Essential Oil-Rich Chinese Formula Luofushan-Baicao Oil Inhibits the Infection of Influenza A Virus through the Regulation of NF-κB P65 and IRF3 Activation

Background Luofushan-Baicao Oil (LBO) is an essential oil-rich traditional Chinese medicine (TCM) formula that is commonly used to treat cold, cough, headache, sore throat, swelling, and pain. However, the anti-influenza activities of LBO and the underlying mechanism remain to be investigated. Methods The in vitro anti-influenza activity of LBO was tested with methyl thiazolyl tetrazolium (MTT) and plaque assays. The effects of LBO on the expressions of viral nucleoprotein and cytokines were evaluated. In the polyinosinic-polycytidylic acid- (Poly I: C-) induced inflammation model, the influences of LBO on the expression of cytokines and the activation of NF-κB P65 (P65) and interferon regulatory factor 3 (IRF3) were tested. After influenza A virus (IVA) infection, mice were administered with LBO for 5 days. The lung index, histopathologic change, the expression of viral protein, P65, and IRF3 in the lung tissue were measured. The levels of proinflammatory cytokines in serum were examined. Results In vitro, LBO could significantly inhibit the infection of IVA, decrease the formation of plaques, and reduce the expression of viral nucleoprotein and cytokines. LBO could also effectively downregulate the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and interferon-β and the activation of P65 and IRF3 in Poly I:C-treated cells. In the IVA-infected mice model, inhalation of LBO with atomizer could decrease the lung index, alleviate the pathological injury in the lung tissue, and reduce the serum levels of IL-1β and IL-6. LBO could significantly downregulate the expression of viral protein (nucleoprotein, PB2, and matrix 2 ion channel) and the phosphorylation of P65 and IRF3 in the lungs of mice. Conclusion The therapeutic effects of LBO on treating influenza might result from the regulation of the immune response of IVA infection. LBO can be developed as an alternative therapeutic agent for influenza prevention.


Introduction
e influenza virus is one of the most common respiratory pathogens, which is a substantial threat to the world [1]. Influenza virus infections could affect the upper and lower respiratory tract, inducing cough, fever, sore throat, rhinorrhea, and pneumonia [2]. Influenza virus can induce seasonal epidemics and worldwide pandemics. ere have been three influenza pandemics in the past hundred years (1957, 1968, and 2009). First reported in Mexico and the United States, the 2009 H1N1 pandemic caused significant morbidity and mortality; about half a million people were dead from it [3,4]. Children, old people, pregnant women, and people with chronic illness are considered to be highrisk groups that can be easily infected by the influenza virus [5]. With the outbreak of the worldwide pandemic induced by coronavirus (SARS-CoV-2) at the end of 2019, evidence shows that the influenza A virus (IVA) could aggravate SARS-CoV-2 infection. Hence, the prevention of influenza infection is of great significance [6]. Neuraminidase inhibitor, oseltamivir, is the most commonly used antiviral drug in treating influenza. However, with the widespread oseltamivir-resistance gene, the limitation of oseltamivir is getting more serious [7,8].
Influenza virus can be recognized by many pattern recognition receptors (PRRs), such as retinoic acid-inducible gene I (RIG-I) and Toll-like receptors (TLRs). Expressed in airway epithelial cells, TLR3 is able to detect the dsRNA intermediate by influenza virus [9,10]. e activation of PRRs initiates innate immune responses, resulting in the releasing of interferons (IFNs), interleukins (ILs), and chemokines, leading to clearance of viruses and infected cells. However, the excessive activation of innate immune also results in lung injury [11]. In recent years, the attenuating of proinflammatory responses and limiting influenzainduced tissue damage has been considered as an alternative strategy for treating influenza virus infection [12,13].
Luofushan-Baicao Oil (LBO) is a unique essential oilrich traditional Chinese herbal (TCM) formula composed of seventy-nine kinds of herbs and related extractions: methyl salicylate, peppermint oil, camphor oil, turpentine oil, eucalyptus oil, camphor, menthol, cinnamon oil, Ocimum gratissimum oil, borneol, star anise oil, and Baicaojing extraction (tea seed oil extraction of the rest of sixty-eight herbs). LBO originated from Ge-Hong, a famous pharmacist in Jin Dynasty. In the Taoism prescription in Ming Dynasty, the formula of LBO was described. e preparation skill of LBO had been listed in the Chinese intangible cultural heritage in recent years.
According to the theory of TCM, influenza and other epidemics belong to the category of "plague (Wenyi)." e "fragrant repelling foulness (Fangxiang Pihui)" is considered to be a therapeutic strategy against epidemic since the Ming Dynasty. In TCM, many fragrant herbs can be used for the prevention and treatment of epidemics through burning, smelling, sneezing, and bathing [14]. LBO is commonly used to treat cold, cough, headache, sore throat, swelling, and pain. However, due to the complex composition, the bioactivity investigation and mechanism study of LBO is very limited. In this research, we evaluate in vitro and in vivo antiinfluenza effects of LBO and investigate the underlying mechanisms.  Table S1, were extracted with 5fold tea seed oil for 15 days in room temperature, and the solution was filtered to get baicaojing extraction). Methyl salicylate 250 g, peppermint oil 250 g, camphor oil 150 g, turpentine oil 95 g, eucalyptus oil 40 g, camphor 30 g, menthol 27.5 g, cinnamon oil 20 g, Ocimum gratissimum oil 15 g, borneol 2.5 g, and star anise oil 2g were mixed with the baicaojing extraction, with extra tea seed oil added to get 1 liter of LBO (relative density 0.96 mg/mL).

GC-MS and LC-MS Analysis of LBO.
e compositions of LBO were analyzed using GC-MS and LC-MS methods. GC-MS analysis was performed on an Agilent 7890B GC system coupled to an Agilent 7000D triple quadrupole MS (QQQ-MS) (Agilent Technologies, California, USA) and HP-INNOWAX column (30 m × 0.32 mm × 0.25 μm, Agilent Technologies, California, USA). e column temperature was programmed as follows: the initial temperature was 50°C and increased to 90°C at 10°C/min, then raised to 150°C at 15°C/min and held for 5 min, and finally increased to 240°C at 20°C/min and held for 5 min. Mass scanning was examined in ionization mode of EI at 45-500 amu.

Cytotoxicity Assay.
Madin-Darby Canine Kidney (MDCK) cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. e MDCK cells are recommended by ATCC to using as the host cells of influenza [15]. MDCK cells were plated in 96-well plates and cultured overnight at 37°C in 5% CO 2 . e LBO were mixed with tween 20 and diluted with DMEM. e medium was removed and the cells were then incubated with various concentrations of LBO (800-50 μg/mL) for 48 h. MTT was added to each well and further incubated for 4 h. e medium was subsequently removed, and formazan crystals were solubilized with dimethyl sulfoxide (DMSO). e absorbance was tested at 490 nm.
2.6. Antiviral Assay. MDCK cells were infected with influenza virus A/WSN/1933 (H1N1) at 37°C for 2 h. e medium was replaced with various concentrations of LBO (12.5-100 μg/mL) at 37°C in 5% CO 2 for 2 days. MTT was added to each well and further incubated for 4 h. e medium was subsequently removed, and formazan crystals were solubilized with DMSO and tested at 490 nm.

Plaque Reduction Assay. Confluent monolayers of MDCK cells were infected with influenza virus A/WSN/1933
(H1N1) at 37°C for 2 h. After incubation, the cell monolayer was covered with the overlay medium containing LBO (50 μg/mL and 100 μg/mL) and further cultured at 37°C in 5% CO 2 for 72 h. Subsequently, the overlay medium was removed, and the cell monolayer was fixed with 4% paraformaldehyde and stained with 1% crystal violet, and the plaques were visualized with ImmunoSpot S6 (CTL, Ohio, USA).

Immunofluorescence
Staining. MDCK cells were infected with influenza virus A/WSN/1933 (H1N1) at 37°C for 2 h. After incubation, the medium was replaced with LBO (50 μg/mL and 100 μg/mL) and incubation at 37°C in 5% CO 2 for another 24 h; the cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 5 min. After blocking with 3% BSA for 1 h, the cells were incubated with anti-IVA NP antibody overnight at 4°C. e cells were incubated with FITC-labeled secondary antibody for 1 h and further stained with 4′',6diamidino-2-phenylindole (DAPI). e fluorescence was visualized using Axio Observer (Zeiss, Oberkochen, Germany).

Poly I:C Treatment.
e Poly I:C is a synthetic analog of viral double-stranded RNA. A549 cell line is human lung cells.
e Poly I:C-treated A549 cells were employed to investigate the anti-influenza mechanism of LBO, specifically, the influence of LBO on the host immunity during the virus infection [11]. A549 cells were plated in 6-well plates (4 × 10 5 cells/well) and incubated overnight. e medium was replaced with various concentrations of LBO (50 μg/mL and 100 μg/mL); cells were then transfected with Poly I:C (Lipo3000 regent treated) at 37°C in 5% CO 2 for 24 h. A549 cells were harvested for RT-PCR and western blot analysis.

Evidence-Based Complementary and Alternative Medicine
After the infection, mice were treated with LBO (100 μg/mL and 400 μg/mL) with YLS-8B animal atomization device platform (Yiyan, Jinan, China). According to the instructions, 80% of aerosol diameter was 1-5 μm. e mice were treated in the container (285 × 240 × 160 mm), the device speed was set as 1.5 μL/s, each treatment lasted for 30 min, and LBO was administered twice a day for 5 days. e control animals were treated with the solvent. Ribavirin was used as a positive control.
Five days after virus infection, mice were weighed and sacrificed. e lung tissues were removed and weighed. e lung index was calculated (lung index � lung weight/body weight × 100%). Lung tissue was harvested for histopathologic examination and western blotting analysis.
2.14. RT-PCR. MDCK cells and A549 cells were scraped from the plate, and the total RNA of the cells were extracted with TRIzol reagent. e quality of the extracted RNA was determined by optical density measurement at 260 nm on a spectrophotometer ( ermo Fisher Scientific, Massachusetts, USA). Retrotranscription was performed using 2 μg of RNA. e mRNA levels of the target genes were determined by PrimeScript RT-PCR Kit using LightCycler 480 (Roche, Basel, Switzerland). Primer sequences are given in Table S2; the expression of target genes was tested by 2 −ΔΔCt method.

Immunoblotting.
e protein of A549 and lung tissue were extracted with RIPA lysis buffer. Equal amounts of protein were separated using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies (p-P65, P65, p-IRF3, IRF3, NP, M2, and PB2 GAPDH) and secondary HRP conjugated antibody. Western blot bands were examined by FluorChem E (Protein Simple, California, USA).

Enzyme-Linked Immunosorbent Assay (ELISA).
e levels of IL-1β and IL-6 were determined by ELISA kit (Meilian, Shanghai, China) according to the manufacturer's instructions. e absorbance of each well was tested at 450 nm.

Histopathological Analysis.
e lung tissues were harvested on the 6th day after infection and fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. Embedded lung tissues were cut into 5 μm thick sections. e sections were stained with hematoxylin and eosin (H&E). e sections were captured by Axio Observer (Zeiss, Oberkochen, Germany).

Statistical Analyses.
e data were expressed as means ± SEM and performed using GraphPad Prism v.6 (GraphPad Software). Statistical comparisons of the data were analyzed with one-way ANOVA followed by Dunnett's post hoc test. A value of p < 0.05 was considered to be significant.

GC-MS and LC-MS Analysis of LBO.
e GC-MS and LC-MS analysis of LBO is shown in Figure 1.
irteen reference substances were used to identify the compositions in LBO. Seven components (eucalyptol, camphor, menthol, methyl salicylate, trans-anethole, cinnamaldehyde, and eugenol) were identified based on the retention times and product ion by GC-MS (Figures 1(a) and S1(a)). Ligustilide was identified based on the retention times and product ion by LC-MS (Figures 1(b) and S1(b)).

e In Vitro Anti-Influenza Activity of LBO.
To investigate the antivirus activity of LBO, we evaluated the influence of LBO on the cell viability of MDCK. At a concentration of 100 μg/mL or less, LBO exerted no significant cytotoxicity in MDCK cells (Figure 2(a)). In MTT assay, LBO (25-100 μg/mL) could inhibit the reduction of cell viability induced by the influenza virus in a dose-dependent manner (Figure 2(b)). e infection of influenza virus A/WSN/1933 (H1N1) could induce plaque formation in the MDCK infection model. LBO (50 μg/mL and 100 μg/ mL) could effectively decrease the plaque number (Figure 2(c)).

e Influence of LBO on the NP Expression in MDCK Cells.
e expression and location of NP were used to confirm the influence of LBO on virus replication and nuclear export of viral ribonucleoprotein (vRNP). As shown in Figure 3, with the infection of influenza virus A/WSN/1933 (H1N1), a strong immunofluorescence signal, as well as the export of vRNP, could be observed in MDCK cells. LBO (50 μg/mL and 100 μg/mL) could remarkably downregulate the NP expression, while the vRNP export could not be alleviated by the treatment of LBO.

e Influence of LBO on the Replication Cycle of Influenza Virus.
e neuraminidase inhibition test, hemagglutination inhibition test, and viral replication test were performed to investigate the effects of LBO on the influenza virus. As shown in Figure S2, LBO could not inhibit the neuraminidase and hemagglutination activity. During the whole replication cycle of influenza virus A/WSN/1933 (H1N1), LBO could not downregulate the expression of NP protein.
ese results indicated that LBO may not directly interact with IVA.

e Influence of LBO on the mRNA Expressions of IL-1β, IL-6, and IFN-β in Virus-Infected MDCK Cells and Poly I:C-Treated A549 Cells.
e expressions of IL-1β, IL-6, and IFNβ were tested to evaluate the influence of LBO on the cytokine. With the stimulation of the influenza virus A/WSN/ 1933 (H1N1) as well as the Poly I:C, the mRNA expressions of IL-1β, IL-6, and IFN-β in MDCK cells and A549 cells could be strongly upregulated. With the treatment of LBO, the expression of IL-1β, IL-6, and IFN-β could be remarkably inhibited (Figures 4 and 5).    Evidence-Based Complementary and Alternative Medicine

e Influence of LBO on the Levels of NF-κB P65 and Interferon Regulatory Factor 3 in Poly I:C-Treated A549 Cells.
To investigate the underlying mechanisms of the inhibition of LBO on cytokine expression, we tested the phosphorylation and expression levels of NF-κB P65 (P65) and interferon regulatory factor 3 (IRF3) in Poly I:C-treated A549 cells ( Figure 6). With the stimulation of Poly I:C, the phosphorylation of P65 and IRF3 was strongly increased. LBO treatment could significantly alleviate the activation of P65 and IRF3 in A549 cells.  Figure 7).

e Influence of LBO on the Serum Levels of IL-1β and IL-6 in Mice.
We further evaluate the effects of LBO on the expression of proinflammatory cytokines in mice serum. e concentrations of IL-1β and IL-6 in serum were tested with ELISA. e influenza virus A/FM/1/47 (H1N1) infection could substantially upregulate the serum levels of IL-1β and IL-6. e treatment of LBO could significantly reduce the expression of IL-1β and IL-6 in mice (Figure 8).

e Influence of LBO on the Virus Proteins, P65 and IRF3, in
Mice. Nucleoprotein (NP), PB2, and matrix 2 ion channel (M2) are the three important proteins of IVA. We tested the expression of NP, PB2, and M2 to evaluate the virus clearance effect of LBO in the lung tissue. With the application of LBO, the expression of NP, PB2, and M2 could be significantly reduced (Figure 9). To further understand the underlying mechanisms of the treatment of LBO on the pneumonia mice, the activation of P65 and IRF3 was examined. After being infected by the influenza virus A/FM/1/ 47 (H1N1), the P65 and IRF3 in lung tissue were remarkably activated. Inhalation of LBO for five days could strongly inhibit the activation of P65 and IRF3 in mice ( Figure 10).

Discussion
According to the TCM theory of "fragrant repelling foulness "(Fangxiang Pihui)," burning, smelling, sneezing, and bathing the fragrant herbs could be used for the prevention and treatment of epidemic [14]. Many modern medical investigations support this ancient theory. According to the team of Zhang Q, the inhalation of the sachet (putting fragrant herbs into the small pocket) may prevent influenza through the enhancement of innate immune responses in mice [16]. Li Y proved that inhalation of Bing-Xiang-San, a fragrant Chinese formula, with an atomizer could inhibit the IVA infection in mice model [17]. Fragrant herbs are commonly rich in essential oil; many investigations indicated that essential oil could inhibit the infection of influenza both in vitro and in vivo [15,18,19]. e prescription of LBO is complex, but the content of essential oil and related components is more than ninety percent (882 g/L or 91.87%). irteen reference compounds were used to identify the composition of LBO. With the GC-MS method and LC-MS method, eight compounds (eucalyptol, camphor, menthol, methyl salicylate, trans-anethole, cinnamaldehyde, eugenol, and ligustilide) were identified. e bioactive compounds in LBO are uncertain; however, according to other researches, cinnamon oil, eugenol, and eucalyptol have anti-influenza effect. Inhalation and nasal inoculation of cinnamaldehyde, the main component of cinnamon oil, could significantly increase the survival rates of IVA mice [20]. Eugenol, the major component Ocimum gratissimum oil, exerted anti-influenza activity in both liquid and vapor phases via inhibition of ERK, p38, and IKK signal pathways [19,21]. Eucalyptol from eucalyptus oil was able to   [22]. Moreover, trans-anethole from star anise oil could reduce LPS-induced acute lung injury by resolution of pulmonary inflammation [23].
In this research, we demonstrated that LBO significantly inhibits the infection of IVA, decreases the formation of plaques, and reduces the expression of viral nucleoprotein. To explore the mechanism of anti-influenza effects, we examined the neuraminidase inhibition and hemagglutination inhibition as well as viral replication inhibition effects of LBO. However, the present results indicate that LBO could not affect the IVA directly. Many investigations about the anti-influenza effects of herbal formulas found that apart from the direct antivirus effects these herbal formulas could regulate the immune response caused by the viral infection [24,25]. Hence, we hypothesized that the anti-influenza effects of LBO may result from the regulation of host immunity.
After the infection of airway epithelial cells, the influenza virus can be recognized by TLR3 and result in the activation of NF-κB and IRF3 [9,11]. e activation of NF-κB leads to the gene transcription of proinflammatory cytokines. e high levels of IL-1β and IL-6, typical cytokines that contribute to the cytokine storm phenomenon, are proved to be correlated directly with tissue injury [26]. Many findings proved that NF-κB pathway is a prerequisite for IVA infection, and the inactivation of NF-κB pathway can protect the mice from infection [27,28]. Our research found that LBO could reduce the expression of IL-1β and IL-6 through the suppression of P65 phosphorylation. e activation of IRF3 leads to the expression of type I IFNs, IFN-α, and IFN-β, which contribute to the restriction of viral replication [29]. However, excessive type I IFNs in response to acute influenza infection contribute to immune cell-mediated tissue damage [30,31]. Interestingly, when it comes to the activation of IRF3 and transcription of IFN-β, host cells show different responses to different subtypes of IVA [32]. In this research, we found that influenza virus A/ FM/1/47 (H1N1) infection and Poly I:C contribute to the activation of IRF3. Treatment with LBO blocked the activation of IRF3 and inhibited the expression of IFN-β. e different concentrations of LBO were used in cell culture and animal study. e concentrations of LBO in the cell culture study were designed according to the cytotoxicity of LBO. e concentrations of LBO in the animal study were designed according to the results of cell culture study and our in vivo pre-experiment. Moreover, LBO exert more antiinflammatory effects in MDCK cells compared to Poly I:Cstimulated A549 cells (Figures 4 and 5). e different antiinflammatory effects of LBO in two models may result from the different systems. e LBO is an essential oil-rich formula, the anti-influenza effects of which are considered to be contributed by the volatile components according to the TCM theory and modern researches [19,33]. Oral administration of LBO is excluded considering about the traditional application and the great amount of volatile components. e administration of LBO was designed according to its clinical use, the inhalation of which could be used for the treatment of cough and sore throat. Inhalation with atomizer is employed by many researchers to investigate the essential oil related product [17,20]. With the animal atomization device platform, we could control the dosage through the control of LBO concentration, device speed, and the treatment time.
In conclusion, the present study demonstrated that LBO could effectively prevent IVA infection both in vitro and in vivo. e anti-influenza effects of LBO may be attributed to the reduction of proinflammatory cytokines and the blocking of P65 and IRF3 activation. ese findings suggest that LBO can be developed as an alternative therapeutic agent for influenza prevention.

Data Availability
All data generated or analyzed during this study are included in this published article.