Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

Keywords: Anti-inflammatory; Antiviral; Liu Shen capsule; SARS-CoV-2.

Graphical abstract

Fig. 1

Reduction of SARS-CoV-2-induced cytopathic effect by LS. A. Vero E6 cells were not-infected (NC) or infected with SARS-CoV-2 and the inhibitory effect of LS and Remdesivir on virus proliferation was evaluated. Images under a NiKon Eclipse TE300 microscope (NiKon Corporation, Tokyo, Japan) at 100 magnification. B. Inhibiting the activity of SARS-CoV-2 when given different concentrations in vitro. (a) The cytotoxicity effects of LS in Vero E6 cells were detected using MTT assay. (b) The inhibitory effects of LS on SARS-CoV-2 in Vero E6 cells. (c) The cytotoxic effects of LS in Huh-7 cells were detected using MTT assay. (d) The cytotoxic effects of Remdesivir in Vero E6 cells were detected using MTT assay. (e) The inhibitory effects of Remdesivir on SARS-CoV-2 in Vero E6 cells. (f) The cytotoxic effects of Remdesivir in Huh-7 cells were detected using MTT assay. Error bars indicate the range of values obtained from counting in triplicate are represented as the mean ± SD of three individual experiments.

Fig. 2

Dose-dependent reduction of SARS-CoV-2 plaque formation after treatment with LS. (A) Inhibitory effect of LS or Remdesivir on plaque formation of SARS-CoV-2. (B) The quantitative analysis of the plaque formation in different groups was analysed by SPSS ver. 19.0. Data are presented as the mean ± SD obtained from three separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001, compared with SARS-CoV-2-infected cells.

Fig. 3

Effect of LS on virus morphology in Vero E6 cells. (A, E) uninfected cells (NC), (B, F) SARS-CoV-2 infected cells (Virus), (C, G) LS-treated infected cells and Remdesivir-treated infected cells. The red boxes and black arrows indicated changes in the number of virus particles after treatment with or without LS and Remdesivir. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4

Effects of treatment with LS or Remdesivir on the mRNA expression levels of inflammatory mediators in SARS-CoV-2-infected Huh-7 cells. TNF-α, IL-1β, IL-6, CXCL-10/IP-10, CCL-2/MCP-1 and IL-8. Data are presented as the mean ± SD obtained from three separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001, compared with SARS-CoV-2-infected cells.

Fig. 5

LS inhibited the inflammation induced by the virus through regulating the NF-κB/MAPK signaling pathway in vitro. (A) The protein expressions of NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, p-p38 MAPK and p38 MAPK in the cells was detected by western blot analysis; (B) The quantitative analysis of the NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, p-p38 MAPK and p38 MAPK proteins was analysed by Image J. The values are presented as the means ± SD of three individual experiments. *p < 0.05; **p < 0.01; ***p < 0.001, when compared to the viral control.