Liushen Capsules, a promising clinical candidate for COVID-19, alleviates SARS-CoV-2-induced pulmonary in vivo and inhibits the proliferation of the variant virus strains in vitro

Abstract

Background: Coronavirus disease 2019 (COVID-19) causes a global pandemic and has devastating effects around the world, however, there are no specific antiviral drugs and vaccines for the constant mutation of SARS-CoV-2.

Purpose: In this study, we evaluted the antiviral and anti-inflammatory activities of Liushen Capsules (LS) on different novel coronavirus in vitro, studied its therapeutic effects on novel SARS-CoV-2 infected mice and observed the LS's clinical efficacy and safety in COVID-19.

Methods: The antiviral and aiti-inflammatory effects of LS on the 501Y.V2/B.1.35 and G/478K.V1/ B.1.617.2 strains were determined in vitro. A hACE2 mouse model of novel SARS-CoV-2 pneumonia was established. Survival rates, histological changes, inflammatory markers, lung virus titers and the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blotting and immumohistochemical staining in the lungs were measured. Subsequently, the disease duration, prognosis of disease, time of negative nucleic acid and the cytokines levels in serum were used to assess the efficacy of treatment with LS in patients.

Results: The results showed that LS (2, 1, 0.5 μg/mL) could significantly inhibit the replication of the two SARS-CoV-2 variants and the expression of pro-inflammatory cytokines (IL-6, IL-8, IP-10, CCL-5, MIP-1α, IL-1α) induced by the virus in vitro. As for the survival experiment in mice, the survival rate of virus group was 20%, while LS-treatment groups (40, 80, 160 mg/kg) could increase the survival rate to 60, 100 and 100%, respectively. LS (40, 80, 160 mg/kg) could significantly decrease the lung titers in mice and it could improve the pathological changes, inhibit the excessive inflammatory mediators (IFN-α, IFN-γ, IP-10, MCP-1) and the protein expression of p-NF-κB p65 in mice. Moreover, LS could significantly decrease SARS-CoV-2-induced activation of p-NF-κB p65, p-IκBα, and p-p38 MAPK and increase the protein expression of the IκBα. In addition, the patient got complete relief of symptoms after being treated with LS for 6 days and was proven with negative PCR test after being treated for 23 days. Finally, treatment with LS could reduce the release of inflammatory cytokines (IL-6, PDGF-AA/BB, Eotaxin, MCP-1, MIP-1α, MIP-1β, GRO, CCL-5, MCP-3, IP-10, IL-1α).

Conclusion: LS effectively alleviated novel SARS-CoV-2 or variants induced pneumonia in vitro and in vivo, and improved the prognosis of COVID-19. In light of the efficacy and safety profiles, LS could be considered for the treatment of COVID-19 with a broad-spectrum antiviral and anti-inflammatory agent.

Keywords: COVID-19; Liushen Capsules; Pulmonary; SARS-CoV-2; The 501Y.V2/B.1.35 and G/478K.V1/ B.1.617.2 strains.

Fig. 1

The antiviral activities of LS against 501Y.V2/B.1.35 strain and the G/478K.V1/ B.1.617.2 strain. A The inhibitory effects of LS on the 501Y.V2/B.1.35 strain in Vero E6 cells. B The inhibitory effects of remdesivir on the 501Y.V2/B.1.35 strain in Vero E6 cells. C The inhibitory effects of LS on the G/478K.V1/ B.1.617.2 strain in Vero E6 cells. D The inhibitory effects of remdesivir on the G/478K.V1/ B.1.617.2 strain in Vero E6 cells. E Inhibitory effect of LS (2 μg/mL, 1 μg/mL and 0.5 μg/mL) on plaque formation of the 501Y.V2/B.1.35 strain. F Inhibitory effect of LS (2 μg/mL, 1 μg/mL and 0.5 μg/mL) on plaque formation of the G/478K.V1/ B.1.617.2 strain

Fig. 2

Effects of LS (2, 1 and 0.5 μg/mL) or Remdesivir on the mRNA expression levels of inflammatory mediators (IL-6, IL-8, IP-10, CCL-5 and MIP-1α) in the 501Y.V2/B.1.351 strain-infected cells. Data were presented as the mean ± SD obtained from three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the 501Y.V2/B.1.351 strain-infected cells

Fig. 3

Effects of LS (2, 1 and 0.5 μg/mL) on the mRNA expression levels of inflammatory mediators (IL-1α (A), IL-6 (B), CCL-5 (C) and IP-10 (D)) in the G/478K.V1/ B.1.617.2 strain-infected cells. Data were presented as the mean ± SD obtained from three separate experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the G/478K.V1/ B.1.617.2 strain-infected cells

Fig. 4

The therapeutic effect of LS or remdesivir on SARS-CoV-2-infected mice. A The survival rate was observed for 5 days after viral infection. B The pulmonary viral load at the fifth d.p.i. was determined by TCID₅₀. C Histopathological changes of lung tissues at the fifth d.p.i. Scale bar = 100 µm. a Mock infected mice treated with PBS (normal control, NC); b SARS-CoV-2-infected mice treated with PBS (viral control, Virus); c SARS-CoV-2-infected mice treated with remdesivir (50 mg/kg); d–f SARS-CoV-2-infected mice treated with LS (160, 80 and 40 mg/kg). *p < 0.05; **p < 0.01; ***p < 0.001, compared with viral control

Fig. 5

Effects of LS on the expression levels of inflammatory mediators and the expression of the key proteins related to the NF-κB/MAPK signaling pathway in SARS-CoV-2-infected mice. A The mRNA expression of the IFN-α, IFN-γ, MCP-1 and IP-10 in the lung of the infected mice were detected by RT-qPCR analysis at the fifth d.p.i.; B The expression of the key proteins related to the NF-κB/MAPK signaling pathway was examined by western blotting; C The expression of phosphorylation of nuclear translocation of NF-κB p65 was determined by immunohistochemistry at the fifth d.p.i. Scale bar = 400 µm. a mock infected mice treated with PBS (normal control, NC); b SARS-CoV-2-infected mice treated with PBS (viral control, Virus); c SARS-CoV-2-infected mice treated with remdesivir; d–f SARS-CoV-2-infected mice treated with LS (160, 80 and 40 mg/kg). *P < 0.05; **P < 0.01; ***P < 0.001, compared with viral control