5.1 Drugs and reagents

HCoV-229E (VR-740, American Type Culture Collection, Pfu ≥ 5 × 103 per/mL. Isoflurane (R510-22-10, RWD Life science Co.,LTD), TRIzol® Reagent (ET101-01-V2, TransGen Biotech Co., LTD), Paraformaldehyde (P1110, Beijing Solarbio Life Sciences Co., LTD), DNase/RNase-Free Water (R1600, Beijing Solarbio Life Sciences Co., LTD), Roswell Park Memorial Institute 1640 (31800, Beijing Solarbio Life Sciences Co., LTD). PerCP/Cyanine5.5 anti-mouse CD4 Antibody (100434, BioLegend, Inc.), PE/Cyanine7 anti-mouse CD8a Antibody(100722, BioLegend, Inc.)

For the preparation of DYY Extract, the following herbs were used: Areca catechu (300 g), Magnolia officinalis (150 g), Anemarrhena asphodeloides (150 g), Paeonia lactiflora (150 g), Scutellaria baicalensis (150 g), Glycyrrhiza glabra (75 g), and Amomum tsao-ko (75 g). Each herb was weighed and mixed uniformly. The mixture was placed in a 1000 mL round-bottom flask, and water was added at 5, 4, and 3 times the volume of the herbs respectively for each sequential extraction. Each extraction was conducted by reflux heating for 1.5 h. The combined filtrates were then concentrated under reduced pressure to achieve a yield of 25.5%, resulting in a crude extract concentration of 1.85 g/mL.

Lianhua Qingwen granule (LHQW, batch number: Z20100040) was purchased from Beijing Yiling Pharmaceutical Co., Ltd. 2.5g LHQW granule was accurately weighed and mixed with pure water preheated at 70 ℃ to fully dissolve it, so as to obtain 5 g/kg LHQW solution.

5.2 Establishment of the HCoV-229E mouse model and grouping

Male BALB/c mice, weighing 12-14 g, were purchased from Beijing HFK Bioscience Co., Ltd, certificate number SCXK2019-008. Mice were kept at 22 ± 2°C, with free access to food and water, under a half-day light and dark cycle. Mice were randomly divided into six groups: normal control group, HCoV-229E infection group, HCoV-229E + DYY group (0.3, 1, 3 g/kg), and a positive control LHQW (5 g/kg) group. The animal study protocol was approved by the Scientific Ethics Review Board of Qingdao Marine Biomedical Research Institute (Permit NO. E-MBWDYY-2023–24). Both cells and animals are operated in the ASBL-2 Biosafety Laboratory.

Except for the control group, mice in all other groups were lightly anesthetized with isoflurane and subsequently infected intranasally with 1 × 106 PFU/mL of HCoV-229E. Each mouse received 50 µL of the virus solution, with administrations given every other day for a total of two doses. Control mice were given the same volume of DMEM intranasal infusion. On the day of the first infection, the LHQW positive control group was administered 5 g/kg/day by gavage, while the DYY low, medium, and high dose groups received doses of 0.3, 1, and 3 g/kg/day, respectively, also by gavage. The normal control group were administered 0.5% CMC-Na with the same volume, once daily for four days (Fig. 8).

Fig. 8

The experiment protocol of HCoV-229E infected pneumonia mice and treatment with DYY

5.3 Respiratory function assessment

On the second and fourth days of the experiment, to minimize the potential impact of medication on respiratory function, the respiratory functions of mice in each experimental group were assessed one hour before medication administration using a whole-body plethysmograph system (Shanghai TOW Intelligent Technology Co., Ltd.). Prior to commencing the experiments, the equipment was zero-calibrated, and flow parameters were set to 0.3 L/min. A bypass was opened to ensure normal breathing for the mice, who were then placed in the chamber for a 15-min acclimation period to ensure calmness, followed by recording 15 min of respiratory waveforms.

5.4 Sample collection and lung index determination

On the fifth day of the experiment, mice were weighed before blood collection. Mice were deeply anesthetized using 3% isoflurane, and then the eyeballs were removed to take blood. When the blood was exhausted, the mice were euthanized by neck removal. After blood collection, the trachea, lungs underwent saline rinsing to remove excess blood, followed by absorption of any remaining moisture with filter paper, and subsequent lung weighing. The lung index (%) was calculated as the organ weight (g) divided by the body weight (g) multiplied by 100%.

Blood samples were stored in 1.5 mL Eppendorf tubes at room temperature for 2 h to allow for serum separation. Subsequently, they were centrifuged at 4°C in a pre-cooled low-temperature centrifuge at 3000 rpm for 15 min. The resulting supernatant was carefully collected and stored at -80°C for further analysis. The trachea and part of the left lung were fixed in tissue fixative, fresh spleen tissue was processed for cell extraction, and the remaining lung tissue was stored at -80°C for future analysis.

5.5 Lung tissue viral load assessment

The lung tissue samples stored at -80 °C were placed in tissue grinding tubes, and 1 mL of Trizol reagent was added for tissue homogenization using an electric homogenizer. The tubes were then left at room temperature for 20 min before being centrifuged at 4 °C and 12,000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube, to which 0.2 mL of chloroform was added, followed by shaking for 15 s and incubation at room temperature for 3 min to allow phase separation. After centrifugation at 4°C and 12,000 rpm for 15 min, the supernatant was transferred to a new centrifuge tube and mixed with 0.5 mL of isopropanol, then incubated at room temperature for 30 min. This was followed by centrifugation at 4 °C and 12,000 rpm for 10 min. The supernatant was discarded, and the RNA precipitate was washed with 1 mL of 75% ethanol and centrifuged at 4 °C and 7500 rpm for 5 min. The supernatant was removed, and the RNA precipitate was briefly dried for 5-10 min. The precipitate was then dissolved in 20 µL of DEPC water and stored at -80 °C. Viral RNA was detected using the Human Coronavirus 229E (HCoV-229E) SYBR RT-PCR Kit (lot number: 300141tdz, TIANDZ Biotechnology Co., Ltd.) following the manufacturer's instructions.

5.6 Flow cytometric analysis of splenic cells

The freshly rinsed spleen was ground in 1640 medium and filtered through a 70 µm cell strainer. The cell suspension was centrifuged at 4 °C and 3000 rpm for 10 min, and the supernatant was discarded. The cell pellet was resuspended in 1 mL of red blood cell lysis buffer at room temperature for 3 min, followed by another centrifugation at 4 °C and 3000 rpm for 10 min. The cells were then resuspended in 500 µL of PBS and washed twice more. Each tube was then added with PerCP/Cyanine5.5 anti-mouse CD4 antibody and PE/Cyanine7 anti-mouse CD8a antibody and incubated in the dark for 30 min. The cells were washed once with PBS and analyzed using BD FACSCanto II Flow Cytometer.

5.7 Immunohistochemistry

After fixation, the lung tissues of the mice underwent infiltration and embedding in paraffin following a gradient alcohol dehydration and xylene clearance process. Subsequently, the embedded tissues were sectioned and affixed onto slides. Following this, the sections underwent staining with hematoxylin and eosin (H&E). Finally, the stained sections were meticulously examined under a microscope to observe any pathological morphological changes in the lung tissues.

5.8 Quantification of multiple cytokines in serum

Levels of multiple cytokines in serum were quantified using a hypersensitive multiplex electrochemiluminescence analyzer (MESO QuickPlex SQ 120MM, Meso Scale Discovery) and the V-PLEX Proinflammatory Panel 1 Mouse Kit (K15048D, Meso Scale Discovery), which includes ten cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO, and TNF-α. Initially, 25 µL of serum was diluted twofold with Diluent 41. Standards were prepared using a fourfold gradient dilution. Both samples and standards (50 µL each) were then incubated at room temperature for 2 h. Subsequently, the plate was washed three times with 150 µL of wash buffer per well. Next, 25 µL of a 50-fold diluted antibody solution prepared with Diluent 45 was added to each well and incubated at room temperature for an additional 2 h. After three more washes with 150 µL of wash buffer per well, each well was treated with 150 µL of Read Buffer T. The plate was read immediately after the final addition.

5.9 Western blot analysis

Using a BCA protein assay kit (Applygen Technologies Inc, Beijing, China), total proteins were extracted from the lung tissues and quantified. The proteins were then mixed with SDS buffer and boiled for 10 min. SDS-PAGE electrophoresis was performed, followed by the transfer of proteins to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h. The proteins were then incubated with various antibodies overnight at 4 °C. After incubation, secondary antibodies were added and reacted for 2 h. The expression of various proteins was observed using a high-sensitivity chemiluminescence detection kit. The expressions of MAS1 (Abcam, ab235914, 1:2000 dilution), Ras (Abcam, ab180772, 1:1000 dilution), Raf1 (Abcam, ab181115, 1:1000 dilution), MEK1/2 (proteintech, 11049-1-AP, 1:5000 dilution), p-MEK1/2 (Cell Signaling Technology, 9121S, 1:1000 dilution), ERK1/2 (proteintech, 11257-1-AP, 1:5000 dilution), and p-ERK1/2 (proteintech, 28733-1-AP, 1:4500 dilution) in the lung tissues were assessed.

5.10 Statistical analysis

Measurement data were expressed as mean ± standard deviation. Data were analyzed using GraphPad Prism software version 7.05. Comparisons were made using one-way ANOVA or two-way ANOVA followed by Tukey's post-hoc test. A P-value of less than 0.05 was considered statistically significant.