Antiviral effects and mechanism of Qi pi pill against influenza viruses

Abstract

Background

Qi pi pill (QPP), which contains Renshen, Baizhu, Fuling, Gancao, Chenpi, Shanyao, Lianzi, Shanzha, Liushenqu, Maiya, and Zexie, was recommended for preventing and treating COVID‐19 in Shandong Province (China). However, the mechanism by which QPP treats infectious diseases remains unclear. This study aims to investigate the therapeutic effect of QPP in vitro and on acute influenza infection in mice, exploring its mechanism of action against influenza A virus (IAV).

Methods

The in vitro activity of QPP was assessed using dose–response curve analysis and titer reduction assay, and its antiviral mechanism was identified in vitro by real‐time polymerase chain reaction (PCR), time‐of‐addition, and enzymatic assays. The antiviral efficacy of QPP was further evaluated in vivo using BALB/c mice infected with IAV. At the same time, each single Chinese herbal medicine in QPP was evaluated to preliminarily identify those with antiviral effects.

Results

In vitro results showed that QPP exhibited a higher potency antiviral effect against both influenza A and B viruses, inhibiting viral RNA replication and release by targeting RNA‐dependent RNA polymerase and neuraminidase. Additionally, QPP significantly decreased the expression of inflammatory cytokines in A549 cells. In vivo study revealed that QPP significantly reduced the lung index and viral load in lung tissue of mice infected with IAV. Renshen, Gancao, Zexie, and Lianzi were the Chinese herbal medicines from QPP that showed anti‐IAV activity.

Conclusion

The antiviral activity of QPP targets IAV replication and release, cytokine modulation in host cells, and provides protection in mice with acute influenza infection.

Qi pi pill presents anti‐influenza effect and inhibits viral replication in vitro by targeting both the viral RNA replication and the release of new viral particles. Furthermore, QPP modulates the production of proinflammatory cytokines induced by influenza infection in host cells. QPP also exhibits a protective effect in mice with acute influenza infection, providing preclinical data to support the clinical investigation of QPP as treatment for other viral diseases.

1. INTRODUCTION

Influenza virus is one of the most important etiological agents of acute respiratory infections, with high morbidity and mortality, imposing a high public health burden. Each year, approximately 1 billion people are infected by seasonal influenza viruses, resulting in 3–5 million severe cases and 290 000–650 000 deaths globally. ¹ Influenza viruses belong to the Orthomyxoviridae family and are divided into four types: A–D. Both influenza A and B viruses cause seasonal epidemics in humans, whereas only influenza A is known to have pandemic potential. Influenza A viruses (IAVs) are subtyped based on the combination of their surface glycoproteins proteins, hemagglutinin (HA), and neuraminidase (NA). IAV possesses 18 HA and 11 NA subtypes and eight negative‐sense RNA genome segments, which can be reassorted resulting in genetic shifts. ² , ³ This phenotype and genetic variability cause rapid and continuous emergence of novel influenza A variants that pose threats of pandemics. Influenza B viruses (IBVs) are further categorized into two lineages: Victoria and Yamagata.

The life cycle of IAV can be summarized into the following steps: adsorption and entry, genome replication and transcription, assembly, budding, and release. ⁴ The HA of IAV binds to the host cellular sialic acid receptors, and the virus is endocytosed. The low pH of the endosome induces a conformational change in the HA protein, allowing the release of viral ribonucleoprotein (vRNP) complexes in the cell cytoplasm. The vRNPs are composed of the RNA segments, viral nucleoproteins, and the RNA‐dependent RNA polymerase (RdRp). RNA replication and transcription occur in the cell nucleus, and after the newly formed negative vRNPs reach cell cytoplasm again, viral proteins are assembled, and the viral particles bud from the cell membrane. Finally, NA cleaves terminal sialic acid residue, releasing the virions from the cell. ⁵

Currently, three classes of anti‐influenza drugs have been clinically approved and used: viral matrix protein 2 (M2) ion channel blockers, such as amantadine and rimantadine; neuraminidase inhibitors (NAIs), including oseltamivir phosphate (OSP), zanamivir, and peramivir; and RdRp inhibitors, such as favipiravir and baloxavir marboxil (BXM). ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ However, the current seasonal IAVs in circulation exhibit resistance to M2 inhibitors and OSP (such as H275Y ¹¹ /N295S ¹² and E119V ¹² ). Moreover, viral variants carrying the I38T substitution have been found to reduce the effectiveness of BXM in clinical practice. ¹³ Therefore, the development of novel antiviral drugs against influenza viruses is urgently needed.

Traditional Chinese medicines (TCMs) exhibit a synergistic effect due to their multiple components and diverse targets. Compared to chemical antivirals, TCM is less likely to result in drug resistance and offers distinct advantages in the treatment of various diseases. During the SARS‐CoV‐2 pandemic, six most effective TCM recipes have been shown to be effective in the treatment of COVID‐19 patients in China, including Qingfei Paidu decoction (QFPD), Xuanfei Baidu formula (XFBD) and Huashi Baidu formula (HSBD), Jinhua Qinggan granules (JHQG) and Lianhua Qingwen capsules (LHQW), and Xuebijing injections (XBJ). ¹⁴ Some formulas among the six TCM recipes have been shown to attenuate influenza virus–induced pneumonia, such as HSBD, ¹⁵ LHQW, ¹⁶ and XBJ. ¹⁷ Therefore, based on the TCM principle “Treating different diseases with the same method” when the diseases have similar pathological changes, ¹⁸ and taking into consideration that SARS‐CoV‐2 and IAV are both RNA viruses and cause diseases with similar clinical signs and symptoms, ¹⁹ we screened the TCMs from the “Diagnosis and Treatment Program of TCM for Novel Coronavirus Pneumonia in Shandong Province (Second Edition, 2020),” using a reporter influenza A/H3N2 virus (A/NY‐HiBiT) system previously described by us, ²⁰ and identified Qi pi pill (QPP) as the most effective TCM against IAV based on the selectivity index (SI) value.

QPP is a patented Chinese herbal formulation consisting of Renshen (Ginseng Radix et Rhizoma), Baizhu (Atractylodis Macrocephalae Rhizoma), Fuling (Poria), Gancao (Glycyrrhizae Radix et Rhizoma), Chenpi (Citri Reticulatae Pericarpium), Shanyao (Dioscoreae Rhizoma), Lianzi (Nelumbinis Semen), Shanzha (Crataegi Fructus), Liushenqu (Massa Medicata Fermentata), Maiya (Hordei Fructus Germinatus), Zexie (Alismatis Rhizoma), and honey (Mel) (as excipient), having functions of treating spleen and stomach weakness, indigestion, and abdominal distension with loose stools. Shandong Province recommends QPP for the treatment of children in the recovery phase of COVID‐19. This study describes the antiviral screening of seven TCMs, including Fangfeng Tongsheng pills (FFTS), Tongxuan Lifei pills (TXLF), Lingqiao Jiedu pills (LQJD), Qingwen Jiedu pills (QWJD), Suhexiang pills (SHX), Angong Niuhuang pill (AGNH), and QPP. It also investigates the antiviral effects of QPP both in vitro and in vivo along with its mechanisms of action against IAV. These findings provide preclinical data to support the clinical investigation of QPP as treatment for other viral diseases.

2. MATERIALS AND METHODS

2.1. Cells and viruses

Human embryonic kidney (293 T), Madin‐Darby canine kidney (MDCK), and human lung epithelium (A549) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1000 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO₂ incubator.

Recombinant influenza reporter viruses A/NY‐HiBiT (H3N2), PR8‐NS^CE2‐FLuc (H1N1), and influenza viruses A/Puerto Rico/8/1934 (A/PR8 H1N1) were propagated and stored as previously described. ²⁰ , ²¹ Influenza A/Brisbane/10/2007 (H3N2) was kindly provided by the Institute of Pathogen Biology, Chinese Academy of Medical Sciences. OST‐resistant influenza A/H1N1/pdm09 (H275Y) virus was kindly provided by Beijing CDC. Influenza B/Yamagata and B/Victoria lineages were kindly provided by Shandong CDC. In vitro infection assays were performed in Opti‐MEM (Gibco) containing 1.5 μg/mL N‐tosyl‐l‐phenylalanine chloromethyl ketone (TPCK)‐trypsin (Sigma‐Aldrich, St. Louis, MO, USA).

2.2. Drugs

Oseltamivir acid (OA) (GS 4071), OSP (GS 4104), and Baloxavir acid (BA) (S‐033447) were purchased from MedChemExpress (MCE).

QPP is a Chinese patent medicine (CPM) purchased from Beijing Tongrentang Pharmaceutical Group Co., Ltd. (Beijing, China). In this study, QPP (3 g) or other CPMs were prepared in 10‐mL RNase‐free H₂O. Then, ultrasonic treatment was performed for 30 min followed by a centrifugation at 9000 × g for 10 min. The supernatant was filtered through a 0.22‐μm syringe filter and diluted in DMEM medium to reach the indicated concentrations for the cell‐based assays.

2.3. Dose–response curve assays

MDCK cells were infected with A/NY‐HiBiT or PR8‐NS^CE2‐FLuc at a multiplicity of infection (MOI) of 0.01 and treated with threefold serial dilutions of CPMs or TCMs in three replicates. After 24 h postinfection (h.p.i.), luciferase activity was measured using a Nano‐Glo HiBiT Lytic Detection System (Promega, Madison, WI, USA) or Britelite plus Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA). Cell viability was evaluated with Cell Counting Kit‐8 (CCK‐8) (MCE) to determine the cytotoxicity. The 50% inhibitory concentration (IC₅₀) and 50% cytotoxic concentration (CC₅₀) of the tested medicines were calculated by fitting dose–response curves using GraphPad Prism 5.0 software. ²²

2.4. Viral titer reduction assay

MDCK cells were seeded at a density of 2 × 10⁵ cells per well in 24‐well plates and then infected with the indicated wild‐type viruses at a MOI of 0.01. Subsequently, the cells were treated with various concentrations of QPP. The culture supernatants were harvested at 24 h.p.i., and virus titers were determined by plaquing the supernatants in MDCK cells. This assay was performed in triplicate runs within 24‐well plates.

2.5. Quantitative reverse transcription PCR reactions

MDCK cells seeded on 6‐well plate (5 × 10⁵ cells/well) were infected with influenza virus A/PR8 H1N1 (MOI = 0.1) at 4°C for 1 h and then transferred to 37°C during an additional 1 h. Cells were washed and treated with different concentrations of QPP or 100 nM BA (positive control) for 24 h. Total RNA was extracted by Simply P Total RNA Extraction Kit (Bioflux, Zhejiang, China) and reverse transcribed into complementary DNA (cDNA) using the SPARKscript II RT Plus Kit (with gDNA Eraser) (SparkJade, Shandong, China). Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) was performed by 2× SYBR Green qPCR Mix (with ROX) (SparkJade, Shandong, China). Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the internal control. The primers used in the PCR reactions are listed in Table S1. The relative expressions of NP gene were calculated by delta–delta Ct (2^−ΔΔCt) method. This assay was performed in triplicate runs within 6‐well plates.

2.6. Western blotting assay

MDCK cells were seeded on a 6‐well plate (5 × 10⁵ cells/well), infected with A/PR8 H1N1 (MOI = 0.1) at 4°C for 1 h and incubated at 37°C for another 1 h. Cells were treated with serial dilutions of QPP, lysed after 24 h, and protein content was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated by 10% SDS‐PAGE gel and transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies anti‐NP (GeneTex, USA) and anti‐GAPDH (ABclonal, Wuhan, China), washed with TBST buffer and incubated with horseradish eroxidase (HRP)‐conjugated anti‐rabbit immunoglobulin G (IgG) (H + L) secondary antibody (ABclonal, Wuhan, China). Protein signal was detected using an GE Amersham Imager 600.

2.7. Indirect immunofluorescence assay

MDCK cells were infected with A/PR8 H1N1 (MOI = 0.1) for 1 h at 4°C and then incubated at 37°C for an extra 1 h. After incubation, the cells were washed and treated with increasing concentrations of QPP or BA (100 nM). After 24 h.p.i., the cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X‐100, and incubated with blocking buffer containing 5% albumin from bovine serum. Subsequently, cells were probed with primary antibody anti‐NP (1:300) (GeneTex, USA) and CoraLite 488‐conjugated goat anti‐rabbit IgG secondary antibody (H + L) (Proteintech, USA). After counterstaining with DAPI, images were collected using a fluorescence microscope (OLYMPUS).

2.8. Time‐of‐addition assay

MDCK cells were seeded at a density of 2 × 10⁵ cells/well in 24‐well plates and infected with A/PR8 H1N1 at an MOI of 0.1 at 4°C for 1 h and then incubated at 37°C for 1 h. The cells were then washed with PBS and incubated with fresh culture media containing 1.5 μg/mL of TPCK‐trypsin at 37°C. The time point when the plates were transferred to 37°C was considered time zero. The cells were treated with 15 mg/mL QPP or 25 μM OA at indicated time points (−2 to 12, −2 to 0, 0–12, 2–12, 4–12, 6–12, and 8–12 h). The supernatants were harvested at 12 h.p.i. and viral titers were determined by plaquing the supernatants in MDCK cells. Each condition was tested in triplicate.

2.9. Mini‐replicon assay

The mini‐replicon assay was performed as previously described. ²³ Briefly, 293 T cells were transfected with viral RNA polymerase plasmids (pCAG‐VN04‐NP, pCAG‐VN04‐PA, pCAG‐VN04‐PB1, and pCAG‐VN04‐PB2), the minigene plasmid pPolI‐NS‐Fluc, and a control plasmid pRL‐TK. At 6 h post‐transfection, cells were seeded into white, flat‐bottom, 96‐well plates with serially diluted QPP solutions. At 36 h post‐transfection, luciferase activity was measured using Dual‐Glo Luciferase Assay System (Promega, Madison, WI, USA). This assay was performed in triplicate runs within 96‐well plates.

2.10. NA inhibition assay

The NA inhibition assay was performed using Neuraminidase Inhibitor Screen Kit (Beyotime, Shanghai, China), as previously described. ²⁴ Briefly, reaction buffer solution, neuraminidase enzyme, and various concentrations of QPP were added to each well of 96‐well black plates. OA was used as a positive control. Then, the fluorescent substrate was added to the plate and incubated at 37°C for 30 min. The fluorescence intensity was measured using a fluorescence microplate reader. This assay was performed in triplicate runs within 96‐well black plates.

2.11. Measurement of proinflammatory cytokine

The effects of QPP in the expression of proinflammatory cytokines were evaluated in A549 cells by RT‐qPCR. A549 cells were infected with A/PR8 (MOI = 1.0) and treated with various concentrations of QPP. At 48 h.p.i., cells were lysed, RT‐qPCR was performed to measure the relative expressions of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and IL‐1β gene. Human GAPDH was used as the internal control. The primers used are listed in Table S1. BA was used as a positive control. This assay was performed in triplicate runs within 6‐well plates.

2.12. In vivo antiviral activity of QPP

A total of 24 specific pathogen‐free (SPF) BALB/c female mice (4‐5 weeks old) were purchased from Beijing Charles River Animal Breeding Company Limited. This study was approved by the Ethic Committee of Shandong University of Traditional Chinese Medicine (approval number: SDUTCM20231113511).

For mouse model with acute influenza infection, mice were anesthetized with isoflurane and intranasally inoculated influenza virus A/PR8 H1N1 at a dose of 0.8 LD₅₀ (n = 6). Mice were treated orally with QPP (2.34 g/kg/day) or OSP (30 mg/kg/day) twice a day. The double human equivalent clinical dose of the drug was chosen as the oral gavage dose for mice, and it was calculated using a standard conversion formula for translating the dose to mice, as previously described. ²⁵ All treatments were administered consecutively over a 5‐day period following infection. Daily monitoring of weight fluctuations was conducted throughout this period, after which the mice were euthanized on day 5 postinfection. Subsequently, samples were collected for further analysis. Figure 1 illustrates the timeline of this experiment. For detailed operations, please refer to the Data S1.

FIGURE 1.

Schematic illustration of the experimental timeline.

2.13. Statistical analysis

Statistical analysis was performed using GraphPad 5.0 software (GraphPad Software Inc.). The statistical significance was determined using Student's t‐test and one‐way analysis of variance (ANOVA). p‐values <0.05 were considered significant and are represented as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001.

3. RESULTS

3.1. QPP demonstrated higher potency against IAV in vitro

Seven CPM pills were screened against influenza virus using the reporter A/NY‐HiBiT virus. Among these, six pills with antiviral activity were identified as hits and inhibited virus replication by more than 80% without significant cytotoxicity (>80% cell viability; Table 1). To further assess the anti‐IAV activity of these six hits, dose–response curve analysis was performed using MDCK cells infected with A/NY‐HiBiT reporter virus. As shown in Table 1, QPP exhibited a higher potency against influenza A/NY‐HiBiT virus, demonstrated by its lower IC₅₀ value of 0.73 ± 0.17 mg/mL and higher SI value (>34.25) in comparison to the other CMPs tested. QPP also demonstrated antiviral activity against influenza A/H1N1 reporter virus (A/PR8‐NS^CE2‐FLuc), with IC₅₀ value of 0.35 ± 0.10 mg/mL (Figure 2). Because QPP was the most promising CMPs, we further evaluated its mechanism of inhibition of IAV replication.

TABLE 1.

Primary screening of the antiviral activity of Chinese patent medicines (CPMs) against influenza virus.

CPM pills	Percentage inhibition a	Percentage cell viability a	IC50 (mg/mL)	CC50 (mg/mL)	SI
FFTS	99.55	108.66	2.07 ± 0.51	22.22 ± 4.24	10.73
TXLF	93.38	109.45	1.91 ± 0.28	27.12 ± 0.36	14.20
LQJD	99.69	86.67	0.91 ± 0.21	26.04 ± 3.40	28.62
QWJD	99.38	105.38	1.39 ± 1.00	30.59 ± 1.51	22.00
SHX	99.55	102.38	1.37 ± 0.34	24.10 ± 1.16	17.59
QPP	98.68	108.58	0.73 ± 0.17	>25.00	>34.25

Abbreviations: CC₅₀, 50% cytotoxic concentration; FFTS, Fangfeng Tongsheng pills; IC₅₀, 50% inhibitory concentration; LQJD, Lingqiao Jiedu pills; QPP, Qi pi pill; QWJD, Qingwen Jiedu pills; SHX, Suhexiang pills; SI, selectivity index; TXLF, Tongxuan Lifei pills.

^a Screening of CPMs at 10 mg/mL.

FIGURE 2.

Dose–response inhibition of Qi pi pill (QPP) against influenza A/NY‐HiBiT (H3N2) and influenza A/PR8‐NS^CE2‐FLuc (H1N1) reporter viruses. QPP was examined against A/NY‐HiBiT (H3N2) (A) or influenza A/PR8‐NS^CE2‐FLuc (H1N1) (B) viruses for dose–response evaluation (red) and cytotoxicity assay (blue). The IC₅₀ and CC₅₀ values were determined by fitting the dose–response curves with four‐parameter nonlinear regression analysis in Prism GraphPad 5.0. Data were expressed as the mean ± standard deviation (SD) (n = 3).

3.2. QPP displays antiviral activity against different types and subtypes of influenza virus

To further study the antiviral activity of QPP, we tested its effects against different subtypes of wild type or OSP‐resistant influenza A and B viruses. QPP effectively inhibited virus replication in MDCK cells of wild‐type influenza A subtypes H3N2 (A/Brisbane/10/2007) (Figure 3A) and H1N1 (A/Puerto Rico/8/1934) (Figure 3B), and influenza B lineages Yamagata (Figure 3D) and Victoria (Figure 3E), demonstrated by the reduction in virus titers quantified by TCID₅₀/mL. QPP was also capable of inhibiting the replication of an OSP‐resistant strain of influenza A H1N1 virus (A/H1N1/pdm09‐H275Y) (Figure 3C). Although QPP was most effective against IAVs in comparison with IBVs, shown by its lower IC₉₀ values depicted in Figure 3, these results suggest that QPP has a broad‐spectrum inhibitory effect against different types and subtypes of influenza viruses.

FIGURE 3.

Antiviral effects of Qi pi pill (QPP) against different influenza viruses. QPP was examined against influenza virus A/Brisbane/10/2007 (H3N2) (A), A/Puerto Rico/8/1934 (H1N1) (B), oseltamivir phosphate (OSP)‐resistant A/pdm09‐H275Y (H1N1) (C), B/Yamagata (D), or B/Victoria (E) viruses in a yield reduction assay. Data were expressed as means ± standard deviation (SD) (n = 3).

3.3. QPP mechanisms of inhibition of IAV replication

3.3.1. QPP inhibits multiple rounds of IAV replication by targeting different steps of its life cycle

To start investigating the inhibitory mechanism of QPP, we evaluated whether QPP would inhibit several rounds of viral replication in vitro. We treated MDCK cells infected with the influenza A/PR8 H1N1 virus using QPP. Viral NP mRNA levels were quantified through RT‐qPCR, whereas NP protein levels were evaluated through WB and IFA. After IAV infection, the expression of viral NP mRNA and protein increased in MDCK cells (Figure 4). The treatment with QPP significantly reduced NP messenger RNA (mRNA) (Figure 4A) and protein levels (Figure 4B,C) in a dose‐dependent manner. At the highest concentration tested (20 mg/mL), QPP resulted in a 95.05% reduction in viral NP mRNA levels (Figure 4A) and a 60.18% decrease in NP protein levels (Figure 4B). Additionally, IFA revealed a significant reduction in NP protein expression at this maximum concentration (Figure 4C).

FIGURE 4.

Qi pi pill (QPP) inhibited multiple rounds and different steps of influenza virus replication. The levels of viral NP messenger RNA (mRNA) were quantified by reverse transcription quantitative polymerase chain reaction (RT‐qPCR) (A) or the expression of NP protein was detected by Western blot (B) or by immunofluorescence microscopy (C) in infected Madin‐Darby canine kidneys (MDCKs), BA was used as a positive control. (D) Time‐of‐addition experiments were conducted with QPP and oseltamivir acid (OA) (used as a positive control). The control group represents the infected and nontreated controls. Student's t‐test and one‐way analysis of variance (ANOVA), ***p < 0.001, **p < 0.01, *p < 0.05, compared to the control group. Data are expressed as the mean ± standard deviation (SD).

Next, to identify which possible steps throughout influenza virus life cycle are being affected by QPP, time‐of‐addition (TOA) assays were performed, as previously described. ²² , ²³ We selected different time points (−2, 0, 2, 4, 6, and 8 h.p.i) to initiate the treatment with QPP in MDCK cells infected with influenza A/PR8 H1N1. As shown in Figure 4D, no significant inhibitory activity was observed when cells were treated with QPP during the infection (−2 to 0 h time interval), suggesting that QPP did not inhibit virus attachment, neither when QPP was added at very‐late‐release stages (8–12 h). On the contrary, QPP significantly reduced IAV replication when used at other time intervals, from 2, 4, or 6 to 12 h.p.i. (Figure 4D), suggesting that the medicine could be targeting different steps of the viral life cycle, such as RNA replication/transcription (2–6 h) and the release stages (>6 h). QPP showed a similar inhibitory profile to OA (Figure 4D), a known NAI in clinical use against IAV. ²⁶

In summary, altogether, these results suggest that QPP maintains its inhibitory activity during several rounds of IAV infection in MDCK cells and also that the medicine targets different steps during influenza virus life cycle in vitro.

3.3.2. QPP interferes with viral RNA replication and the release stage of viral life cycle by targeting RdRp and NA, respectively

During IAV life cycle, several viral proteins present crucial roles for the replication to be successfully completed. ²⁷ Among them are the viral RdRp, composed of three subunits (PA, PB1, and PB2), and found in the virion complexed with the viral NPs and RNA segments forming the vRNPs that are essential for viral RNA transcription and replication processes, ²⁸ and the viral NA, the enzyme responsible for the release of the newly formed virus particles from the host cell plasma membrane. ²⁹ The results of our TOA assay suggest that QPP maybe interfere with both viral RNA replication/transcription and viral release (Figure 4D). Therefore, to confirm that these proteins could be targeted by QPP, we tested its activity against both of them.

To evaluate the activity of QPP against viral RdRp, we measured viral genome replication by using a cell‐based influenza virus RdRp mini‐replicon assay in 293 T cells. After the cytotoxicity of QPP in this cell line was tested, we determined the CC₅₀ to be 29.92 mg/mL (Figure S1A). We also observed significant cytotoxicity of QPP against 293 T cells at a concentration of 30 mg/mL compared to the solvent control group (Mock, Figure 5A). Therefore, we used the highest nontoxic concentration of 15 mg/mL in the subsequent RdRp inhibition assay and NA inhibition assay. QPP demonstrated a dose‐dependent inhibition of both RdRp and NA activities, as indicated by the observed reductions in luciferase activity (Figure 5B,C). BA and OA were used as positive controls for inhibition of viral RdRp and NA, respectively.

FIGURE 5.

Mechanistic studies of the antiviral effect of Qi pi pill (QPP) against influenza virus. (A) Cytotoxicity of QPP was determined after 293 T cells were treated with increasing concentrations of the medicine with the CCK‐8. (B) The inhibitory effect of QPP on viral RdRp activity was tested using a cell‐based mini‐replicon RdRp assay in 293 T cells. (C) The inhibitory effect of QPP on viral neuraminidase (NA) activity was tested in a cell‐free experiment using a Neuraminidase Inhibitors Screen Kit (Beyotime, Shanghai, China). Student's t‐test and one‐way analysis of variance (ANOVA), ***p < 0.001, **p < 0.01, *p < 0.05, compared to the control group. Values are expressed as means ± standard deviation (SD) (n = 3).

Overall, these results show that QPP inhibits influenza virus replication by targeting RdRp and NA activities, confirming that QPP acts in more than one step of the virus life cycle, the RNA replication, and the release stages.

3.4. QPP reduces the expression of proinflammatory cytokines in cells infected with IAV

Influenza virus infection can cause excessive expression of proinflammatory cytokines and chemokines, leading to severe lung damage and high disease severity. ³⁰ The expression levels of proinflammatory cytokines were measured in A549 cells infected with IAV. As expected, IAV infection significantly upregulated the expression of IL‐6, TNF‐α, and IL‐1β compared to the noninfected group (Mock, p < 0.001) (Figure 6B–D). Subsequently, we determined that the CC₅₀ value of QPP in A549 cells is 74.22 mg/mL (Figure S1B). Therefore, we selected the nontoxic concentration of QPP to treat A549 cells infected with influenza A/PR8 H1N1. The results showed that QPP, at a concentration of 20 mg/mL, significantly reduced the mRNA expression levels of these three cytokines (Figure 6B–D) and viral NP gene (Figure 6A) in comparison to the virus‐infected control group (control, p < 0.001) (Figure 6). Because BXM ameliorated lung inflammation via prevention of virus replication, ³¹ we chose BA as a positive control in our in vitro studies (BXM is a precursor of BA). These results suggest that QPP also possesses immunomodulatory properties in human lung epithelium cells infected with IAV.

FIGURE 6.

Qi pi pill (QPP) reduced the messenger RNA (mRNA) levels of proinflammatory cytokines induced by influenza infection in A549 cells. A549 cells were infected with influenza A/PR8 H1N1 virus in the presence of 10 or 20 mg/mL of QPP. The mRNA levels of viral NP gene (A), interleukin‐6 (IL‐6) (B), tumor necrosis factor‐α (TNF‐α) (C), and IL‐1β (D) were determined by reverse transcription quantitative polymerase chain reaction (RT‐qPCR) in the cells at 48 h postinfection. Student's t‐test and one‐way analysis of variance (ANOVA), ^### p < 0.001 , compared to the Mock group; ***p < 0.001, **p < 0.01, *p < 0.05, compared to the control group. Values are expressed as means ± standard deviation (SD) (n = 3).

3.5. QPP exhibits a protective effect in mice with acute influenza infection

Next, we investigated the protective effect of QPP in vivo in mice with acute influenza infection. We observed a rapidly decreased body weight after the third day of infection in the IAV‐infected group (control) compared to the noninfected group (Mock), indicating that the animals were successfully infected (Figure 7A). QPP treatment improved the weight loss observed in infected mice when compared to the control group, with this effect being most pronounced on day 5 postinfection (p < 0.05) (Figure 7A). Consequently, on that same day, the mice were humanely euthanized, and the lungs were collected for evaluation of lung index, viral loads, and inflammatory cytokines. As shown in Figure 7, the lung index of mice, along with the relative expression levels of viral RNA in the lungs, exhibited a reduction following the treatment with QPP and OSP. Furthermore, analysis of inflammatory cytokines in lung tissues revealed that only the expression level of TNF‐α mRNA was significantly reduced after QPP treatment (Figure 7E). Nevertheless, QPP did not improve the survival rates of mice infected with lethal doses of the IAV (Figure S2). The results indicate that symptoms related to milder IAV infections may be alleviated following QPP treatment.

FIGURE 7.

Antiviral effects of Qi pi pill (QPP) in vivo. The body weight of mice was monitored for 5 days (A) and, at day 5 postinfection, lung indexes (B), viral load (C), interleukin‐6 (IL‐6) (D), tumor necrosis factor‐α (TNF‐α) (E), and IL‐1β (F) in the lungs were evaluated. Student's t‐test, ^### p < 0.001, ^## p < 0.01, compared to the Mock group; ***p < 0.001, ^** p < 0.01 and *p < 0.05, compared to the control group. Values were expressed as means ± standard deviation (SD) (n = 6 animals per group).

3.6. Anti‐IAV effects of each QPP components individually

To explore the antiviral activity of each single Chinese herbal medicine in QPP, we screened the herbs and the excipient honey in MDCK cells infected with the influenza A/NY‐HiBiT reporter virus and treated with 10 mg/mL of each medicinal material. The results indicated that, at the tested concentration, with the exception of Shanzha, all of the other Chinese herbs and the excipient honey displayed no or very low cytotoxicity (Table 2). Among them, Renshen, Gancao, Lianzi, and Zexie showed a very good anti‐IAV activity, with percentage inhibition of viral replication higher than 90% (Table 2). The excipient (honey) did not affect the antiviral activity of QPP components (percentage inhibition equals to 8.2%) (Table 2).

TABLE 2.

Anti‐influenza effect of Qi pi pill (QPP) components.

TCM	Percent Inhibition a	Percent Cell Viability a	IC50 (mg/mL)	CC50 (mg/mL)	SI
Renshen	92.62	101.80	0.38 ± 0.130	> 4.56	12.00
Gancao	96.92	95.23	0.18 ± 0.020	> 4.56	25.33
Lianzi	98.40	94.97	0.04 ± 0.006	> 4.56	114.00
Zexie	99.85	99.44	0.02 ± 0.003	> 4.56	228.00
Baizhu	40.52	103.06	‐	‐	‐
Fuling	38.20	102.67	‐	‐	‐
Chenpi	26.51	101.90	‐	‐	‐
Shanyao	3.10	101.47	‐	‐	‐
Shanzha	98.83	51.06	‐	‐	‐
Liushenqu	75.99	98.92	‐	‐	‐
Maiya	75.48	93.23	‐	‐	‐
Honey	8.20	102.00	‐	‐	‐

Abbreviations: CC₅₀, 50% cytotoxic concentration; IC₅₀, 50% inhibitory concentration; SI, selectivity index; TCM, traditional Chinese medicines.

^a Screening of QPP individual components at 10 mg/mL.

4. DISCUSSION

In recent years, there has been an abundance of reports on the utilization of TCMs to treat influenza virus–induced pneumonia. ³¹ In this study, we screened seven CPMs from “Diagnosis and Treatment Program of TCM for Novel Coronavirus Pneumonia in Shandong Province, China in 2020 (Second Edition)” against influenza virus. QPP was the CPM that displayed better percentage of inhibition of viral replication and also had significant inhibitory effects on different types and subtypes of influenza viruses, including influenza A subtypes H1N1 and H3N2 virus, OSP‐resistant influenza A/H1N1/pdm09‐H275Y, and influenza B Yamagata and Victoria lineages. IAVs and IBVs are both responsible for causing seasonal influenza. However, they exhibit significant differences in terms of antigenic diversity, ammino acid sequence, and host range. ²³ , ³² Based on the IC₉₀ value, QPP demonstrated a more potent inhibitory effect on IAV. Considering that IAV is responsible for the majority of human and animal flu infections compared to IBV, ³³ the superior inhibition of IAV by QPP holds significant importance. Therefore, this study aimed to investigate the in vivo and in vitro inhibition of QPP on IAV and elucidate its mechanism of action.

TCMs‐based formulas and their active ingredients usually act through a variety of molecular mechanisms, presenting direct antiviral effects and modulating host response to infections. ³⁴ According to the in vitro infectious and TOA results, we demonstrated that QPP inhibited multiple rounds of IAV replication and interfered on virus life cycle in different stages, most likely during RNA replication/transcription process and new viral particles release stage. By further investigating QPP viral targets, we confirmed, through a cell‐based RdRp mini‐replicon and NA inhibition assays, that QPP can decrease both RdRp and NA enzymatic activities, corroborating our TOA data. Additionally, our results demonstrated that QPP indeed modulates host cell immune response to IAV infection, reducing the expression of proinflammatory cytokines IL‐6, IL‐1β, and TNF‐α in infected A549 cells. Because disease severity caused by influenza virus is mainly correlated to the host dysregulated immune response that provokes large lung damage, ³⁵ , ³⁶ a medicine with both antiviral and immunomodulatory effects has the potential for clinical development.

We also evaluated the effects of QPP in mice with acute influenza infection. The clinical signs of influenza virus infection in mice manifest as weight loss and pathological changes in the lungs, which can be demonstrated as increased lung index, a reflection of the degree of inflammation of the lungs. ²⁵ The treatment of infected mice with QPP resulted in the maintenance of body weight and a lower lung index compared to the infected control group. These results may be attributed to the reduction in influenza virus replication and proinflammatory cytokines in the lungs of treated animals. However, the detailed mechanistic effects of QPP in vivo still need further research.

QPP is a Chinese medicinal preparation composed of 11 Chinese herbs, based on “si jun zi decoction” (SJZD) of the prescription book “Taiping Huimin He Ping Bureau Formula” during Song dynasty government. QPP has the effect of strengthening the spleen. The theory behind TCM believes that the spleen prompts the lungs—“the essence distributed by spleen qi flowing upward into the lung,” meaning that in the clinical treatment of lung diseases, we can strengthen the spleen to restore the weakened lungs. For instance, SJZD is a TCM prescription with the effect of strengthening the spleen, which has been proved to treat lung injury diseases in “Yifang Jijie.” Zhao et al. reported that SJZD can inhibit the growth of lung cancer. ³⁷ Modified SJZD decoction effectively treats severe pneumonia in children with ventilator‐associated pneumonia, improving lung function, cure rate, and reducing inflammation. ³⁸ All these data provide insights for the clinical development of TCM as treatment for viral pneumonia.

In addition, we investigated the antiviral activity of the 11 components of QPP individually and identified four of them as potent anti‐influenza substances, including Renshen, Gancao, Lianzi, and Zexie. Ginsenosides, the important components of Renshen, have been reported to be effective in fighting IAV and reducing symptoms associated with influenza. The compounds capable of inhibiting IAV include ginsenoside rk1 (G‐rk1), G‐rg5, Rg1, Rg3(S), Rk1, and Rg5. ³⁹ , ⁴⁰ Glycyrrhizin, a compound derived from Gancao, exhibited anti‐IAV activity. ⁴¹ Additionally, other chalcones extracted from Gancao, along with glycyrrhizic acid and its derivatives, have also shown anti‐IAV activity. ⁴² , ⁴³ The compounds demonstrating anti‐IAV activity in the other two Chinese medicinal herbs need further investigation.

We demonstrated that QPP possesses antiviral activity, both in vitro and in vivo, against influenza virus, providing experimental evidence for the pharmacological effects of QPP and for its mechanisms of action. QPP is a promising medicine for further clinical studies as preventive therapy and as treatment for influenza. Future research will focus on TCM theory and investigate QPP mechanisms of action, both in vivo and in vitro effects, of its most active substances.

AUTHOR CONTRIBUTIONS

Chengcheng Zhang: Investigation; methodology; writing – original draft. Jing Gao: Resources. Meiyue Dong: Data curation; formal analysis; investigation. Carolina Q. Sacramento: Writing – original draft. Ping Li: Data curation; formal analysis. Xiangyu Lian: Investigation. Lingyuan Fan: Investigation. Lijun Rong: Conceptualization. Ruikun Du: Conceptualization. Jingzhen Tian: Conceptualization. Qinghua Cui: Conceptualization; writing – review and editing.

FUNDING INFORMATION

This work was supported by the Major Basic Program of Natural Science Foundation of Shandong Province (ZR2021ZD17); Project for Development of TCM Science and Technology of Shandong Province (M‐2022145); Special Emergency research and development of Social Benefiting Technology Program, Qingdao (grant number: 23‐7‐8‐smjk‐3‐nsh); Jinan City Funding for University Innovation Teams (2021GXRC028).

CONFLICT OF INTEREST STATEMENT

Qinghua Cui is an editorial board member of Animal Models and Experimental Medicine (AMEM) and a corresponding author of this article. To minimize bias, he was excluded from all editorial decision‐making related to the acceptance of this article for publication.

ETHICS STATEMENT

This study was approved by the Ethic Committee of Shandong University of Traditional Chinese Medicine, and all animals received humane care in compliance with the Chinese Animal Protection Act and the National Research Council Criteria (approval number: SDUTCM20231113511).

Supporting information

Figure S1.

Figure S2.

Data S1.

Zhang C, Gao J, Dong M, et al. Antiviral effects and mechanism of Qi pi pill against influenza viruses. Anim Models Exp Med. 2025;8:1364‐1375. doi: 10.1002/ame2.12511

DATA AVAILABILITY STATEMENT

All relevant data that support the findings of this study are presented in the manuscript and Supporting Information file. Data are available from the corresponding authors upon reasonable request.