Antiviral Effect of Hyunggaeyungyo-Tang on A549 Cells Infected with Human Coronavirus

Background Herbal medicine is widely recommended to treat viral infectious diseases. Over 123,000,000 individuals have been infected with the coronavirus since a worldwide pandemic was declared in March 2020. We conducted this research to confirm the potential of herbal medicine as a treatment for coronavirus. Methods We infected the A549 cell line with betacoronavirus OC43 and then treated it with 100 μg/mL Hyunggaeyungyo-tang (HGYGT) or distilled water with a control of HGYGT. We measured the mRNA expression levels of proinflammatory cytokines and interferon stimulated genes (ISGs) to confirm the effectiveness of HGYGT upon coronavirus infection. Results We found that the effects of HYGYT decrease the expression level of pPKR, peIF2α, IFI6, IFI44, IFI44L, IFI27, IRF7, OASL, and ISG15 when administered to cells with coronavirus infection. The expressions of IL-1, TNF-α, COX-2, NF-κB, iNOS, and IKK mRNA were also significantly decreased in the HGYGT group than in the control group. Conclusion Through the reduction of the amount of coronavirus RNA, our research indicates that HGYGT has antiviral effects. The reduction of IKK and iNOS mRNA levels indicate that HGYGT reduces coronavirus RNA expression and may inhibit the replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. In addition, decreases in mRNA expression levels of proinflammatory cytokines indicate that the HGYGT may relieve the symptoms of coronavirus infections.


Introduction
e COVID-19 Korean Medicine Treatment Recommendation, written based on the National Health Commission of the People's Republic of China's treatment plan, recommends herbal medicine treatment at the appropriate time. Some guidelines recommend that uninsured herbal extracts are administered for symptomatic relief. However, in Korea, uninsured herbal extracts are generally perceived to be ineffective compared with packed herbal medicines. erefore, we determined the effectiveness of an uninsured herbal extract for the treatment of COVID-19.
Hyunggaeyungyo-tang (HGYGT) is an herbal medicine composed of 13 herbs (Table 1). HGYGT is widely prescribed in patients with anhidrosis, excessive thirst, excessive sputum, fever, or sore throat. It has been reported that 88% of patients with COVID-19 had fever, 65% had dyspnea, and 60% had a cough [1]. HGYGT is prescribed for these symptoms. We confirmed the antiviral effect of HGYGT using a cell line infected with OC43, a relatively safe Betacoronavirus.
Protein kinase RNA-activated (PKR) is one of four mammalian serine-threonine kinases that primarily react to double-stranded RNA (dsRNA) during a viral infection and phosphorylate the eukaryotic initiation factor 2α (eIF2α) translation initiator. e phosphorylation of eIF2α inhibits the translation of viral mRNAs, ceasing viral replication [2]. In addition, dsRNA is a potent inducer of type I IFN, which produces interferon stimulated genes (ISGs) and forms a protective antiviral state [3]. Virus infection produces reactive oxygen specifications (ROS), which causes virus replication and oxidative injury. [4] We identified the presence of immune-related proteins such as PKR, phosphorylated PKR(pPKR), eIF2, and phosphorylated eIF2α (peIF2α) using immunocytochemistry and measured the expression of proinflammatory cytokines and ISGs to confirm and understand the antiviral effects of HGYGT in A549 cells infected with human coronaviruses.

Cell
Culture. A549 cells (Korean Cell Line Bank, Korea) were cultured in RPMI medium (Welgene, Korea) and 10% fetal bovine growth serum (RMbio, USA). e cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 .

Virus
Infection. OC43 (1.0 MOI) was added to 5 × 10 5 A549 cells, and the cells were incubated for three days. Cells were then treated with HGYGT (100 μg/mL) or deionized, distilled water (DW). e cells were cultured for 72 h prior to RNA extraction.

SRB Viability
Assay. Two days after drug treatment, the cells were fixed during 1 hr with 10% TCA solution, washed twice with DPBS, and dried at room temperature. e cells were stained with a 0.05% SRB solution. e stained cells were washed four times with 1% acetic acid and dried at room temperature. After washing four times with DPBS, the cells were then reconstituted 1 hr at room temperature using 10 mM Tris (pH � 10.5). e supernatant of Tris washed samples are analyzed with a microplate reader at an absorbance of 510 nm.

RNA Extraction.
e total RNA was extracted using TRIsure (Bioline, England). Prepared cells were distributed into an 8 pi medium consisting of 1 × 10 6 cells on 100 pi plates. A549-OC43 cells (OC43 group) and A549-OC43 cells treated with HGYGT (HGYGT group) were cultured for 72 h. All prepared groups were incubated for 5 min at room temperature before treatment with 1 mL of TRIsure per 5 × 10 5 cells.
e lysate was passed several times with a pipette tip. After incubation for 5 min at room temperature, chloroform was added, and the mixture was vortexed for 15 min and then centrifuged for 1 h at 15,000 rpm. After the supernatant was discarded, the pellet was blended with cold isopropyl alcohol to precipitate the RNA. e sample was incubated at room temperature for 10 min and centrifuged at 12,000 rpm at 4°C for 10 min. e pellet was washed using 75% ethanol, air-dried, and dissolved in PCR water. To remove the genetic DNA, purified nucleic acids were treated with DNA enzyme I (Takara, Japan). RNA was reversetranscribed using RevertAid reverse transcriptase ( ermo Fisher Scientific, USA). e sample was centrifuged for 10 min at 12,000 rpm and room temperature, and the supernatant was discarded. After it was washed twice with 75% ethanol and dried, the pellet was dissolved in DW.

cDNA Synthesis and RT-PCR.
For reverse transcription (RT) reactions, PCR was performed with 800 ng of total RNA prepared with 1 μL of random primer. e sample was denatured at 65°C for 5 min, and then, the temperature  Table 2. e forward/reverse primer mixture (2 μL at 3 μM) was combined with 7.5 μL SYBR, 4.5 μL DW, and 1 μL cDNA. In addition, predenaturation was performed for 40 cycles at 5 min each at 95°C, 40 cycles at 95°C, and 1 min at 60°C. e Cq values were calculated.

Immunocytochemistry.
e cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. en, the cells were permeabilized with 0.1% Triton X-100 in BSA buffer and blocked for 1 h at room temperature. Cells were incubated with primary antibodies diluted with 1% BSA for 2 h and then washed four times with 0.1% (v/v) Tween-20 in PBS and incubated with Alexa Fluorconjugated secondary antibodies. e cells were imaged using a Zeiss LSM 760 confocal microscope with a C-Apochromat 20x objective lens (NA � 1.40). PKR and pPKR primary antibodies were purchased from Santa Cruz Biotechnology; eIF2α and peIF2α antibodies were obtained from Cell Signaling Technology.

Statistical Analysis.
Variables are expressed as mean-± SEM. Unpaired one-tailed Student's t-tests were used to compare the data. Statistical significance was set at p < 0.05.

Cell Visualizing.
Although the difference was not significant, the expression of PKR was higher in the cells treated with OC43 than cells treated with both OC43 and HGYGT. e expression of pPKR was significantly increased in the cells treated only with OC43 compared with the expression in cells treated with both OC43 and HGYGT. e expression of pPKR in the HGYGTgroup and uninfected A549 cells was not significantly different ( Figure 2). e expression of peIF2α was significantly higher in the control group than in the cells that were infected with OC43 and treated with HGYGT ( Figures 3 and 4).

Discussion
In December 2019, a number of pneumonia cases of unknown origin were diagnosed in Wuhan City, Hubei Province, China. From this pneumonia, a coronavirus with new genetic information, which has not yet been identified, was detected [5]. e World Health Organization named the virus SARS-CoV-2 as COVID-19 and declared a global pandemic [6]. Evidence-Based Complementary and Alternative Medicine SARS-CoV-2 is a positive-sense single-stranded RNA (ssRNA) virus, enclosed in an 80-220 nm envelope [9]. e virus recognizes a specific protein in the host cell membrane and binds to it, resulting in absorption by the cell. After the virus and the host cell are fused, the uncoated virus RNA invades the host cell. e RNA is replicated using host cell ribosomes and enzymes, and capsids are synthesized. Viruses formed by viral proteases are fused with the host cell membrane and released externally via budding (Figure 9). Antiviral drugs aim to phase out the life cycle of the virus to weaken or extinguish the invading virus by interfering with RNA transcription or inhibiting virus budding [10].
Clinical signs of COVID-19 typically begin within one week after infection and include persistent fever, cough, nasal congestion, fatigue, and other symptoms of upper respiratory infections. Symptoms of the gastrointestinal tract have also been reported, and some cases are asymptomatic [11]. Symptoms may progress to difficulty breathing or extreme chest pain related to pneumonia, decreased oxygen saturation, and physiological changes observed through imaging, such as chest X-ray or CT [12].
According to a study by Son et al. [13], 58.19% of Korean medicine doctors use uninsured herbal extracts. Insured herbal extracts are widely prescribed due to the low cost to patients but differ from packaged herbal medications because they contain extracts of a single herb [14]. For this reason, uninsured herbal extracts, which are most similar to packed herbal medicines, are widely prescribed despite the cost burden of patients. In this study, uninsured herbal extracts were used for standardization and quality control of drugs.

Evidence-Based Complementary and Alternative Medicine
pPKR is activated to inhibit global translation by preventing the transition of eIF2 to eIF2B [22]. Existing studies have reported that PKR is a signaling sensor for cellular metabolism [2]. e change in pPKR is proportional to the change in dsRNA. In this study, the expression of pPKR decreased as the amount of dsRNA decreased (Figure 2). Decrease of dsRNA expression inhibits the phosphorylation of PKR, thus inhibiting the activation of eIF2α. is reduces global translation and reduces protein synthesis (Figure 9). Recently, it has been reported that SARS-CoV-2 activates pPKR [23]. In this study, we found that HGYGT could block phosphorylation of PKR with a decrease of virus RNA expression. is result signifies that HGYGT could be used as a drug to help treat COVID-19.
We confirmed that IFI6, IFI44, IFI44L, IFI27, IRF7, OASL, and ISG15 mRNA expressions were decreased in cells treated with HGYGT compared with those that were not treated with HGYGT (Figure 7). e reduced expressions of these proteins may affect the inactivation of type I interferon (IFN) pathway ( Figure 9). Type 1 IFN pathway is a defense mechanism that induces an innate immune response, and excessive production of IFN-α/β can lead to the development of autoimmune diseases [24]. e reduction of ISGs that involved in type 1 IFN with HGYGT means that it could be used for the treatment of various immune and inflammatory diseases. Previous studies on hepatitis C virus and RNA viruses have reported that IFI44, IFI44L, and IRF7 are involved in translation [25]. In particular, IFI44L has been reported to be directly involved in the IFN response and acts as a regulator of antiviral responses [26]. It has been reported that IFI27 is involved in replication [27] and OASL is involved in translation [25] and replication [28].
SARS-CoV-2 promotes excessive ROS. As ROS increases, H 2 O 2 accumulates in tissues. e increase in ROS level promotes viral replication and causes oxidative inflammation and cell apoptosis due to DNA damage. Recent studies have reported that catalase regulates cytokine in COVID-19, protects oxidative injuries, and inhibits replication of SARS-CoV-2 [29]. Viral infection increases free radicals, including nitrite oxide (NO), and depletes antioxidants [30]. RNA viruses cause oxidative stress, presumably due to the production of ROS in mitochondrial dysfunction, which occurs during virus infection with cells [31]. e production of iNOS involved in the oxidative injury involves activation of NF-kB/Rel [32]. Other studies have reported that inhibiting NF-kB pathways reduces iNOS   injury [35]. Inactivated NF-kB/Rel fails to induce transcription at the NF-kB/Rel bond site in the TATA box of iNOS gene [36]. e expression of iNOS, IKK, and NF-κB was decreased in cells treated with HGYGT compared with those that were not treated with HGYGT ( Figure 8). Inflammation with OC43 increases IKK and iNOS mRNA levels in OC43 group. HGYGT reduces IKK activity, and it appears that NF-kB/Rel is inactivated due to reduced IκB phosphorylation, resulting in iNOS expression reduction. e reduction of IKK and iNOS mRNA levels after HGYGT treatment is believed to be likely to reduce levels of dsRNA and replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. e results of this study suggest that HGYGT may have antiviral effects. We evaluated the expression of several proteins and mRNAs to determine the mechanism of HGYGT. HGYGT reduces the expression of mRNAs for cytokines, such as IL-1, TNF-α, COX-2, NF-κB, iNOS, and IKK, and the expressions of mRNAs for ISGs involved in the type I IFN pathway, resulting in a decreased immune response and fewer clinical symptoms. We suggest that HGYGT of uninsured herbal extracts manufactured by pharmaceutical companies effectively reduces the virus via regulation in NF-kB/Rel pathways and reduces the symptoms of the coronavirus infection. However, our study is limited due to its in vitro design and an insufficient sample size. More studies, including in vivo and clinical studies, are needed to determine the effects of HGYGT on coronavirus infections. e preprint of this manuscript [37] can be found in https://www.biorxiv.org/content/10.1101/2021.06.02. 446680v1.

Conclusions
HGYGTreduced the mRNA expression levels of coronavirus and inhibited the phosphorylation of PKR. It reduced mRNA expression level of iNOS and IKK. It means HGYGT may regulate NF-kB/Rel pathways and has an antioxidative effect. HGYGT blocks a series of processes of ISG expression and reduces the inflammatory response in Betacoronavirusinfected A549 cells.
us, HGYGT acts as a potential therapeutic agent for various clinical symptoms caused by coronavirus.
Data Availability e datasets generated or analyzed in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.