg/ml, Supplementary Fig. S2b) showed low cytotoxithành phố to lớn Vero cells, as determined by standard neutral red assay. These findings suggest that these extracts have some biologically active sầu components in comtháng. The MOMW và MOME were dissolved in DMSO, và subject to liquid chromatography coupled with mass spectrometry (LC/MS) analysis. The base peak chromatogram is shown (Fig. 2c). The compound with mass-to-charge ratio (m/z) of 359.07 was most abundant in the analyte derived from aqueous fraction, và was present in similar quantity in the analyte derived from EtOAc fraction. The compound was further identified using LC/MS/MS (Supplementary Fig. S2c,e). The mass spectrum of this compound matched that of rosmarinic acid (RA) (Supplementary Fig. S2d,f). The compound was purified from MOMW using column chromatography (Supplementary Fig. S3a), and analyzed by nuclear magnetic resonance spectroscopy (NMR). The 1H-NMR spectrum of this compound (Supplementary Fig. S3b) was identical to lớn that of RA (Supplementary Fig. S3c).


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Identification of RA as an antiviral constituent of MOM. (a) The procedure for extraction và fractionation of MOM is shown in the flow chart. (b) Vero cells were mock- or infected with BrCr, 1743 & 4643 strains for 1 h, & were overlaid with 0.3% agarose in DMEM/2% FBS containing 0, 39, 78 μg/ml of MOMW or 0, 10, or 20 μg/ml of MOME. Vero cells were incubated for 5 days. Cells were fixed in 10% formalin and stained with 1% crystal violet solution. Representative plates are shown here. (c) MOMW and MOME, as well as the solvent DMSO were subject to UPLC-MS analysis. The base peak chromatogram is shown. The retention time và m/z for some of base peaks are shown. A representative sầu experiment out of three is shown.

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RA inhibits EV71 replication

Plaque formation was effectively inhibited by treatment with 39 or 78 μg/ml of RA for Vero và RD cells and for all viral strains tested (Fig. 3a). At these concentrations, RA was not cytotoxic to RD cells (CC50 = 209.8 ± 1.04 μg/ml, Supplementary Fig. S4a) và Vero cells (CC50 = 593.7 ± 1.08 μg/ml, Supplementary Fig. S4b). Consistent with this, expression of enteroviral protein 3Dp°l decreased by 78% & 91% in infected RD cells treated with 78 and 156 μg/ml RA (Fig. 3b). The levels of VP2 declined by 73% and 86%, respectively, in BrCr-infected cells upon treatment with 19 và 39 μg/ml RA (Fig. 3c & Supplementary Fig. S4c). The levels of VP0 were 42% và 63% lower in similarly treated BrCr-infected RD cells compared khổng lồ cells infected alone. The discordant decrease in levels of these proteins implies that the processing of VP0 to VP2 may be affected. The ratio of the cấp độ of VP2 lớn that of VP0 decreases in MOM- or RA-treated cells in a dose dependent manner (Supplementary Fig. S4d) Additionally, RA inhibited synthesis of viral genomic synthesis in EV71-infected RD and Vero cells in a dose-dependent manner (Fig. 3d và Supplementary Fig. S4e). The màn chơi of EV71 RNA in infected RD cells decreased by 71% and 78%, respectively, upon treatment with 78 & 156 μg/ml RA (Fig. 3d). Similarly, its cấp độ in infected Vero cells declined by 79% after treatment with 312 μg/ml RA (Supplementary Fig. S4e). Decreases in viral RNA replication và protein synthesis is accompanied by reduction in progeny vi khuẩn. Extracellular and intracellular viral particles decreased by 91% và 90% in infected RD cells upon treatment with 78 μg/ml RA (Fig. 3e). The IC50 of RA for inhibitory effect on EV71 in RD cells was 43.07 ± 1.05 μg/ml (Supplementary Fig. S4f). These findings validate the anti-EV71 activity of RA.


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RA represses EV71 infection. (a) RD & Vero cells were mock- or infected with BrCr, 1743 và 4643 strains for 1 h, & were overlaid with 0.3% agarose in DMEM/2% FBS supplemented with 0, 39 or 78 μg/ml RA. The samples were processed as described in the legend of Fig. 1. Representative sầu plates are shown here. (b) RD cells were infected EV71 at an m. o. i. of 0.05 in absence or presence of 39, 78 or 156 μg/ml RA for 16 h. Cellular protein was harvested, và was subject to western blotting with antibodies to 3 chiều & β-actin. The cropped images of the blots are shown. The full-length blots are presented in Supplementary Fig. S9. A representative sầu experiment out of three is shown. (c) RD cells were mock- or infected BrCr at an m. o. i. of 0.05 in the absence or presence of 19, 39, 78 or 156 μg/ml RA for 16 h. Cellular protein was harvested, & was subject lớn western blotting with antibodies to lớn VP2 & β-actin. The cropped images of the blots are shown. The full-length blots are presented in Supplementary Fig. S10. A representative experiment out of three is shown. (d) RD cells were similarly infected as described in (b), & total RNA was isolated. The level of EV71 genomic copy was determined by quantitative sầu reverse transcription PCR, and normalized khổng lồ the cấp độ of β-actin. Data are expressed relative khổng lồ that of untreated cells. The results are means ± SD of three separate experiments. ***P Full form size image

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