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 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.

Methods
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.

背景 草药疗法被广泛推荐用于治疗病毒性传染病。自2020年3月全球宣布新冠肺炎大流行以来，已有超过1.23亿人感染冠状病毒。本研究旨在验证草药用于冠状病毒感染治疗的潜力。

方法 本研究以β冠状病毒OC43感染A549细胞系，随后分别用100 μg/mL的亨桂宁汤（Hyunggaeyungyo-tang，HGYGT）或蒸馏水处理细胞，以HGYGT处理组为实验组，蒸馏水处理组为对照组。通过检测促炎细胞因子（pro-inflammatory cytokines）及干扰素刺激基因（interferon stimulated genes，ISGs）的mRNA表达水平，验证HGYGT抗冠状病毒感染的效果。

结果 本研究发现，在冠状病毒感染的细胞中施加HGYGT后，pPKR、peIF2α、IFI6、IFI44、IFI44L、IFI27、IRF7、OASL及ISG15的表达水平均下调。与对照组相比，HGYGT处理组中白细胞介素-1（IL-1）、肿瘤坏死因子-α（TNF-α）、环氧合酶-2（COX-2）、核因子κB（NF-κB）、诱导型一氧化氮合酶（iNOS）及IκB激酶（IKK）的mRNA表达水平亦显著降低。

结论 通过降低冠状病毒RNA水平，本研究证实HGYGT具有抗病毒活性。IKK和iNOS mRNA水平的下调表明，HGYGT可抑制冠状病毒RNA表达，并可能通过作用于NF-κB/Rel通路抑制病毒复制，从而减轻氧化损伤。此外，促炎细胞因子mRNA表达水平的下调提示HGYGT可缓解冠状病毒感染的相关症状。

2.1 试剂与仪器
本实验使用的HGYGT购自韩国全州的韩丰制药（Hanpoong Pharm）。实验前计算HGYGT的有效成分含量以控制活性成分浓度，并用生理盐水将其稀释至100 μg/mL。
本实验使用的试剂包括：TRIsure试剂（Bioline，英国）、SYBR荧光染料（Bioline，英国）、反转录酶（赛默飞世尔科技，美国）、dNTP混合物（Takara，日本）、DNase酶（Takara，日本）、RNase抑制剂（Takara，日本）、蛋白酶抑制剂（Takara，日本）、一抗（Cell Signaling Technology，美国）、二抗（赛默飞世尔科技，美国）、乙酸钠（Invitrogen，美国）、乙醇（Sigma，德国）。
本实验使用的仪器包括：实时荧光定量PCR（real-time quantitative PCR，RT-PCR）、酶标仪（赛默飞世尔科技，美国）、PCR扩增仪（赛默飞世尔科技，美国）、化学发光成像系统（chemidoc，赛默飞世尔科技，美国）。

2.2 细胞培养
A549细胞培养于RPMI培养基（Welgene，韩国）及10%胎牛血清（RMbio，美国）中，置于37℃、含5% CO₂的饱和湿度培养箱中培养。

2.3 病毒感染
将1.0 MOI的OC43病毒加入至5×10^5个A549细胞中，孵育3天。随后分别用100 μg/mL的HGYGT或去离子蒸馏水（DW）处理细胞，继续培养72小时后提取RNA。

2.4 磺酰罗丹明B（sulforhodamine B，SRB）细胞活力检测法
药物处理2天后，用10% TCA溶液固定细胞1小时，用DPBS洗涤2次，室温晾干。随后用0.05% SRB溶液染色，染色后的细胞用1%乙酸洗涤4次，室温晾干。再用DPBS洗涤4次后，加入10 mM Tris溶液（pH=10.5），室温静置1小时使细胞充分溶解。取上清液置于酶标仪中，于510 nm波长处检测吸光度值。

2.5 RNA提取
采用TRIsure试剂（Bioline，英国）提取总RNA。将制备好的细胞以1×10^6个细胞接种于100 mm培养板中，培养基体积为8 mL。将A549-OC43细胞（OC43组）及经HGYGT处理的A549-OC43细胞（HGYGT组）继续培养72小时。每5×10^5个细胞加入1 mL TRIsure试剂，室温孵育5分钟后，用移液枪枪头反复吹打裂解细胞。室温孵育5分钟后加入氯仿，涡旋震荡15分钟，15000 rpm离心1小时。弃去上清液，向沉淀中加入冷异丙醇以沉淀RNA。室温孵育10分钟后，4℃、12000 rpm离心10分钟。用75%乙醇洗涤沉淀，室温晾干后用PCR级水溶解沉淀。为去除基因组DNA污染，纯化后的核酸用DNA酶I（Takara，日本）处理。随后用RevertAid反转录酶（赛默飞世尔科技，美国）进行RNA反转录。反转录产物4℃、12000 rpm离心10分钟，弃去上清液，用75%乙醇洗涤2次后晾干，沉淀用去离子蒸馏水溶解。

2.6 cDNA合成与实时荧光定量PCR
对于反转录（RT）反应，取800 ng总RNA，加入1 μL随机引物进行PCR反应。样品于65℃变性5分钟，降温至4℃后，加入4 μL 10 mM dNTP混合物、1 μL反转录酶、1 μL RNase抑制剂（20 U/μL）及4 μL 5×RT缓冲液（250 mM Tris-HCl，pH=8.3，375 mM KCl，15 mM MgCl₂）。反转录反应条件为：25℃ 10分钟，42.5℃ 60分钟，70℃ 10分钟。以合成的cDNA为模板，使用正/反向引物及SensiFAST SYBR Lo-Rox试剂盒（Bioline，英国）进行RT-PCR反应（Bio-Rad，美国）。本实验所用引物序列详见表2。反应体系为：2 μL 3 μM正/反向引物混合物、7.5 μL SYBR染料、4.5 μL去离子蒸馏水及1 μL cDNA。预变性步骤为95℃ 5分钟，随后40个循环：95℃ 30秒，60℃ 1分钟。计算Cq值。

2.7 免疫细胞化学
细胞用PBS洗涤2次，4%多聚甲醛室温固定15分钟。随后用含0.1% Triton X-100的BSA缓冲液透化细胞，室温封闭1小时。用1% BSA稀释一抗，将细胞与一抗共同孵育2小时，随后用含0.1%（v/v）Tween-20的PBS洗涤4次，再与Alexa Fluor标记的二抗共同孵育。使用蔡司LSM 760激光共聚焦显微镜及C-Apochromat 20倍物镜（数值孔径NA=1.40）进行成像。PKR及pPKR一抗购自Santa Cruz Biotechnology；eIF2α及peIF2α抗体购自Cell Signaling Technology。

2.8 统计学分析
计量资料以均值±标准误（mean ± SEM）表示。采用独立样本单尾t检验进行组间比较，以p<0.05作为统计学显著性阈值。