Background
Influenza is a contagious respiratory illness causing seasonal epidemics and occasional pandemics. The death toll of influenza epidemics is between 250,000 to 500,000. The frequent reassortment of influenza virus may cause high mortality and over-burden the healthcare system [
1]. For example, the outbreak of 2009 H1N1 pandemic (swine flu) caused around 185,000 of people death [
2]. The most recent 2013 H7N9 is the largest annual epidemics in China also caused significant morbidity and mortality [
3].
To date, two classes of anti-influenza drugs are commonly used [
4]. One consists of inhibitors of the M2 ion channel, such as amantadine and rimantadine. Treatment with these drugs results in the emergence of resistant strains thus it is not recommended for general use. The other consists of neuraminidase inhibitors, such as oseltamivir, zanamivir, laninamivir and peramivir. In addition, ribavirin and favipiravir (T-705) show anti-viral RNA polymerase effect [
5,
6]. However, resistance against these drugs has already emerged in recent years [
7]. These highlight the urgent need for new anti-influenza agents.
Traditional herbal medicine remains an under-explored, yet potentially fruitful basis for antiviral discovery [
8]. In ancient China, some Chinese prescriptions were used to treat Wen Bing (Warm Disease), which is considered as influenza in modern time, with influenza-like symptoms, such as high fever, thirsty and anxiety [
9]. To date, these prescriptions are still used in clinics by traditional Chinese medical practitioners. Also, in South China, multi-herb drink or “cooling herbal tea” is a convenient folk treatment against normal cold or mild influenza [
10]. However, the efficacy of most of these products has not been vigorously tested. There is no conclusive experimental evidence to support the clinical efficacies of these prescriptions in treating influenza. Nevertheless, researchers have begun to evaluate the therapeutic values and underlying mechanism of selected prescriptions, including Chinese patent drugs and traditional Chinese prescriptions [
11‐
13]. For example, Lianhua Qingwen capsule [
14] was shown to have broad-spectrum efficacy on a number of influenza virus strains, through regulating the immune responses after virus infection. Kang Bing Du oral liquid [
15] was found to reduce the susceptibility to influenza virus via mitochondrial antiviral signaling.
Sheng Jiang San (SJS) is a famous Chinese prescription that was originally recorded in a Traditional Chinese Medicine Classic Shanghan Wenyi Tiaobian of Qing Dynasty. SJS is composed of Rhei Radix et Rhizoma, Bombyx Batryticatus, Cicadae Periostracum and Curcumae Longae Rhizoma in a ratio of 4:2:1:3 (w/w/w/w). It has been prescribed in treating “Warm Disease”. In modern time, traditional Chinese medical practitioners use it to treat seasonal influenza. However, there is no proper statistics on its clinical efficacy and revelation of the anti-influenza virus mechanism. Our preliminary test showed that it could indeed inhibit influenza A/WSN/33 (H1N1) in cell culture. As a contribution to increase the clinical value and the modernization of Chinese medicine, we set forth to examine the influenza inhibitory effect of SJS.
Currently, influenza virus infected mouse model is frequently used to test the in vivo influenza therapeutic efficacy of a drug [
11‐
16]. In this study, except examining the inhibitory effect of SJS against different influenza virus strains on Madin-Darby canine kidney (MDCK) cells, we also used influenza virus infected BALB/c mice as an in vivo model to investigate the therapeutic action of SJS. The underlying anti-influenza virus mechanisms were studied by a battery of biological assays, which include viral absorption and release, and the function of viral polymerase complex.
Methods
Reagents
Rhei Radix et Rhizoma, Bombyx Batryticatus, Cicadae Periostracum and Curcumae Longae Rhizoma were purchased from Zisun Chinese Pharmaceutical Co., Ltd. (Guangzhou, China). Standard compounds of rhein, chrysophanol, emodin, aloe emodin and curcumin were purchased from Chengdu Pufeide Biotechnology Co., Ltd. (Chengdu, China). Oseltamivir was purchased from Yichang Changjiang Pharcaceutical Co., Ltd. (Wuhan, China). Minimum essential medium (MEM), Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Gibco, NY, USA). Neuraminidase inhibitors screen kit (no. P0309) was purchased from Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). Chicken erythrocytes were purchased from Lampire Biological Laboratories (PA, USA). Tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) treated-trypsin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse TNF-α, IFN-α and IL-2 Enzyme-linked immunosorbent assay (Elisa) kit were purchased from Invitrogen (Carlsbad, CA, USA). Water used in this study was purified by a Milli-Q system (Millipore, MA, USA). All culture plates were obtained from Greiner (Cellstar, Germany).
The identities of Rhei Radix et Rhizoma, Bombyx Batryticatus, Cicadae Periostracum and Curcumae Longae Rhizoma were confirmed by an expert at the Institute of Chinese Medicine, The Chinese University of Hong Kong, by referring to their organoleptic characteristics. The voucher specimens were kept at Li Dak Sum Yip Yio Chin R & D Centre for Chinese Medicine, The Chinese University of Hong Kong. The aqueous extract of SJS was prepared by boiling the herbs at 4:2:1:3. The four ingredients in proportion were boiled twice with deionized water for 1 h each time. The aqueous extract was filtered and concentrated by a rotary evaporator under vacuum in a 60 °C water bath. Then the concentrated extract was lyophilized into powder under vacuum of 105 × 10− 3 mbar and − 40 °C. The freeze-dried powder was dissolved in culture medium or water before used.
Quality control is important in Chinese prescription, as the consistency will affect the repeatability of experiments and clinical efficacy. In light of this, a large amount of freeze-dried powder of SJS was prepared only once for studies to avoid composition differences between different batches of herbs. SJS powder was analyzed by high-performance liquid chromatography (Additional file
1) and the chemical profile is shown in Additional file
2: Figure S1. By comparing with reference compounds, rhein, chrysophanol, emodin, aloe emodin and curcumin were found.
Cells, viruses and animals
MDCK cells and human embryonic kidney 293 T (293 T) cells were obtained from American Type Culture Collection and routinely cultured in MEM and DMEM, respectively, supplemented with 10% FBS and incubated at 37 °C with 5% CO2. Influenza A/WSN/33 (H1N1) (WSN), A/PR/8/34 (H1N1) (PR8), A/GZ/GIRD07/09 (H1N1), A/HK/8/68 (H3N2), A/Aichi/2/1968 (H3N2), A/HK/Y280/97 (H9N2), A/China/24/96 (H7N3), B/Lee/1940 (Flu B) were provided by Dr. Zifeng Yang (Guangzhou Institute of Respiratory Disease, China). All in vitro tests were performed in class II biosafety cabinet.
Specific-pathogen-free Balb/c mice weighing 14–16 g were used in this study. Mice were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China). The animal experiments were carried out according to the Guidelines of Guangdong Regulation for the Administration of Laboratory Animals. The mice were kept in biosafety level 3 housing and provided with standard laboratory diet and water ad libitum.
Cytotoxicity assay
Cytotoxic effect of SJS was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. MDCK cells (2 × 105) were seeded on a 96-well culture plate in MEM with 10% FBS. After overnight culture, cells were treated with different concentration of SJS in MEM. After 24 h incubation at 37 °C, MTT (5 mg/ml) in phosphate buffered saline (PBS) was freshly prepared, 10 μl of MTT solution was added to each well and the plates were incubated at 37 °C for 4 h. The medium was then removed and formazan crystal was dissolved in dimethyl sulfoxide (DMSO) (100 μl/well). Then the absorbance at 570 nm was read by a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany). The 50% toxic concentration (TC50) was calculated as the concentration required to decrease 50% of cell viability.
Cytopathic effect inhibition (CPE) assay
80% confluent MDCK cells in a 96-well plate were infected with 0.01 MOI of influenza virus for 1 h at 37 °C. Afterwards, the viral inoculum was removed, and cells were washed twice with PBS. 100 μl SJS at different concentration in serum-free MEM with 1 μg/ml TPCK treated-trypsin (TPCK treated-trypsin was absent when MDCK cells were infected by WSN virus) was added to the cells. After incubating at 37 °C for 48 h, 10 μl of 5 mg/ml fresh MTT solution in PBS was added to each well and the plates were incubated at 37 °C for 4 h. The medium was then removed and formazan crystal was dissolved in DMSO (100 μl/well). Absorbance at 570 nm was read by a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany). The concentration that inhibited 50% of virus-induced cytopathic effect was determined as IC50.
Plaque reduction assay
Confluent MDCK cells were seeded in 6-well plates in MEM with 10% FBS. Cells were infected with around 200 pfu per well of different viral strains for 1 h at 37 °C. The inoculum was aspirated to remove unbound viral particles, followed by washing with PBS. The MDCK monolayer was then overlaid with 1% low melting agarose (Cambrex) in MEM which contained different concentration of SJS and 1 μg/ml of TPCK treated-trypsin (TPCK treated-trypsin was absent when MDCK cells were infected by WSN virus). After incubating for 72 h at 37 °C, the agarose was removed and the cell monolayers were stained with staining solution (0.25% coomassie blue, 10% acetic acid, 50% methanol). The number of plaques was counted and the percentage of plaque inhibition relative to the control (no drug treatment) was calculated.
Multicycle growth assay
80% confluent MDCK cells were seeded in a 24-well plate. After infecting with 0.001 MOI of WSN for 1 h at 37 °C, the inoculum was removed and 500 μl of SJS (500 μg/ml, 125 μg/ml, 60 μg/ml) or oseltamivir (100 μM) in MEM, or 500 μl MEM only were added to the cells and incubated at 37 °C. The supernatants were then collected at 12, 24, 48 and 72 h post infection. The virus titers were determined by plaque assay as described previously [
17].
Hemagglutination inhibition assay
Twofold serial dilution of SJS was prepared in 25 μl of PBS in a 96-well U-bottom plate. WSN in 25 μl of PBS (4 HA units) was added to each dilution and mixed well, and the plate was incubated for 30 min at room temperature. Then, 50 μl of chicken erythrocytes in PBS (0.05%
v/v) was added to each well and mixed thoroughly. The reaction was observed after incubating the plates at room temperature for another 30 min. Pentagalloyglucose (PGG) was used as a positive control [
18] while oseltamivir was a negative control [
19].
Ribonucleoprotein (RNP) reconstitution assay
2 × 106 of 293 T cells were seeded on a 6 cm dish and incubated overnight in DMEM with 10% FBS. Plasmids pcDNA3a-PB1, pcDNA3a-PB2, pcDNA3a-PA, pcDNA3a-NP, pPOL-NS-Luci (kindly provided by Dr. Ervin Fodor, University of Oxford, UK) were transfected to 293 T cells with Lipofectamine 2000 (Invitrogen, CA, USA) to reconstitute the RNP complex. The RNP complex consisted of WSN polymerase proteins PA, PB1 and PB2, NP and a luciferase reporter gene. Plasmid pEGFP was also co-transfected to 293 T cells as an internal control to normalize the transfection efficiency. After 6 h of transfection, transfected cells were trypsinized and aliquoted into a 96-well plate. SJS at different concentration dissolved in DMEM was added into each well. After incubating for 24 h at 37 °C, the cell lysates were harvested and the luciferase activity was assayed with a luciferase reporter assay system kit (Promega, No. E1910). The luminescence was read by a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany).
Neuraminidase (NA) inhibition assay
A neuraminidase inhibitors screen kit was employed to evaluate the inhibition of SJS on the NA enzymatic activity. The assay followed the instruction manual. 70 μl of reaction buffer, 10 μl of NA and 10 μl of SJS at different concentration were well mixed in a black 96-well microplate. After incubating at 37 °C for 2 min, 10 μl of substrate was added into each well, mixed thoroughly and incubated for 1 h. Fluorescence was measured with a CLARIOstar multi-mode microplate reader (BMG Labtech, Germany) at an excitation wavelength of 322 nm and an emission wavelength of 450 nm. Oseltamivir acid [
19] was used as a positive control. The NA activity inhibitory percentage was calculated as follow:
$$ \mathrm{NA}\ \mathrm{inhibition}\ \left(\%\right)=\left({\mathrm{F}}_{\mathrm{control}}\hbox{--} {\mathrm{F}}_{\mathrm{SJS}}\right)/\left({\mathrm{F}}_{\mathrm{control}}\hbox{--} {\mathrm{F}}_{\mathrm{blank}}\right)\times 100\%\left(\mathrm{F}:\mathrm{Fluorescence}\ \mathrm{intensity}\right). $$
Anti-influenza virus test in mouse model
Mice were randomly divided into vehicle group, SJS group, oseltamivir group and untreated group, with 16 mice in each group. Except the vehicle group, other groups were anaesthetized with ethyl ether and inoculated intranasally with 3 LD50 (50% lethal dose) of mouse-adapted PR8 virus in a volume of 50 μl. At 4 h after inoculation, SJS group and oseltamivir group were treated by gavage feeding with SJS solution (dissolved in water at a dose of 1 g/kg/day) or oseltamivir solution (dissolved in water at a dose of 90 mg/kg/day) in a volume of 200 μl, respectively. Then these two groups were administrated orally once daily for seven consecutive days. Untreated group and vehicle group were fed with water. Parameters of mice such as mortality, body weight and general conditions were monitered for consecutive 14 days.
Three mice from each group were randomly selected and sacrificed on the fourth day post-inoculation for lung index calculation, lung viral load titer and lung cytokine expression analysis. Another three mice from each group were also sacrificed on the sixth day post-inoculation for histopathologic observation. The sacrificed mice were euthanized by cervical dislocation after fully anaesthetization by inhaling diethyl ether. The remaining ten mice in each group were monitored continuously for 14 consecutive days to study their mortality and body weight changes.
Lung index
Four days after virus infection, mice were weighed and their lung tissues were extracted and washed with PBS, dried by gauze and then weighed. Lung index was calculated as follow:
$$ \mathrm{Lung}\ \mathrm{index}=\mathrm{lung}\ \mathrm{weight}/\mathrm{body}\ \mathrm{weight}\times 100\%. $$
Lung viral load titer
After weighing the lung tissues, they were homogenized in MEM by a refiner (Qiagen, TissueRuptor) and centrifuged at 12000 rpm for 5 min at 4 °C. The lung homogenates were aliquoted and stored at − 80 °C. The virus titer of these homogenates were determined by plaque assay [
17] on MDCK cells.
Lung cytokine expression analysis
Part of the lung homogenates was used to perform lung cytokine expression analysis with mouse cytokines Elisa kits of TNF-α, IFN-α and IL-2 (Invitrogen). The content of TNF-α, IFN-α and IL-2 were assessed according to the manufacturer’s protocol. Absorbance at 450 nm was read by a spectro-photometer (Thermo Scentific).
Histopathologic observation
Six days after virus infection, lung tissues were extracted from three randomly sacrificed mice from each group. The lungs were immersed in 10% formaldehyde solution immediately and embedded in paraffin. Then the lung tissue was cut into 4 μm-thick sections. Tissue sections were stained with hematoxylin and eosin for observating the histopathologic changes under microscope.
Statistics
All statistical analyses were conducted with Graphpad Prism 6.0 (Graphpad, San Diego, CA, USA) and data were presented as mean ± SD. One-way ANOVA was used for multiple-group comparison. Differences were considered statistically significant when p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).
Discussion
SJS is a classical Chinese formulation for treating influenza virus infection. However, there has been no systematic study to substantiate the anti-influenza efficacy. The mechanism of SJS action also remained unclear.
We first found that SJS could inhibit flu A and B strains on MDCK cell line at less than 750 μg/ml, showing that SJS has a broad-spectrum inhibitory activity to influenza viruses. We also showed that SJS could protect mice from PR8 infection. Compared with the untreated group, SJS could significantly improve the survival rate and prolong the average survival day, also help the body weight of the mice to rebound. SJS treated mice not only had a lower lung viral load but also had reduced lung index and alleviated histopathology, suggesting that SJS might work on the virus directly and SJS could also decrease the lung injury induced by PR8 infection. Compared with oseltamivir, SJS is less effective in protecting PR8 infected mice. However, considering that the formulation containing many phytochemicals, with each in a minute quantity, there is a high chance to have some potent fractions or phytochemicals in the extract.
Previous reports pointed out that over-expression of TNF-α and IFN-α induced by influenza virus is a cause of lung inflammation and can in turn result in tissue injury [
20,
21]. In contrast, IL-2 decreases after infected by influenza virus and lower expression of IL-2 causes dysfunction of immune system [
11]. In this study, the expression level of three cytokines (TNF-α, IFN-α, IL-2) after PR8 virus infection was examined. Our data showed SJS was beneficial to PR8 infected mice, as SJS could down-regulate TNF-α and up-regulate IL-2. The expression level of IFN-α under treatment of SJS also showed a down-regulatory trend, although there was no statistically significant difference with the untreated group. Meanwhile, compared with the treatment of oseltamivir, SJS showed a stronger regulation on the expression of these cytokines (Fig.
5). Thus, besides inhibiting virus replication directly through targeting on NA, SJS might also protect the mice through regulating the expression of cytokines. We are in the process of carrying out bioassay-guided fractionation to find the pure phytochemicals that inhibit NA and regulate the concerned cytokines.
As influenza infection is a common disease, there are many cold and flu herbal remedies in the market, such as Chinese patent drugs, traditional Chinese prescriptions, or even cooling herbal tea. However, their efficacy in treating influenza is not well investigated. Our research has provided an example on the evidence-based research on an anti-influenza formulation, which involves virus inhibitory studies in cell culture, animal model and mechanism elucidation. The work may be extended to other traditional or folk medicine, which will strengthen the confidence on their clinical uses and for downstream development of these formulations.
Acknowledgements
The authors would like to extend their sincere appreciation to Dr. Zifeng Yang (Guangzhou Institute of Respiratory Disease, China) and Dr. Ervin Fodor (University of Oxford, UK) for providing viruses and plasmids respectively.