Reference substances of Gallic acid (Lot: 1019C023), 3'-Hydroxypuerarin (Lot: 1019C02333), Puerarin (Lot: 1202A022), Paeoniflorin (Lot: 918B021), Daidzin (Lot: 1215A022), Daidzein (Lot: 715A021), Cinnamic acid (Lot: 510A022) and Glycyrrhizic acid (Lot: 419B021) were purchased from Solaibao technology Co., Ltd. (Beijing, China). Protocatechuic acid (Lot: Z30M6L1), Albiflorin (Lot: Y15D8H50784), 3'-Methoxypuerarin (Lot: P22A8F42194), Puerarin 6"-O-xyloside (Lot: P09J7F8771), Mirificin (Lot: P09J7F8770), Rutin (Lot: Y16M9S61523), Genistin (Lot: P09M8F31018), Ononin (Lot: R28O8F46957) and 6-Gingerol (Lot: Y02J9H52043) were bought from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Ephedrine hydrochloride (Lot: 171241-201508) and Pseudoephedrine hydrochloride (Lot: 171237-201510) were obtained from the National Institutes for Food and Drug Control (Beijing, China). Acetonitrile and methanol (HPLC grade) were purchased from TEDIA (Ohio, USA). Formic acid (HPLC grade) was purchased from China National Pharmaceutical Group Corporation (Beijing, China). Oseltamivir Phosphate was purchased from MedChemExpress LLC (NJ, USA). The herbal materials of GGD were purchased from the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, Shandong, China) and authenticated by Professor XU Ling-Chuan (Shandong Traditional Chinese Medicine University, Jinan, China). The quality of these herbs reached the standards of Chinese Pharmacopoeia. The voucher specimens of the herbal materials were kept in the Collaborative Innovation Center.
According to the method in "Chinese Pharmacopoeia", Pueraria lobate (Wild.) (12 g), Ephedra sinica (9 g), Cinnamomum cassia (6 g), Paeonia lactiflora (6 g), Glycyrrhiza uralensis (6 g), Zingiber officinale (9 g) and Ziziphus jujuba (22 g) were decocted separately with 700 mL and 560 mL water for 30 minutes each time. The decoction was concentrated and freeze-dried to obtain the lyophilized extract powder (yield: 34.7%).
Stock solutions of the 19 reference substances were prepared by dissolving the powders in 50% methanol. The stock solutions were stored at 4 ℃. The standard working solutions were obtained by mixing or diluting the stock solutions.
The lyophilized extract powder (equivalent to 1 g raw material) was dissolved in 10 mL 50% methanol. The solution was centrifuged at 7000 r·min-1 for 10 min. The supernatant was filtered through a 0.22 μm membrane before LC and LC/MS analysis.
HPLC-Q-TOF-MS/MS analysis was performed on an Agilent 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization source. Chromatographic separation was carried out on a ZORBAX SB-C18 column (5 μm, 4.6 mm × 250 mm, Agilent Technologies) with column temperature set at 30 ℃. The mobile phase was composed of A (0.1% formic acid-water) and B (acetonitrile), a gradient elution was performed as follows: 0-30 min, 7% B; 30-45 min, 7%-11% B; 45-55 min, 11% B; 55-80 min, 11%-16% B; 80-120 min, 16%-30% B; 120-140 min, 30%-60% B. The sample injection volume was 10 μL, and the flow rate was 1.0 mL·min-1. The DAD detector scanned from 200 to 400 nm, and the samples were detected at 245 nm. For MS detection, the operating parameters in both negative and positive ion modes were set as follows: drying gas (N2) flow rate, 12 L×min-1; drying gas temperature, 350 ℃; capillary voltage, 3500 V; nebulizer pressure, 40 psi. The collision energy was automatically optimized according to the ion type. The MS range was set at m/z 100-1000.
An Agilent 1260 HPLC-DAD instrument (Agilent Technologies, CA, USA) was used for HPLC determination. The chromatographic separation conditions were the same as LC/ MS mentioned above.
Madin-Darby canine kidney (MDCK) cells were acquired from Cell Resource Center of Shanghai Academy of Life Sciences of the Chinese Academy of Sciences (Shanghai, China), which cultured in DMEM (Gibco, CA, USA) contained 10 % FBS (Gibco, CA, USA), 1000 U·mL-1 penicillin, and 100 μg·mL-1 streptomycin (Gibco, CA, USA). Infections were carried out in Opti-MEM containing 2 μg·mL-1 TPCK- Trypsin (Sigma-Aldrich, MO, USA).
Mouse-adapted influenza A virus (IAV) H1N1 [A/PR/8/ 34] was acquired from Chinese Center for Disease Control and Prevention (Beijing, China), which multiplied in the 10-day-old embryonated eggs. The 50% tissue culture infective dose (TCID50) of the virus in the MDCK cells and 50% lethal dose (LD50) of the virus in the mice were determined by the method of Reed-Muench  (1 × 105.7 TCID50/mL, 6.7 × 105 LD50/mL).
Various concentrations of GGD extract and Oseltamivir phosphate (OP) were inoculated into MDCK cells for 48 h. The 50% cytotoxic concentration (CC50) for MDCK cells was determined using a CCK-8 assay kit. The maximum drug concentration which caused cell mortality rate to be lower than 10% was chosen as drug non-cytotoxic concentration.
OP was used as a reference control. Briefly, 1 × 104 cells/ well were plated in 96-well plates and incubated for 18-24 h. The cells were supplemented with various concentrations of GGD or OP immediately after virus (TCID50/well) inoculation in triplicate. After 48 h, the virus inhibition rate was determined using CCK-8 assay  and calculated as Equation (1). The 50 % inhibitory concentration (IC50) values of GGD and OP were calculated according to the Reed-Muench method .
Antiviral activity of the GGD extract was examined before and after viral inoculation [18-19]. Briefly, the MDCK cells grown in 96-well plates were inoculated with 100 TCID50/ well virus. Various concentrations of GGD extract were supplemented at −4 h or −2 h (4 h or 2 h before viral inoculation), 2 h or 4 h (1 h or 2 h after viral inoculation). After 48 h incubation at 37 ℃, the virus inhibition rate was determined as described above.
The effect of GGD extract on viral attachment and internalization was also examined as described previously [18-19]. Briefly, for the attachment assay, the cells were pre-chilled at 4 ℃ for 1 h and inoculated with a mixture of virus (100 TCID50/well) and different concentrations of GGD extract at 4 ℃ for 3 h. Then, the cells monolayer was firstly washed by PBS buffer (4 ℃), then covered with fresh medium and incubated at 37 ℃ for another 48 h. For the internalization assay, the cells were pre-chilled at 4 ℃ for 1 h, then inoculated with the virus (100 TCID50/well) and incubated at 4 ℃ for 3 h. The medium was replaced with fresh medium containing various concentrations of GGD extract. The cells were shifted to the culture at 37 ℃ for 1 h. Then, the cells monolayer was washed by PBS, covered with medium, and incubated at 37 ℃ for another 48 h.
The effect of GGD on the later stage of viral biosynthesis in a single infectious cycle was evaluated by qRT-PCR . Briefly, the cells (3.5 × 105 cells/well) grown in 6 well plates were inoculated with virus (0.01 MOI). After incubation for 2 h at 37 ℃, the medium was replaced with a fresh medium containing GGD (6.25 mg·mL-1) and OP (62.5 μg·mL-1) for three times. At 10 h post-infection, the viral RNA in the supernatants was isolated by using a Simply P Virus RNA Extraction Kit (Bio Flux, China) on a Bio-Rad CFX Connect apparatus. The primers are M-protein forward primer 5'-CTT CTAACCGAGGDCGAAAC-3' and M-protein reverse primer 5'-CGTCTACGCTGCAGTCCTC-3'. Recombinant plasmid including the M-protein gene of influenza virus (A/PR/8/34) was selected as a reference substance. In each experiment, a standard curve (R2> 0.99 within the range of 103 to 108 copies) was drawn to convert CT into the number of viral-genome copies. The reaction conditions were as follows: 95 ℃ for 30 s, followed by 35 cycles of denaturation at 95 ℃ for 5 s, annealing and extension at 62 ℃ for 34 s.
Based on a previous report , female ICR mice (18 to 20 g) were inoculated intranasally with 20 μL of a viral suspension (5 LD50 or 0.8 LD50) or PBS (n = 6) under anesthesia. The daily dosage of GGD extract (3.5 g·kg-1·d-1) or OP (21.2 mg×kg-1·d-1) was based on human-mouse equivalent dosage conversion. Mice were administered GGD, OP, or distilled water orally for 10 days from 3 days before infection. Mortality, weight and clinical features were monitored daily in the next two weeks. Mice with a weight loss of more than 20% were considered dead.
Another group of mice was inoculated with 0.8 LD50 of virus. From 3 days before infection, mice were administered GGD, OP, or distilled water orally. These mice were euthanized at the prescribed time. Lung and peripheral blood were harvested for further analysis. Based on previous reports, the viral load in the lung tissue was determined by RT-PCR assay .
Animals experiments were processed in line with approved regulations by Animal Protection and Use Committees, Shandong Traditional Chinese Medicine University (Approval: SDUTCM-2017024, 23 October 2017). Animals were maintained in the ABSL-2 laboratory, and all efforts were made to minimize animal suffering and the number of animals used in the experiment.
The lung index assessed pulmonary edema. The pathological changes were observed on routine paraffin sections that were stained with hematoxylin (HE).
The total RNA of the lung tissue was isolated by using the RNAiso Plus reagent (Takara bio, China). The cDNA samples were prepared by using the PrimeScript RT reagent Kit (Takara bio, China). RT-PCR was performed with the SYBRs Premix Ex Tap TM Ⅱ (Takara bio, China) on a Bio-Rad CFX Connect apparatus. The primers are described in Table 1. The reaction conditions were as follows: 95 ℃ for 30 s, followed by 35 cycles of denaturation at 95 ℃ for 5 s, annealing at 56 ℃ for 30 s and extension at 72 ℃ for 30 s. CT value was analyzed using the comparative CT (ΔΔCT) method. The results were normalized to GAPDH.
Target Gene Direction Sequence (5'- 3') IL-1α For GTTCCTGACTTGTTTGAAGACC Rev GTTGGACATCTTTGACGTTTCA IL-6 For GCTACCAAACTGGATATAATCAGGA Rev CCAGGTAGCTATGGTACTCCAGAAC TNF-α For TGTAGCCCACGTCGTAGC Rev TTGAGATCCATGCCGTTG IFN-γ For ATCTGGAGGAACTGGCAAAA Rev TTCAAGACTTCAAAGAGTCTGAGG IL-4 For TACCAGGAGCCATATCCACGGATG Rev TGTGGTGTTCTTCGTTGCTGTGAG TLR7 For CTGGAGTTCAGAGGCAACCATT Rev GTTATCACCGGCTCTCCATAGAA MyD88 For CAGCGAGGDTTGCATCTTCTT Rev TCACTTTCTTGGGGACTCAGG TRAF6 For GCAGTGAAAGATGACAGCGTGA Rev TCCCGTAAAGCCATCAAGCA IRF7 For CTGGAAGCATTTCGGDCG Rev CCTGTGTGGGCAGAGCA NF-κB For ACGCGGATTCCTGTACACCT Rev CAGGAGCTCCACAGGACAGA GAPDH For CTTCTAACCGAGGDCGAAAC Rev CGTCTACGCTGCAGTCCTC
Table 1. Primer sequence for RT-PCR
Proteins were extracted from the lung tissue by RIPA buffer (Beyotime, Shanghai, China) containing PMSF (Beyotime, Shanghai, China) and Phosphatase inhibitor cocktail (Beyotime, Shanghai, China). A BCA protein concentration assay kit (Servicebio technology Co., Ltd., Wuhan, China) was used to measure protein concentration. Protein (40 μg) was separated by 8% (TLR7), 10% (virus-NP, MyD88, TRAF6, IRF7, p-IRF7, and IKBα, ) and 15% (IL-6, TNF-α, IFN-γ, and IL-4) SDS-PAGE. Proteins were transferred to PVDF membranes (0.22 μm, Millipore, Darmstadt, Germany). The membranes were separately incubated with various primary antibodies against IL-6, TNF-α (Proteintech Group, Illinois, USA), IFN-γ, IL-4 (Affinity Biosciences, Ohio, USA), virus-NP, TLR7, TRAF6, IKBα (Abcam, Shanghai, China), MyD88, IRF7, phospho-IRF7 (Ser477) (Affinity Biosciences, Ohio, USA), and β-actin (Abcam, Shanghai, China) at 4 ℃ overnight. The secondary antibody incubated the membranes for 30 min at 37 ℃. Protein bands were detected by an ECL kit (Wuhan servicebio technology Co., Ltd., Wuhan, China) and visualized by an Alpha Innotech imaging system (San Leandro, CA, USA). If the different proteins on the blot need to be detected by the second set of specific probes, the primary and secondary antibodies will be cleaned with antibody stripping buffer (Beyotime, Jiangsu, China) and detected again. The results were normalized to β-actin.
PBMCs were isolated from the peripheral blood by lymphocyte separation medium (Solarbio, Beijing, China). PBMCs were stimulated with phorbol myristate acetate (PMA, 30 ng·mL-1, Beyotime, Shanghai, China) and ionomycin (1 μg·mL-1, Beyotime, Shanghai, China) in the presence of monensin (1.7 μg×mL-1, Beyotime, Shanghai, China) for 4 h. Then the cells were washed and resuspended in FACS staining buffer (PBS + 1% FBS). FC Receptor Blocker (BD Biosciences, NJ, USA) was used to block Fc receptors on the surface of cells. The cells were stained with anti-CD4-FITC (BD Biosciences, NJ, USA) antibodies in the dark at 4 ℃ for 30 min, then fixed with 4% Paraformaldehyde at 4 ℃ for 30 min. Next, the cells were washed and permeabilized by permeabilization solution (Solarbio, Beijing, China), then incubated with anti-IFN-γ-APC (BD Biosciences, NJ, USA) and anti-IL-4-PE (BD Biosciences, NJ, USA) antibodies at 4 ℃ for 1 h. Appropriate isotype controls were performed. The cells were washed by permeabilization solution and FACS staining buffer respectively, then suspended in FACS staining buffer and detected by flow cytometry (BD flow cytometer C6, BD Biosciences, NJ, USA).
The results were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using a two-tailed Independent-Samples t test. The Wilcoxon Mann- Whitney test conducted the statistical analysis of mortality. All data were analyzed by SPSS25 (IBM, Armonk, NY, USA).
HPLC-Q-TOF-MS/MS analysis and HPLC analysis
Cells and virus
In vitro antiviral determination
Attachment assay and internalization assay
The effect of GGD on the viral replication
In vivo anti-influenza virus experiments
Pathological changes in lungs
Western Blot analysis
Immunofluorescence labeling and flow cytometry
By analyzing the retention time, molecular ion, and fragment ions of the chromatographic peaks, and comparing the results with reference substances and previous literature data [22-25], 31 compounds were identified in GGD (Table 2, Figs. 1a and 1b), which included nineteen flavonoids, three organic acids, three monoterpenoids, two triterpenoid saponins, two alkaloids, and two other types of compounds. The contents of seven components with antiviral, antimicrobial, and anti-inflammatory activities [10-14, 26-27] in GGD were determined by high-performance liquid chromatography (HPLC) as 0.63 mg·g-1 gallic acid, 21.46 mg·g-1 puerarin, 3.74 mg·g-1 paeoniflorin, 3.08 mg·g-1 daidzin, 0.48 mg·g-1 daidzein, 0.24 mg·g-1 cinnamic acid, and 1.8 mg×g-1 glycyrrhizic acid (Fig. 1c).
Compound Formula Quasi-
Measured value Error/ppm MS/MS(m/z) 1a Gallic acidL C7H6O5 [M - H]- 169.0142 169.0145 1.78 125.02 2a Protocatechuic acidC/L C7H6O4 [M - H]- 153.0193 153.0195 1.31 109.03 3a EphedrineE C10H15NO [M + H]+ 166.1226 166.1223 1.81 148.11, 133.22, 115.05 4a PseudoephedrineE C10H15NO [M + H]+ 166.1226 166.1221 3.01 148.11, 133.08, 115.05 5 3′-Hydroxy-4′-O-glucosyl-PuerarinP C27H30O15 [M + H]+ 595.1657 595.166 0.50 433.14, 313.27, 148.11 6 Puerarin-4′-O-glucosideP C27H30O14 [M + H]+ 579.1708 579.1701 1.21 459.12, 417.12, 297.07 7 KakkalideP C28H32O15 [M + H]+ 609.1814 609.1816 0.33 300.19, 285.28, 277.17, 247.13 8 Daidzein-4′, 7-diglucosideP C27H30O14 [M + H]+ 579.1708 579.1702 1.04 255.06, 417.11, 121.05 9a 3′-HydroxypuerarinP C21H20O10 [M + H]+ 433.1129 433.1126 0.69 313.07, 261.08 10a PuerarinP C21H20O9 [M + H]+ 417.118 417.1178 0.48 399.10, 381.09, 297.07 11a AlbiflorinL C23H28O11 [M + H]+ 481.1704 481.1704 0.00 341.09, 319.04, 197.08, 161.09, 133.08 12a 3′-MethoxypuerarinP C22H22O10 [M + H]+ 447.1286 447.1282 0.89 327.08, 342.16 13a Puerarin 6"-O-xylosideP C26H28O13 [M + H]+ 549.1603 549.1607 0.73 417.11, 399.11, 366.24 14a MirificinP C26H28O13 [M + H]+ 549.1603 549.1614 2.00 417.11, 399.09, 351.14, 297.07, 267.26 15a PaeoniflorinL C23H28O11 [M + H]+ 481.1704 481.1701 0.62 381.15, 341.11, 219.08 16a DaidzinP C21H20O9 [M + H]+ 417.118 417.1182 0.48 255.23 17 3′-MethoxydaidzeinP C16H12O5 [M + H]+ 285.0757 285.0751 2.11 270.05 18 Genistein-7-O-apiosyl-glucosideP C26H28O14 [M + H]+ 565.1552 565.1551 0.18 433.11, 415.19, 379.10, 313.07, 283.17 19a RutinZJ C27H31O16 [M + H]+ 611.1607 611.1577 4.91 465.10, 303.05 20a GenistinP C21H20O10 [M + H]+ 433.1129 433.1131 0.46 271.05 21 Formononetin-7-xylosyl-glucosideP C27H30O13 [M + H]+ 563.1759 563.1775 2.84 431.13, 413.19, 395.16, 311.08, 281.23 22 6''-O-malonyldaidzinP C24H22O12 [M + H]+ 503.1184 503.1204 3.98 459.21, 417.14, 255.06 23 Pueroside DP C24H26O10 [M + H]+ 475.1599 475.1597 0.42 313.10, 257.07, 207.06, 107.04 24a OnoninP C22H22O9 [M + H]+ 431.1337 431.1336 0.23 269.08 25a DaidzeinP C15H10O4 [M + H]+ 255.0652 255.0652 0.00 199.06, 181.10, 153.13 26a Cinnamic acidC C9H8O2 [M - H]- 147.0451 147.0455 2.72 119.04, 117.05, 103.05 27 BenzoylpaeoniflorinL C30H32O12 [M + COOH]- 629.1876 629.1851 3.97 583.18, 553.16, 535.21, 165.01, 121.01 28 Licorice saponin G2G C42H62O17 [M + H]+ 839.406 839.4056 0.48 487.34, 469.33 29a Glycyrrhizic acidG C42H62O16 [M + H]+ 823.4111 823.4103 0.97 647.37, 471.34, 453.33 30 FormononetinP C16H12O4 [M + H]+ 269.0808 269.0801 2.60 254.17, 213.16 31a 6-GingerolZR C17H26O4 [M - H]- 293.1758 293.1765 2.39 137.01 Not: P: Pueraria lobate, E: Ephedra sinica, C: Cinnamomum cassia, L: Paeonia lactiflora, G: Glycyrrhiza uralensis, ZR: Zingiber officinale, ZJ: Ziziphus jujube.a compared with reference substances
Table 2. Identification of chemical constituents in GGD
In vitro experiments showed that GGD and oseltamivir phosphate (OP) were dose-dependently effective against H1N1 (A/PR/8/34) in MDCK cells (Figs. 2a and 2b). The calculated IC50 values of GGD and OP were 1.81 mg·mL-1 and 13.20 μg·mL-1, respectively. The CC50 of GGD was 10.88 mg·mL-1, whereas that of OP was more than 250 μg·mL-1.
Figure 2. In vitro anti-IAV H1N1 assays. Antiviral activity of (a) GGD and (b) OP against H1N1 in vitro. (c) GGD extract showed dose-dependent and time-dependent activity against H1N1 in MDCK cells. (d) GGD inhibited viral attachment in a dose-dependent manner. (e) GGD did not affect viral internalization. (f) Effect of GGD on the later stage of viral biosynthesis in a single infectious cycle evaluated by qRT-PCR. Data are presented as the means ± SD (n = 3). *P < 0.05, **P < 0.01 vs the model group
A preliminary time-of-addition assay explored the possible antiviral mechanism of GGD. The drugs were added at different time points before and after viral inoculation. The activity of GGD against H1N1 in MDCK cells was dose-dependent and time-dependent (Fig. 2c). GGD was more effective when it was given before viral infection. The IC50 values were 1.42 mg·mL-1 (4 h before), 1.58 mg·mL-1 (2 h before), and 4.96 mg×mL-1 (2 h after). No noticeable difference was observed between 4 h before infection and 2 h before infection, which suggested that GGD might interfere with the attachment or internalization of the virus. A subsequent attachment assay confirmed this hypothesis. GGD inhibited viral attachment in a dose-dependent manner (Fig. 2d) with an IC50 of 2.59 mg·mL-1. However, GGD did not affect viral internalization (Fig. 2e). Viral genome copies in the supernatants were analyzed by quantitative RT-PCR (qRT-PCR) to determine whether GGD affected the later stage of viral biosynthesis. At 10 h post-infection, the number of viral genome copies in the GGD treatment group was 5-fold lower than that in the model group (Fig. 2f).
In summary, GGD showed anti-IAV activity against H1N1 in vitro with an IC50 of 1.81 mg·mL-1 and targeted the viral attachment and replication stages rather than the internalization stage.
Next, we investigated the effect of GGD in IAV-infected mice. The model group began to lose body weight, became inactive, and showed respiratory distress on day 3 post-infection (5 LD50). The peak in the number of deaths appeared on day 6 to 7 post-infection. Although there were similar clinical features in the GGD group, they were less severe than those in the model group. Compared with the model group, mortality was significantly reduced by GGD treatment (3.5 g·kg-1·d-1, adult equivalent dose), whereas none died in the OP group (21.2 mg×kg-1·d-1, adult equivalent dose) (Fig. 3a).
Figure 3. Changes in mortality and body weight. (a) Mortality of mice inoculated with a lethal dose of H1N1 and treated with GGD (3.5 g·kg-1·d-1) or OP (62.5 μg·mL-1) for 10 days from 3 days before infection. Mortality was monitored over two weeks (n= 6). (b) Changes in weight in mice inoculated with a sub-lethal dose of H1N1 and monitored over two weeks (n= 6). Data are presented as the means ± SD. *P < 0.05, **P < 0.01 vs the model group
Another group of mice was inoculated with a sub-lethal dose of H1N1 (0.8 LD50). The body weight in the model group decreased markedly in the primary phase of infection and gradually recovered in the later phase (Fig. 3b). Compared with the model group, the body weight reduction was inhibited after GGD and OP treatment.
In summary, GGD reduced the mortality of mice infected with a lethal dose of virus and decreased the body weight loss of mice infected with a sublethal dose of virus. Although the protective effect of GGD was lower than that of OP for a lethal dose of H1N1 (5 LD50), the curative effect of GGD was comparable to the OP for a sublethal dose of H1N1 (0.8 LD50). The results suggested that the infection dosage (0.8 and 5 LD50) would affect the therapeutic efficacy of GGD. We reasoned that this might be the cause of the discrepancies surrounding the anti-IAV effects of GGD [3-6].
We measured the relative expression of H1N1 in the lung tissue of virus-infected mice (0.8 LD50). An RT-PCR assay showed that the virus titers of lung tissue in the model group peaked on day 4 post-infection and fell again on day 6 post-infection (P < 0.05) (Fig. 4a). The virus titers of the lung tissue in the GGD and OP groups were significantly reduced (P < 0.01). On day 2 post-infection, the virus titers in the GGD group were higher than those in the OP group (P < 0.05); however, there was no significant difference in virus titers between in the GGD group and the OP group on day 4 and 6 post-infection (P > 0.05). The expression of the viral nucleoprotein in lung tissue on day 4 post-infection also confirmed this result (Figs. 4c and 4d).
Figure 4. Effects of GGD on virus titers in lung tissue and on lung lesions. (a) Virus titers in lung tissue measured by RT-PCR on day 2, 4, and 6 post-infection (0.8 LD50) (n = 5). (b) Lung indexes on day 4 post-infection (0.8 LD50) (n = 6). (c) Expression of viral nucleoprotein in lung tissue measured by western blotting on day 4 post-infection (0.8 LD50) (n = 3). (d) Quantification of viral nucleoprotein expression relative to β-actin (n = 3). (e) Histological observation of lung tissue on day 4 post-infection (0.8 LD50) (200 ×). (e-i) Alveolar structure of the control group is clear and interstitial alveolar walls show no inflammatory cells infiltration. (e-ii) Differences in the lung tissue of the model group, such as narrowed alveolar space (black arrow), thickened alveolar wall, lung congestion (red arrow), and infiltration of inflammatory cells (green arrow). The degree of lung lesions is reduced greatly in treated groups. Alveolar morphology following GGD (e-iii) and OP (e-iv) treatment is normal. Data are presented as the means ± SD. *P < 0.05, **P < 0.01 vs the model group. #P < 0.05, ##P < 0.01 vs the control group
These results indicated that influenza resolved on its own when mice were infected with a sub-lethal dose (0.8 LD50) of H1N1. However, lung indexes (Fig. 4b) and pathological changes (Fig. 4e) in the model group showed that the immune system would inevitably cause inflammatory injury in the process of removing the virus, which is consistent with clinical observations. The treatment with GGD (3.5 g·kg-1·d-1) or OP (21.2 mg·kg-1·d-1) relieved the lung injury. Compared with the model group, GGD and OP treatment improved the lung indexes (Fig. 4b) and histological changes (Fig. 4e) on day 4 post-infection.
In summary, GGD had direct anti-IAV activity in vitro and in vivo. GGD treatment significantly reduced the virus titers in lung tissue and alleviated pathological damage in mice infected with H1N1 (0.8 LD50). Some components in GGD have immunomodulatory and anti-inflammatory activities [10-15]. Thus, further studies are required to verify the anti-inflammatory and immunomodulatory hypothesis of GGD in the treatment of influenza.
The expression of cytokines in lung tissue was measured by RT-PCR and western blotting on day 4 post-infection. The RT-PCR assay showed that the mRNA expression of IL-1α in the model group was significantly higher than that in the control group. Compared with the model group, the expression of IL-1α mRNA was reduced after GGD and OP treatment, which is consistent with previous research  (Fig. 5a).
Figure 5. Effects of GGD on the expression of IL-1α, IL-6, and TNF-α in lung tissue. mRNA expression of (a) IL-1α, (b) IL-6, and (c) TNF-α determined by RT-PCR on day 4 post-infection (n = 5). (d) Protein expression of IL-6 and TNF-α determined by western blotting on day 4 post-infection (n = 3). Quantification of (e) IL-6 and (f) TNF-α protein expression relative to β-actin. Data are presented as the means ± SD. *P < 0.05, **P < 0.01 vs the model group. #P < 0.05, ##P < 0.01 vs the control group
The RT-PCR assay indicated that the mRNA expression of IL-6 and TNF-α in the model group was significantly higher than that in the control group. The mRNA expression of IL-6 and TNF-α in the GGD and OP groups was lower than that in the model group (Figs. 5b and 5c). Notably, the expression of TNF-α mRNA in the GGD group was lower than that in the OP group (P < 0.01) (Fig. 5c). The protein expression of IL-6 and TNF-α was consistent with the mRNA data (Figs. 5d, 5e, and 5f).
The important pro-inflammatory cytokines IL-6 and TNF-α cause severe damage to the lungs during influenza infection , and the overexpression of pro-inflammatory cytokines increases the pathogenicity of influenza virus [9, 29]. Compared with the model group, the expression of IL-1α, IL-6, and TNF-α in lung tissue was decreased significantly after treatment with GGD and OP. However, compared with the OP group, the GGD group exhibited a unique immunomodulatory effect. The level of TNF-α in lung tissue after treatment with GGD was lower than that in the OP group. Because TNF-α is the main cytokine that causes acute lung injury , we propose that the decreased expression of TNF-α in the GGD group helped to relieve inflammatory injury in IAV-infected mice.
To explore whether GGD exerts an anti-inflammatory effect by regulating the Th1/Th2 immune balance, we measured the expression of the Th1 and Th2 cytokines IFN-γ and IL-4, respectively, in lung tissue by RT-PCR and western blotting. Compared with the control group, the expression of IFN-γ was increased, whereas the expression of IL-4 was decreased in the model group on day 4 post-infection. However, compared with the model group, the expression of IFN-γ was decreased (Figs. 6a, 6c, and 6d), whereas the expression of IL-4 was increased (Figs. 6b, 6c, and 6e) after treatment with GGD and OP. Compared with the OP group, the level of IFN-γ in the GGD group was closer to those of the control group. This result indicates that GGD regulates the host immune system to maintain the balance of Th1/Th2.
Figure 6. Effects of GGD on Th1/Th2 balance. mRNA expression of (a) IFN-γ and (b) IL-4 determined by RT-PCR on day 4 post-infection (n = 5). (c) Protein expression of IFN-γ and IL-4 in lung tissue determined by western blotting (n = 3). Quantification of (d) IFN-γ and (e) IL-4 protein expression relative to β-actin. (f) Changes in the ratio of Th1/Th2 cells in peripheral lymphocytes determined by flow cytometry (n = 6). Representative FACS plots for (g) CD4+IFN-γ+ T cells and (h) CD4+IL-4+ T cells. Data are presented as the means ± SD. *P < 0.05, **P < 0.01 vs the model group. #P < 0.05, ##P < 0.01 vs the control group
To determine the effect of GGD on the Th1/Th2 balance, the percentages of CD4+IFN-γ+ and CD4+IL-4+ cells in peripheral lymphocytes were determined by flow cytometry. Compared with the control group, the Th1/Th2 ratio in the model group was significantly increased (P < 0.01), suggesting that the Th1/Th2 balance was pushed towards Th1 after infection (Figs. 6f, 6g, and 6h). Interestingly, compared with the OP group, the Th1/Th2 ratio in the GGD group was closer to that of the control group. These results suggest that the anti-inflammatory effect of GGD may be related to the improvement of the Th1/Th2 immune balance.
Mouse CD4+ Th cells are divided into the Th1 and Th2 subgroups with different functions according to the types of cytokines they secrete . In healthy individuals, the Th1/Th2 cell ratio maintains a dynamic balance. The Th1/Th2 imbalance is related to the induction of inflammation and the occurrence of various immune diseases . Our study shows that the Th1/Th2 cell ratio was skewed toward Th1 after infection. Although cell-mediated anti-infective immune responses are essential in the clearance of the influenza virus [8, 33], Th1 cytokines, such as IFN-γ and TNF-α, also mediate inflammatory damage in the early stage of influenza . IFN-γ and IL-4 are a pair of mutually inhibitory cytokines that indirectly reflect the degree of the Th1 and Th2 immune responses. The immunomodulation function of GGD can be inferred by comparing the changes in IFN-γ and IL-4 expression in the GGD and OP groups. Compared with the OP group, the level of IFN-γ in lung tissue and the CD4+IFN-γ+/ CD4+IL-4+ ratio in the peripheral lymphocytes of the GGD group were closer to those of the control group. Thus, we believe that GGD regulates the immune system to maintain the Th1/Th2 balance, avoiding the over-activation of inflammation.
Toll-like receptor 7 (TLR7) is a protein that recognizes the influenza virus and triggers an immune response . Thus, we examined the effect of GGD on the TLR7 signaling pathway. We measured the mRNA and protein expression of key proteins in the TLR7 signaling pathway.
The RT-PCR assay showed that the expression of TLR7, myeloid differentiation primary response 88 (MyD88), TNF receptor associated factor 6 (TRAF6), and interferon regulatory factor 7 (IRF7) mRNA in the model group was significantly higher than that in the control group on day 4 post-infection (Fig. 7a). Compared with the model group, the mRNA expression of TLR7, MyD88, TRAF6, and IRF7 decreased after GGD and OP treatment. However, the expression of nuclear factor (NF)-κB mRNA was similar in the GGD and OP groups.
Figure 7. mRNA and protein expression of the TLR7 signaling pathway in lung tissue. (a) Expression of TLR7, MyD88, TRAF6, IRF7, and NF-κB mRNA in lung tissue (n = 5). (b) Western blot of the protein expression of TLR7, MyD88, TRAF6, IRF7, P-IRF7, and IκBα in lung tissue (n = 3). (c) Quantification of TLR7, MyD88, TRAF6, P-IRF7/IRF7, and IκBα protein expression relative to β-actin. Data are presented as the means ± SD. *P < 0.05, **P < 0.01 vs the model group. #P < 0.05, ##P < 0.01 vs the control group
Similar to mRNA levels, the expression of TLR7, MyD88, TRAF6, and phospho (P)-IRF7 protein in the model group increased, the P-IRF7/IRF7 ratio increased, and the expression of IκBα protein decreased (Figs. 7b and 7c). Although there was no noticeable difference in the mRNA expression of NF-κB among these groups, the reduced expression of IκBα protein indicated that H1N1 triggered IκBα degradation, indicating the activation of the NF-κB pathway. Compared with the model group, the expression of TLR7, MyD88, TRAF6, and P-IRF7 protein in the GGD and OP groups was reduced, the P-IRF7/IRF7 ratio decreased, and the expression of IκBα protein recovered. Moreover, the P-IRF7/IRF7 ratio in the GGD group was lower than that in the OP group (P < 0.05). However, the expression of total IRF7 protein was similar in the GGD and OP groups, which suggests that changes in the expression of P-IRF7 protein did not result from the change in total IRF7 protein.
Toll-like receptors, which are pattern recognition receptors, play essential roles in recognizing pathogens and in inducing cascade signaling and immune responses [36-37]. TLR7 recognizes the single-stranded RNA of influenza viruses and triggers an immune response through a series of cascade reactions [35, 38]. The antiviral pathways mediated by the TLR7 signaling pathway may be TLR7-MyD88-TRAF6-NF-κB and TLR7-MyD88-TRAF6-IRF7 . However, the TLR7 signaling pathway also plays an essential role in the development of acute lung injury induced by the influenza virus . The appropriate activation of the TLR7 signaling pathway is necessary for combating the infection. However, when these pathways are not properly controlled, the continuous and bulk release of inflammatory cytokines causes inflammatory injury [9, 41].
In the present study, the expression of TLR7, MyD88, and TRAF6 and the activation levels of IRF7 and NF-κB in lung tissue increased significantly post-infection. After GGD treatment, the mRNA and protein expression of TLR7, MyD88, and TRAF6 decreased, and the activation levels of IRF7 and NF-κB decreased, which was consistent with the alleviation of pathological damage. In addition, the activation of IRF7 after GGD treatment was lower than that in the OP group, but this result requires further explanation.
In summary, we focused on the anti-inflammatory and immunomodulatory activities of GGD. By comparing several immune-related indexes in the GGD and OP groups, we found that GGD treatment decreased the expression of TNF-α to relieve lung injury. GGD treatment also regulated the balance of Th1/Th2 to alleviate inflammation. In addition, GGD treatment decreased the level of IRF7 activation, although the reason for this remains unclear.