We previously reported in randomized controlled trials that maoto, a traditional herbal medicine, showed clinical and virological efficacy for seasonal influenza. In this study, a culturing system for influenza was used to test the effect of maoto. A549 cells in the culture were infected with influenza virus A (PR8) and followed after treatment with maoto; the virus titers in the culture supernatant, intracellular viral proteins, and viral RNA were determined. When infected cells were cultured with maoto for 24 hr, the virus titer and protein were significantly reduced compared with medium only. Other subtypes, A/H3N2, H1N1pdm, and B, were also inhibited by maoto. Proliferation of viral RNA in a 6 hr culture was inhibited by maoto in the early phase, especially in the first 30 min. Focusing on the entry step of the influenza virus, we found that endosomal pH, regulated by vacuolar-type H+ ATPase (V-ATPase) located in the membrane, was increased when treated with maoto. We also found that uncoating of influenza viruses was also inhibited by maoto, resulting in the increase of the number of virus particles in endosomes. These results strongly suggest that the inhibition of endosomal acidification by maoto results in blocking influenza virus entry to cytoplasm, probably through the inhibition of V-ATPase. The present study provides evidence that supports the clinical use of maoto for the treatment of influenza.
Influenza remains an important infectious disease that causes major pandemic outbreaks worldwide. The currently available anti-influenza drugs, the M2 proton channel inhibitors amantadine and rimantadine and the neuraminidase inhibitors oseltamivir, zanamivir, and laninamivir, have made a remarkable contribution to the treatment of influenza [
Traditional herbal medicines have long played an important role in countries in the Far East, especially Japan, China, and Korea. Maoto (Ma-Huang-Tang in Chinese) is traditionally prescribed for acute febrile diseases or upper respiratory tract infection [
In spite of its clinical advantages, the anti-influenza mechanism of maoto remains unclear. We studied the anti-influenza effect of maoto using a culturing system in which lung epithelial cells were experimentally infected with influenza virus in the presence of maoto. After attachment to cell surface receptors, influenza viruses are trapped by the endosome, where the envelope of the influenza virus fuses with the endosome membrane in an acidified condition; then nucleocapsids are released into the cytosol. The intraluminal space of the endosome and lysosome is acidified by vacuolar-type H+ ATPase (V-ATPase) at the endosomal membrane. We examined whether or not the function of V-ATPase at the endosomal membrane was inhibited by maoto, with a quick increase of endosomal pH possibly resulting in the influenza virus being unable to enter the cytoplasm. We show here evidence that maoto has an anti-influenza drug effect on the host’s endocytic system and defense.
The following cell lines were used for experimental infection: human lung carcinoma cell line A549, human keratinocyte cell line HaCat, human cervix carcinoma cell line HeLa, and mouse embryonic fibroblast cell line MEF. Madin-Darby canine kidney (MDCK) cells were used for the titration of infective influenza virus in the culture supernatant. For the main experiments the Puerto Rico/8/34 (PR8) influenza A virus strain was used, and for other experiments A/Victoria/210/2009 (H3N2), A/California/7/2009 (H1N1, pdm09), and B/Brisbane/60/2008 were used. The latter three strains were the components of the influenza vaccine used in the 2011-2012 winter season in Japan. PR8 was provided by Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan), and all other viruses were provided by the National Institute of Infectious Diseases (Tokyo, Japan). The virus titer in supernatant was determined on MDCK cells according to the methods of Reed and Muench [
For the attachment of virus to the cell surface, suspended virus at a moi of 1–100 was added to confluent monolayers of A549 cells in 12-well tissue culture plates for 1 hr, on ice. Cells were washed twice with cold PBS to remove the extracellular viruses and then cultured for the indicated amount of time with or without antivirus reagents at 37°C.
Maoto is a multicomponent formulation extracted from four plants: Ephedra Herb, Apricot Kernel, Cinnamon Bark, and Glycyrrhiza Root (Table
Maoto extract.
Plants | Weight ratio (%) | Major components |
---|---|---|
Ephedra Herb | 32.3 | Ephedrine, Tannin |
Apricot Kernel | 32.3 | Amygdalin |
Cinnamon Bark | 25.8 | Cinnamic aldehyde, Tannin |
Glycyrrhiza Root | 9.6 | Glycyrrhizin |
To visualize the influenza protein, the cells were stained with anti-matrix protein 2 (M2) (Thermo Scientific, Hudson, NH), anti-haemagglutinin (HA) (Santa Cruz Biotechnology, Dallas, TX), or anti-nucleoprotein (NP) (ViroStat, Portland, ME) mAb and visualized by FITC-conjugated secondary mAb followed by staining with DAPI nucleic acid stain (invitrogen) for 1 min; then the cover glasses were mounted on microscope slides. The cells were analyzed on a Keyence all-in-one fluorescence microscope BZ-9000 with a 60x lens (Osaka, Japan) and confocal microscopy (Zeiss LSM710, Oberkochen, Germany). Acidified organelles were stained with dyes, LysoSensor green (ThermoFisher, Yokohama, Japan) and Acridine orange (ThermoFisher), and were assayed by confocal microscopy (Zeiss LSM710, Oberkochen, Germany).
Cultured cells were collected and stained with anti-M2 (Thermo Scientific) mAb for 1 hr, washed with PBS, and stained with FITC-conjugated mouse mAb (Invitrogen). After the cell wash, cells were gated in forward and side scatter using flow cytometry, FACSCanto (BD, Franklin Lakes, NJ): up to 30,000 gated cells were acquired for the analysis. For the toxicity assay of maoto, the cells were stained with 7-amino-actinomycin D and Annexin V.
RNA extraction was carried out using ISOGEN II (Nippon Gene, Tokyo, Japan) and cDNA synthesis with a prime script RT reagent kit with gDNA eraser (Takara, Tokyo, Japan), according to the manufacturer’s instructions.
Real-time PCR analysis was performed using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) with SYBR Green Kit (Takara). The following primers were used: GAPDH-Fw: 5′-TCCACCACCCTGTTCCTGTA-3′, GAPDH-Rv: 5′-ACCACAGTCCATGCCATCAC-3′, PA-Fw: 5′-GCTTCTTATCGTTCAGGCTCTTAGG-3′, PA-Rv: 5′-CCGAGAAGCATTAAGCAAAACCCAG-3′, NS-Fw: 5′-CAGGACATACTGATGAGGATG-3′, NS-Rv: 5′-GTTTCAGAGACTCGAACTGTG-3′, MxA-Fw: 5′-TTCAGCACCTGATGGCCTATC-3′, MxA-Rv: 5′-TGGATGATCAAAGGGATGTGG-3′, PKR-Fw: 5′-TCTCTGGCGGTCTTCAGAAT-3′, PKR-Rv: 5′-ACTCCCTGCTTCTGACGGTA-3′, IFN-beta-Fw: 5′-TGCTCTCCTGTTGTGCTTCTCC-3′, IFN-beta-Rv: 5′-CATCTCATAGATGGTCAATGCGG-3′, IFN-lambda1-Fw: 5′-GAAGCAGTTGCGATTTAGCC-3′, and IFN-lambda1-Rv: 5′-GAAGCTCGCTAGCTCCTGTG-3′.
All samples were done at least in triplicate, with each experiment repeated at least twice. Results are expressed as means ± standard deviation. Differences between groups were determined using the unpaired Student
The cytotoxic effect of maoto was first assayed by flow cytometry after cell staining with 7-amino-actinomycin D and Annexin V in which the double negative population is determined as viable cells. When A549 cells were cultured with 0 to 1,000
Maoto is not toxic for cell lines and influenza viruses. (a) A549 cells were cultured with 0 to 1,000
In order to investigate the anti-influenza virus effect of maoto, we cultured A549 cells infected with PR8 in various concentrations of maoto, laninamivir, or amantadine, and the culture supernatant was assayed for the titer of extracellular infectious virus. The virus titer without antivirus reagent was 6.54
Maoto inhibited influenza virus propagation in vitro. All cell lines infected with influenza viruses (moi = 1) were cultured for 24 hr; then culture supernatants were assayed for the titer of infectious virus. (a) A549 cells infected with PR8 were cultured with various concentrations of maoto, laninamivir, or amantadine. (b) Left: A549 cells infected with PR8 were cultured with the components of maoto, Ephedra Herb (130
To generalize this inhibitory effect of maoto on the virus titer, experiments were performed using other subtypes of influenza virus and different cell lines. When A549 cells were infected with A/California/7/2009 (H1N1, pdm09), A/Victoria/210/2009 (H3N2), or B/Brisbane/60/2008, the virus titer in wells cultured with maoto was significantly lower than the titers with the control medium (Figure
To visualize the components of the influenza virus, cells were cultured for 24 hr in the presence of maoto, stained with mAb to M2 or NP of PR8, and imaged by immunofluorescence microscopy (Figure
Maoto inhibits intracellular protein derived from PR8. A549 cells infected with PR8 (moi = 1) were cultured with or without maoto for 24 hr. Proteins derived from PR8 were visualized by labeled mAb. (a) Cells were stained with mAb to M2 or NP (green) and assayed by fluorescence microscopy. Nuclei were dyed blue. (b) Cells were stained with mAb to M2, and fluorescent intensity was assayed by flow cytometry. (c) Cell lysates were assayed by western blot analysis for M2 protein.
In influenza virus infection, type I interferons (IFN
Maoto did not induce an intracellular antiviral molecule related to type I or type III IFNs in influenza virus infection. A549 cells infected with PR8 were cultured with or without maoto, and mRNA levels of IFN-
Second, we hypothesized that maoto blocked the entry step of influenza virus into the cytoplasm. Because the replication cycle of influenza virus needs 6–8 hrs from attachment to shedding, the assay used to determine virus entry into the cytoplasm of host cells requires short-term culturing to avoid the influence of progeny viruses. We next examined viral RNA replication (NS and PA segments) under the short-term culture condition, resulting in the viral RNA being remarkably inhibited by maoto throughout 6 hrs after infection (Figure
The effect of maoto was observed in the very early phase of influenza virus infection. A549 cells infected with PR8 (moi = 1) were cultured for 6 hr. The length of maoto treatment was varied in each experiment. Levels of mRNA for NS and PA segment of PR8 were assayed by real-time PCR analysis. (a) Cells were treated with maoto for 6 hr. Open and closed circles indicate control and maoto, respectively. (b) Maoto treatment for 0–2, 2–4, or 4–6 hr. (c) Maoto treatment for 0–30, 30–60, 60–90, or 90–120 min.
It has been reported that an acidic condition in endosomes is essential for the uncoating process of influenza virus infection and that it triggers viral envelope fusion activity [
Endosomal acidification was inhibited by maoto. A549 cells were incubated with PR8 for 1 hr at 4°C (moi = 1), washed by PBS, and then cultured at 37°C with or without maoto in the presence of LysoSensor green (a) or Acridine orange (b). Acidified endosomes and lysosomes at 0, 30, and 60 min were visualized by fluorescence microscopy. The former can be visualized as green fluorescence and the latter as orange fluorescence in high concentration whereas it was green in low concentration.
We further investigated whether or not influenza viruses could be uncoated, following the inhibition of endosomal acidification by maoto. The number of influenza virus particles visualized by HA-staining after treatment with maoto showed a greater increase when compared to the control (Figure
A549 cells were incubated with PR8 for 1 hr at 4°C (moi = 100), washed by PBS, and then cultured at 37°C with or without maoto. Intracellular influenza virus at 30 and 60 min after infection was visualized by the staining of mAb to HA (green) followed by staining with DAPI.
The present study demonstrates that maoto, a traditional Japanese herbal medicine, has the capacity to inhibit experimental infection with influenza virus PR8, as well as the subtypes A/H1N1(2009 pdm), A/H3N2, and B. Of the four plants included in maoto extracts, only Ephedra Herb and Cinnamon Bark had a significant antiviral effect. The antiviral effects of maoto were mainly explained by blocking the uncoating process of the influenza virus through the inhibition of V-ATPase, a proton pump located in the endosome and lysosome membranes.
Recently, several new reports related to Kampo and Chinese medicines for influenza infection have been focused on the molecular level [
Our results correspond with a previous study showing an anti-influenza effect of Ephedra Herb, one of the plants included in maoto extracts, through the inhibition of endosomal acidification in vitro [
V-ATPase is a proton pump that acidifies the lumen of endomembrane organelles, such as lysosomes, endosomes, Golgi apparatus, and secretory granules and that is required for the entry of influenza virus into cells [
Maoto extracts consist of four plants and include hundreds of molecules [
Antiviral drugs mainly target viral molecules, such as neuraminidase and M2. These drugs are at risk of losing effectiveness by viral mutation. Kampo medicine targets the host cellular processes and enhances the defense system against various microorganisms, which can reduce the risk of viral mutation. In addition, maoto may have an effect on other viruses that use endosomes in the infection process, such as coronavirus, adenovirus, and togavirus. Interestingly, maoto has traditionally been used not only for influenza, but also for other acute febrile diseases. Our results confirm the common wisdom built over hundreds of years that the use of maoto is effective in the treatment of acute febrile diseases caused by various pathogens.
The authors declare that they have no conflicts of interest.
The authors thank Dr. Takeshi Ichinohe (The Institute of Medical Science, The University of Tokyo) for technical advice, Dr. Kazuhiko Kimachi (Chemo-Sero-Therapeutic Research Institute) for providing PR8, and Dr. Takato Odagiri (National Institute of Infectious Diseases) for A/Victoria/210/2009 (H3N2), A/California/7/2009 (H1N1, pdm09), and B/Brisbane/60/2008. This work was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science (13200657).