Pudilan xiaoyan oral liquid (PDL) inhibit the replication of inuenza A virus through regulating TLR3/MyD88 signaling pathway

The in vitro inhibitory effect of PDL on inuenza A virus was investigated using MDCK cell model. The in vivo inhibitory effect on inuenza virus pneumonia was evaluated with the ICR female mice (14-16 g) model infected by inuenza A virus (A/FM/1/47, H1N1, mouse-adapted). Moreover, expression levels of inammatory cytokines including TNF-α, IP10, IL-10, IL-1β, IL-6 and IFN-γ in lung tissue were measured by qRT-PCR. The potential mechanism of PDL against acute lung injury caused by inuenza A virus was investigated by RT-PCR and Western blot. Our results indicated that in vitro PDL has a broad-spectrum on different of and vivo PDL could dose-dependently prevent weight loss increase H1N1 the acute lung reduce mRNA inammatory factors TNF-α, IP10, IL-10, IL-1β, IL-6, IFN-γ.


Background
Pudilan Xiaoyan Oral Liquid (PDL) as a famous Chinese patent medicine has been widely used for treating upper respiratory tract infection. However, the antiviral effect of PDL remain unclear. Here, the antiviral effect of in vitro and in vivo of PDL against in uenza A virus were for the rst time investigated.

Methods
The in vitro inhibitory effect of PDL on in uenza A virus was investigated using MDCK cell model. The in vivo inhibitory effect on in uenza virus pneumonia was evaluated with the ICR female mice (14-16 g) model infected by in uenza A virus (A/FM/1/47, H1N1, mouse-adapted). Moreover, expression levels of in ammatory cytokines including TNF-α, IP10, IL-10, IL-1β, IL-6 and IFN-γ in lung tissue were measured by qRT-PCR. The potential mechanism of PDL against acute lung injury caused by in uenza A virus was investigated by RT-PCR and Western blot.

Results
Our results indicated that in vitro PDL has a broad-spectrum inhibitory effect on different subtypes of in uenza A viruses and in vivo PDL could dose-dependently prevent weight loss of mice, increase food intake and reduce mortality caused by in uenza A H1N1 virus. Furthermore, PDL could markedly improve the acute lung injury caused by in uenza A virus and signi cantly reduce the mRNA levels of in ammatory factors such as TNF-α, IP10, IL-10, IL-1β, IL-6, and IFN-γ. Mechanistic research indicated that the protective effect of PDL on viral pneumonia might be achieved by inhibiting TLR3/MyD88/IRAK4/TRAF3 signaling pathway.

Conclusion
PDL not only showed a good inhibitory effect on in uenza A virus in vitro, but also exhibited a signi cant protective effect against lethal in uenza virus infection in vivo. These ndings provide evidence for the clinical treatment of in uenza A virus infection with PDL.

Background
In uenza (Flu) is a common human respiratory disease caused by in uenza A virus, which could result in pandemic outbreaks worldwide [1][2][3][4][5]. In 1918, the severe seasonal u epidemic (H1N1) caused more than 50 million deaths in worldwide [6,7]. Due to the high mutation rate of in uenza A virus and poor cross-protective effect of different subtype in uenza vaccines, the protective effect of vaccine is very limited [8]. Furthermore, the newly emerging cross-species transmission of avian in uenza viruses such as H5N1 and H7N9, which could cause high mortality in patients, poses great challenges to human health [9][10][11][12]. An emerging of researches has reported that the acute lung injury induced by excessive in ammatory response caused by in uenza A virus is the main cause of high mortality in patients with in uenza A virus infection [6,8]. Therefore, modulating of the excessive in ammatory response may be an effective therapy for u treatment.
Pudilan xiaoyan oral liquid (PDL) as a famous Chinese patent medicine, which is composed of Taraxacum mongolicum Hand.-Mazz., Scutellaria baicalensis Georgi, Corydalis bungeana Turcz., and Radix Isatidis seu Baphicacanthii, has been widely used to treat upper respiratory tract infection diseases, pharyngitis, tonsillitis in clinical [13][14][15]. A large number of clinical studies have indicated that antiin ammatory is the main mechanism of PDL for treating these above diseases [13]. However, whether PDL has anti-in uenza virus activity remains unclear. In this study, the antiviral effect of PDL on in uenza A virus in vitro and in vivo was for the rst time investigated. Our results showed that PDL could directly inhibit the replication of seasonal in uenza A virus (H1N1 and H3N2) and showed a good protective effect against cell pathogenic effect induced by in uenza A viruses. Furthermore, PDL effectively increased the survival rate, prolonged the median survival time of mice, and reduced lung injury by suppressing the in ammatory cytokine expression via inhibition of the TLR3/MyD88/NF-κB signaling.
These ndings provide evidence for the clinical treatment of in uenza A virus infection with PDL in the future.
Ribavirin injection (100 mg/ml, 19022581) was purchased from Jiangsu Lianshui Pharmaceutical Co., Ltd (Lianshui, China). Working concentrations of oseltamivir phosphate and ribavirin were obtained by dilution with MEM as previously described [8].
Virus and cells h, the medium was removed and 150 µl of DMSO was added into each well to dissolve formazan. The absorbance per well was measured at 570 nm with a microplate reader (BioTek, USA). The non-toxic concentration and 50% cytotoxic concentration (CC 50 ) of drugs was determined as previously reported [17].

Anti-in uenza virus activity of PDL in vitro
The anti-in uenza viral effect of PDL was determined as previously described [18]. Brie y, MDCK cells (2×10 4 cells/well) were plated into 96-well plates for 12, and then were inoculated with 10TCID 50 viruses for 1 h. After inoculation, the plates were exposed with various concentrations of drugs for 96 h at 35℃ under 5% CO 2 . After 96 h, 30 µl of cell supernatant per well was taken to 96-well V-type hemagglutination plate and 25 µl of detected by HA assay according to WHO guidelines for hemagglutination test. Plates were xed with 100 µl of a 10% formaldehyde solution. After removing the solution, the plates were stained with a 0.1% (w/v) crystal violet staining. The minimum dilution without obvious toxicity was taken as the maximum non-toxic concentration (TC 0 ) of drugs, and 50% cytotoxic concentration (TC 50 ) was calculated according to the Reed-Muench method [16].
Anti-in uenza virus effects of PDL in vivo ICR female mice (14-16 g) were purchased from Comparative Medical Center for Yangzhou University and were raised in IVC on a 12 h light/dark cycle and maintained at 22±2°C. All animal experiments were approved by the Ethics Committee of Yangzhou University (202010002) according to the Chinese Animal Protection Act and the National Research Council Criteria. To investigate the anti-in uenza effect of PDL against H1N1 virus in vivo, all mice were anesthetized with ether inhalation, and were inoculated intranasally with 25 µl of viral suspension containing 5 LD 50 of in uenza virus (A/FM/1/47, H1N1, mouse-adapted) or normal saline. After infection, the mice were orally administered with PDL (8.0, 6.0, 4.0 ml/kg/day), ribavirin (80 mg/kg/day), or saline daily for 7 days. All mice were observed daily for 15 days.
The weight loss, food intake, and mortality in each group were recorded daily. The protective effects of PDL against in uenza A virus were evaluated by the survival time and mortality. Furthermore, to explore the potential mechanism of PDL against in uenza A virus in vivo, mice were intranasally inoculated with 5 LD 50 of in uenza A virus and were administrated by gavage with PDL (5.5 or 4 ml/kg/day), ribavirin (80 mg/kg/day), or saline daily for 6 days. After treatment for 6 days, all mice were sacri ced and the lung tissues were harvested. The lung index (the ratio of the lung weight to the body weight) was recorded in each mouse. Every lung tissue of each mice was divided into two parts: one for the hematoxylin and eosin (H&E) staining, and the other for analyzing the expression of in ammatory cytokines by RT-qPCR. H&E staining was performed as previous described [19]. The histopathological scores of the lung were evaluated as previously described [20].

RT-qPCR analysis of the expression of in ammatory cytokines
Total RNA was extracted using RNA extraction reagent (R401) (Vazyme, China) according to the manufacturer's instructions. cDNA synthesis was performed with HiScript II Q RT Supermix (R222) (Vazyme, China) according to the manufacturer's instructions. qPCR was conducted with AceQ Universal SYBR qPCR Master Mix (Vazyme, China). qPCR was performed as follows: 5 min at 95°C, followed by 42 cycles of 10s at 95°C and 60s at 60°C, and a melting curve step. Primers were synthesized by Genscript (Nanjing, China) and are detailed in Table 1. Relative transcript quantities were calculated using the 2 −ΔΔCt method with GAPDH as a reference. The primer sequences for QPCR were obtained as required.

Western blot analysis
Western blot assay was performed as described previously [17]. Brie y, A549 cells were infected with in uenza A virus and then were exposed to PDL or RBV for 24 h. After treatment, cells were lysed with RIPA lysis buffer (Beyotime, China) to get total protein lysates. Protein samples were separated by 10% or 12% SDS-PAGE gels, and then transferred onto nitrocellulose (NC) membrane (0.45 µm, Millipore) using a tank transfer system (Bio-Rad). The membranes were blocked for 1 h in 5% BSA in Tris-buffered saline (20 mM Tris, 166 mM NaCl, and 0.05% Tween 20, pH 7.5) and then incubated with primary antibodies overnight at 4ºC, and nally incubated with a horseradish peroxidase-conjugated species-speci c secondary antibodies at room temperature for 1 h. Bands were visualized using an enhanced chemiluminescence kit (Millipore) with a Molecular Imager SH-523 System (ShenHua, HangZhou). Quanti cation relative to ACTB by densitometric analysis was performed using Quality One software (Bio-Rad).

Statistical analysis
All data are expressed as the mean ± standard deviation (S.D.). For multiple groups, statistical difference was evaluated by one-way analysis of variance (ANOVA) or Student's t-test. Differences in the survival rate between groups were analyzed using the Log-rank test. P-values of less than or equal to 0.05 were considered statistically signi cant.

Results
HPLC pro le of PDL To con rm whether PDL contains the main bioactive ingredients from four Chinese herbs (Taraxacum mongolicum Hand.-Mazz., Scutellaria baicalensis Georgi, Corydalis bungeana Turcz. and Radix Isatidis seu Baphicacanthii), UPLC-MS analysis was performed as previous described [13]. Compared with the mixed standard materials, the nine effective constituents of PDL including (a) adenosine, (b) (r,s)epigoitrin, (c), chlorogenic acid, (d) caffeic acid, (e) cichoric acid, (f) scutellarin, (g) baicalin, (h) oroxylin A, (i) wogonoside were identi ed and quanti ed ( Fig. 1 and 2). The concentrations of these bioactive components in PDL were 0.3074 mg/ml, 0.0830 mg/ml, 0.0341 mg/ml, 0.0995 mg/ml, 0.3606 mg/ml, 0.0786 mg/ml, 7.7628 mg/ml, 0.3402 mg/ml and 0.3173 mg/ml, respectively. This result is consistent with the reported literatures [13,15], suggesting that the stability and repeatability of ingredients in PDL. In vivo anti-in uenza viral effect of PDL To determine the optimal dose of PDL, the effect of PDL on the growth of mice body weight was analyzed. As shown in Fig. 3A, compared with the control group, 8.0 ml/kg of PDL (H) had obvious effects on the increase of mice body weight, 6.0 ml/kg of PDL (M) showed a slight effect on the growth of body weight of mice, while 4.0 ml/kg of PDL (L) has no obvious effect. However, generally, the effect of PDL (4.0-8.0 ml/kg) on the growth of body weight of mice was markedly less than that the positive drug ribavirin, which has a signi cant effect on mice body weight [3]. Furthermore, we further investigated the effect of PDL against in uenza A virus in vivo according to these above dosages. To evaluate the effect of PDL against in uenza A virus in vivo, the clinical signs, food intake, and weight change of mice treated with different drugs or model were recorded daily. During the observation period, compared with the virus-infected group, the clinical symptoms of the mice-treated with PDL (8.0 and 6.0 ml/kg) were signi cantly improved and the onset time was also markedly delayed. After 3 days post-infection, the food intake of the mice-infected group was signi cantly decreased, while the treatment with PDL or RBV could signi cantly prevent the rapid decline in the diet of mice (Fig. 3B). Moreover, compared with the virus-infected group, PDL treatment could dose-dependently delay the weight loss of mice. As shown in Fig. 3C, 4 day post-infection, the weight body of the virus-infected group or PDL (4.0 ml/kg) began to gradually decrease compared to initial weight, while the body weight of the mice treated with PDL (6.0 ml//kg and 8.0 ml/kg) delay to decline until the 6th day post-infection. Positive drug RBV could signi cantly prevent the weight loss of mice. In addition, we also found that all mice infected with H1N1 virus died at between 8-and 11-days post-infection with 10.1±2.2 days of median survival time (Table 1). However, PDL could dose-dependently promote the survival of the mice-infected with virus and prolonged the survival time (Table 1). Notably, although RBV treatment couldn't prevent the weight loss caused by lethal H1N1 virus, but could provide 100% protection for lethal H1N1 virus (Table 1).

PDL effectively improves acute lung injury caused by in uenza A virus
To further con rm the antiviral effect of PDL on in uenza A H1N1 virus in vivo, the lung edema was evaluated by the lung index at 8 days post-infection. As shown in Fig. 4, the lung index of the virusinfected group was markedly increased, which was signi cantly higher than that of the control group. However, PDL treatment could signi cantly decrease the lung index, suggesting the protective effect of PDL on acute lung injury caused by in uenza A H1N1 virus. Moreover, the inhibition effect of PDL (0.55 ml/kg) on the lung index is equivalent to the positive drug RBV, but signi cantly stronger than the SHL treatment group. H&E staining also indicated that PDL treatment could effectively improve the pathological changes of lung tissue caused by in uenza A virus. As shown in Fig. 4, in uenza A H1N1 virus could cause severe lung injury with a diffuse swelling, severe cell necrosis, alveolar cavity collapse, alveolar thickening, severe in ltration of large in ammatory cells, and hemorrhage in the model group, while PDL treatment could reduce the above pathological changes, which was also been con rmed by pathology scores. These results indicated that PDL treatment showed signi cant protective effects on acute lung injury caused by in uenza A virus.

The in vivo inhibitory effect of PDL on the in ammatory cytokines
It has been demonstrated that the pro-in ammatory cytokines play a crucial role in the acute lung injury and ARDS caused by severe in uenza A virus [21,22]. Therefore, we examined the expression levels of the proin ammatory factors in lung tissue. As shown in Fig. 5, compared with the virus-infected group, PDL and RBV treatment could markedly inhibit the transcription of TNF-α, IP10, IL-10, and IFN-γ mRNA, but the inhibitory effect of PDL treatment on these above cytokines is stronger than that of RBV treatment. Notably, compared with the virus-infected group, PDL and RBV treatment also signi cantly increased the expression of IL-6, but the expression levels of IL-6 in the PDL treatment group was slightly weaker than that in the RBV treatment group. Taken together, these above results indicated that PDL treatment can signi cantly inhibit the expression of in ammatory cytokines caused by in uenza virus.
The potential mechanism of PDL against acute lung injury caused by in uenza A virus Toll-like receptors (TLRs) as the host's rst line of defense against pathogenic microbial infections play a crucial role in clearing virus and inhibiting the excessive in ammatory responses caused by virus [23,24].
In addition, it has been widely reported that TLR3-mediated signaling pathway are involved in regulating host in ammatory responses to in uenza A virus [22][23][24][25][26]. To further investigate the underlying antin ammatory mechanisms of PDL, the expression levels of TLR3, MyD88, IRF7, and TRAF3 mRNA in lung tissue were analyzed. As shown in Fig. 6, compared with the virus-infected group, the mRNA levels of TLR3, MyD88, IRF7, and TRAF3 in the lung tissue were signi cantly decreased in the PDL-treated group.
Notably, the pharmacological effect of PDL on these above in ammatory signaling pathways is almost similar to that of the positive antiviral drug, RBV. To further con rm the above effect of PDL, A549 human lung cancer cells were selected to investigate the anti-in ammatory mechanism of PDL by immunoblotting. As shown in Fig. 7, in uenza A virus infection could markedly increase the expression of TLR3, TLR7, MyD88, IRF7, TRAF3 and TRAF6, while PDL treatment could signi cantly inhibit the expression of these above proteins. Furthermore, the results of viral NP gene expression also indicated that PDL can effectively inhibit the replication of in uenza virus as RBV, further con rming the antiviral effect of PDL. These results suggested that PDL may exert the antiviral or anti-in ammatory effect by suppressing TLR3/MyD88/NF-κB signaling pathway.

Discussion
In uenza A virus as one common respiratory pathogen could cause global pandemics and epidemics in the world [27][28]. Due to the high mortality and morbidity of in uenza A virus infection, in uenza is still a major public health problem. Although preventive administration with antiviral drugs such as oseltamivir, zanamivir, amantadine could effectively improve outcomes in patients infected by in uenza viruses, there are no effective strategies to treat severe seasonal in uenza and highly pathogenic avian in uenza [8,29]. It has been demonstrated that the "cytokine storm" and excessive in ammatory response caused by lethal in uenza virus are responsible for its high mortality and morbidity [21,22,30,31]. Therefore, inhibiting the in ammatory response caused by in uenza A virus may become an effective therapy for treatment of this diseases [8,29]. Traditional Chinese herbal medicine (TCM) such as honeysuckle, forsythia, skullcap and dandelion were widely used in clinical treatment of infectious disease for long time. In addition, Chinese patent medicine, such as Yin-Qiao-San, Gegen-qianliandecotion, and Ching-fang-pai-tu-san, which is composed of traditional Chinese herbal medicine, has been widely used to treat common colds, in uenza, and upper respiratory tract infection [20,[32][33][34]. PDL as a well-known Chinese patent medicine, which is composed of Taraxacum mongolicum Hand.-Mazz, Scutellaria baicalensis Georgi, Corydalis bungeana Turcz, and Radix Isatidis, has been widely used to treat pharyngitis, tonsillitis and upper respiratory tract infection in clinical due to its remarkable antiin ammatory effect [13][14][15]. Sometimes, it is clinically used for the treatment of pandemic colds. However, whether it has an anti-in uenza virus effect has not been reported. In this present study, our results for the rst time demonstrated that PDL could effectively inhibit the replication of seasonal severe in uenza A virus including H1N1 and H3N2 in vitro. Furthermore, our results also indicated that PDL exhibited a good anti-in uenza activity in vivo, which has been con rmed by reducing the mortality and extending the survival time. Moreover, PDL treatment could signi cantly alleviate the pulmonary edema and pathological scores caused by in uenza A virus and markedly prevent the weight loss. In addition, mechanistic study indicated that the anti-in uenza effect of PDL in vivo might be highly associated with the inhibition of the expression of in ammatory cytokines such as TNF-α, IL-6, IP-10, IFN-γ, and IL-1β via TLR3-MyD88-IRAK4-TRAF6 signaling pathway.
An emerging of studies has reported that excessive in ammatory response is the main reason for the high clinical mortality of in uenza A virus infection. For example, high levels of proin ammatory cytokines such as TNF-α, IL-6, and IP-10 in sera of patients infected with severe seasonal in uenza or highly pathogenic avian in uenza viruses were commonly found [11,[30][31]. Therefore, inhibiting the excessive in ammatory responses may effectively improve the prognosis of in uenza A virus infection.
Recently, it has been demonstrated that PDL could effectively suppress the expression of proin ammatory factors TNF-α and IL-6 in LPS-induced respiratory injury model through inhibiting TLR4 signaling pathway or regulating the disturbed metabolic pathway [14]. Similar to the results reported in the above literature, we also found that PDL treatment signi cantly inhibit the expression of proin ammatory cytokines including TNF-α, IP-10, IFN-γ, and IL-10 in the mouse model of in uenza A virus infection. However, contrary to the inhibitory effect of PDL treatment on IL-6 expression observed in the aforementioned LPS-induced acute injury model in mice, our results showed that PDL treatment could signi cantly upregulate IL-6 expression. It has been demonstrated that IL-6 as a well-known cytokine produced by many different cells has a variety of biological functions, including antiviral, promoting acute phase proteins expression, stimulating B cell differentiation to producing antibody, activating human T cells, and enhancing NK cell activity. Compared with the virus-infected group or other positive control, the antibody titers of PDL treatment against H1N1 virus was the highest, while the virus titers in lung tissue was the lowest, suggesting that high levels of IL-6 in PDL treatment group might be associated with the high levels of antibody against in uenza A virus. However, the underlying mechanism of PDL treatment how to induce IL-6 expression and enhance the antibody titer against in uenza A virus remains to be further investigated. The delineation of the relationship of IL-6 and antibody titers will help to understand the potential mechanism of PDL against in uenza A virus.
Toll-like receptors (TLRs) as key pattern recognition receptors for recognizing virus play a critical role in triggering host antiviral responses. In mammals, TLR3, 7, 8 and 9 are widely involved in recognizing viral nucleic acids [22][23][24]. Among them, TLR3 as a major receptor for recognizing viral double-stranded RNA (dsRNA) plays a crucial role in protecting the host against viruses [25]. However, it has also been observed that the activation of TLR3 signaling might be deleterious in some viral infections. For example, it has been reported that TLR3-de cient mice survived in uenza A virus infection better than wild-type mice due to lower levels of in ammatory mediators, suggesting that TLR3 plays a detrimental role of TLR3-mediated in ammatory responses in in uenza virus-induced acute pneumonia [35]. Recently, Huo et al also demonstrated that lethal in uenza A virus preferentially activates TLR3 and triggers severe in ammatory responses [22]. Furthermore, the effect of TLR3 promoting acute lung injury in the development of ARDS-like pathology has also been demonstrated using TLR3-gene de cient mice which exhibited less acute lung injury, activation of apoptotic cascades and reduced neutrophil in ux. In view of this major information, we rst investigated the effect of PDL treatment on the expression of TLR receptors. Notably, compared with the virus-infected mice, PDL treatment signi cantly inhibited the expression of TLR3 mRNA, but markedly promoted the transcription of TLR4 and TLR7 mRNA, suggesting that PDL has different effects on TLRs expression. Consistent with the effect of PDL on TLR3, PDL treatment markedly inhibited the expression of MyD88, IRAK4, and TRAF3 mRNA. In addition, we found that PDL treatment signi cantly upregulated the expression of TRAF6 mRNA. Due to TLR3 signaling pathway is MyD88-independent pathway, the down-regulation of MyD88 caused by PDL treatment suggested that PDL might exert its pharmacological effect by other MyD88-dependent signaling pathway. These above results indicated that the anti-in uenza effect of FFYH against viral pneumonia may be achieved through regulating the TLR7/MyD88 signaling pathway.

Conclusion
In summary, our results showed that PDL not only can effectively inhibit the replication of in uenza A virus, including H1N1, H3N2, H5N1, H7N9, and H9N2 in vitro, but also markedly improve the survival rate of mice caused by lethal in uenza virus. Mechanistic researches indicated that PDL might exert its potential therapeutic effect on acute lung injury caused by in uenza A virus through regulating the TLR3 independent of MyD88 signaling pathway to inhibiting of excessive in ammatory response (Fig. 7). These ndings suggested that PDL might be effective for treatment of in uenza A virus.  Figure 1