Background Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) pose a huge threat to human public health, no specific treatment is available. Jinzhen granule (JZ) is a traditional eight ingredients-Chinese medicine with prominent efficacy for treating viral-induced diseases. However, little is known about the antiviral effect and mechanism of JZ against SARS-CoV-2 and HCoV-229E. Purpose This study aimed to reveal the antiviral effects of JZ against SARS-CoV-2 and HCoV-229E, and to further explore the underlying mechanisms regulating the host immune response. Methods The chromatographic separation of JZ was performed using a Shimadzu analytical high-performance liquid chromatograph with UV detection and Alltech ELSD 2000ES. We conducted cytopathic effect (CPE) and plaque reduction assays to evaluate the antiviral effect of JZ. A lethal human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 was established to determine the protective effect of JZ on mortality and lung virus titers. Real-time quantitative PCR assays were used to analyze the expression of proinflammatory cytokines in vitro and in vivo . Western blotting was further performed to determine the activities on regulating the nuclear factor kappa B (NF-κB)/MAPK pathway. Finally, mitochondrial membrane potential assays, flow cytometry analysis and western blotting were used to assess the anti-apoptotic potency toward HCoV-229E infection. Results The results showed that 13 chemical components were identified and five peaks were determined and quantitated (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g). We found that JZ exerted inhibitory potency against SARS-CoV-2 and HCoV-229E in vitro by using CPE and plaque reduction assays, and it was further found that JZ protected mice infected by SARS-CoV-2 from death and inhibited lung virus titers. JZ also significantly decreased the induction of inflammatory cytokines (IL-1α, IL-6, CCL-5 and MIP-1β), similar to the observed in vitro effect. Moreover, JZ suppressed the release of inflammatory cytokines in vitro and it decreased the protein expression of p-p38 MAPK, p-JNK, p-NF-κB p65 and p-IκBα induced by HCoV-229E and increased the expression of IκBα. Notably, JZ significantly protected HCoV-229E-infected Huh-7 cells from mitochondrial damage and decreased apoptotic cells. The activation of the mitochondria-mediated apoptotic pathway was inhibited by JZ, as shown by the reduced expression of cleaved caspase-9, caspase-3 and p-PARP. Conclusions In conclusion, JZ (gallic acid 1.97 mg/g, baicalin 20.69 mg/g, glycyrrhizic acid 4.92 mg/g, hyodeoxycholic acid 4.86 mg/g, cholic acid 4.07 mg/g) exhibited antiviral activities against SARS-CoV-2 and HCoV-229E by regulating the NF-κB/MAPK pathway and the mitochondria-mediated apoptotic pathway. These findings demonstrated the efficacy of JZ against CoVs and suggested JZ treatment as a novel clinical therapeutic strategy for COVID-19.
【저자키워드】 SARS-CoV-2, Coronaviruses, Anti-inflammatory, Antiviral, COVID-19, Coronavirus disease 2019, HCoV-229E, SARS-CoV, severe acute respiratory syndrome coronavirus, FBS, fetal bovine serum, MERS-CoV, Middle East respiratory syndrome coronavirus, CPE, cytopathic effect, DMSO, dimethyl sulfoxide, MOI, multiplicity of infection, SI, selective index, DMEM, Dulbecco's modified Eagle's medium, PBS, phosphate-buffered saline, HCoV-229E, human coronavirus 229E, G-CSF, granulocyte colony-stimulating factor, TNF-α, tumor necrosis factor-α, IL-6, Interleukin-6, EC50, half maximal effective concentration, TCM, traditional Chinese medicine, IL-1β, interleukin-1beta, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, MIP-1a, Recombinant Macrophage Inflammatory Protein 1 Alpha, MCP-1, Monocyte chemoattracctant protein-1, Jinzhen granule, CC50, 50% cytotoxicity concentration, CCCP, carbonyl cyanide m-chlorophenylhydrazone, cytC, cytochrome c, Huh-7, hepatocellular carcinoma cell line, IC50, 50% inhibition concentrations, IP-10, Interferon-inducible protein-10, JZ, Jinzhen granule, MAPK, mitogen activated protein kinase, NF-κB, nuclear transcription factor-kappa B, NHP, nonhuman primates animal model, PARP, poly (ADP-ribose) polymerase, PFU, plaque forming-unit, RT-qPCR, Real-time Fluorescent Quantitative Polymerase Chain Reaction, TCID50, 50% tissue culture infective dose, Vero E6, African green monkey kidney epithelial cell line, VGM, virus growth medium,