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 beta coronavirus OC43 then treated with 100 μg/mL Hyunggaeyungyo-tang (HGYGT) or distilled water with a control of HGYGT. We measured the mRNA expression levels of pro-inflammatory cytokines and interferon stimulated genes (ISGs) to confirm the effectiveness of HGYGT upon coronavirus infection.

Results. We found 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 inhibits the replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. In addition, decreases in mRNA expression levels of pro-inflammatory cytokines indicate that the HGYGT may relieve the symptoms of coronavirus infections.

2.1. Reagents and Instruments

The HGYGT used in this experiment was purchased from Hanpoong Pharm.(Jeonju, Korea). The amount of HGYGT's validity was calculated to control the concentration of the active ingredient. HGYGT was diluted with physiological saline to 100 μg/mL.

The reagents used are TRIsure (Bioline, England), SYBR (Bioline, England), Reverse transcriptase (Thermo Fisher Scientific, USA), dNTP (Takara, Japan), DNase (Takara, Japan), RNase inhibitor (Takara, Japan), protease inhibitor (Takara, Japan), primary antibody (Cell signaling technology, USA), secondary antibody (Thermo Fisher Scientific, USA), Sodium acetate (Invitrogen, USA), Ethanol (Sigma, Germany).

The devices used are RT-PCR (Thermo Fisher Scientific, USA), micro reader (Thermo Fisher Scientific, USA), PCR machine (Thermo Fisher Scientific, USA), chemidoc (Thermo Fisher Scientific, USA).

2.2. Cell culture

A549 cells were cultured in RPMI medium (Welgene, Korea) and 10% fetal bovine growth serum (RMbio, USA). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

2.3. Virus infection

OC43 (1.0 MOI) was added to 5 x 10⁵ A549 cells and the cells were incubated for three days. Cells were then treated with HGYGT (100 μg/mL) or deionized, distilled water (DW). The cells were cultured for 72 h prior to RNA extraction.

2.4. SRB Viability Assay

Two days after drug treatment, the cells were fixed during 1hr with 10% TCA solution, washed twice with DPBS, and dried at room temperature. The cells were stained with a 0.05% SRB solution. The stained cells were washed 4 times with 1% acetic acid and dried at room temperature. After washing four times with DPBS, the cells were then reconstituted 1hr at room temperature using 10 mM Tris (pH = 10.5). The supernatant of Tris washed samples are analyzed with a microplate reader at an absorbance of 510 nm.

2.5. RNA Extraction

The total RNA was extracted using TRIsure (Bioline, England). Prepared cells were distributed into an 8 pi medium consisting of 1 × 10⁶ 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 × 10⁵ cells. The 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 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. The sample was incubated at room temperature for 10 min and centrifuged at 12,000 rpm at 4°C for 10 min. The 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 reverse transcribed using RevertAid reverse transcriptase (Thermo Fisher Scientific, USA). The 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.

2.6. 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. The sample was denatured at 65°C for 5 min, then the temperature dropped to 4°C before 4 μL of a 10 mM dNTP mixture and 1 μL of reverse transcriptase were added with 1 μL RNase inhibitor (20 U/μL) and 4 μL 5×RT buffer (250 mM Tris-HCl, pH = 8.3, 375 mM KCl, 15 mM MgCl₂). PCR was performed at 25°C for 10 min, at 42.5°C for 60 min, and at 70°C for 10 min. RT-PCR (Bio-Rad, USA) was performed using synthesized cDNA, forward/reverse primers, and the SensiFAST SYBR Lo-Rox Kit (Bioline, England). The sequences of primers used in the experiment are presented in Table 2. The 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, pre-denaturation was performed for 40 cycles at 5 min each at 95°C, 40 cycles at 95°C, and 1 min at 60°C. The Cq values were calculated.

2.7. Immunocytochemistry

The cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Then, 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 two h then washed four times with 0.1% (v/v) Tween-20 in PBS and incubated with Alexa Fluor-conjugated secondary antibodies. The 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.

2.8. 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.