NRICM101 ameliorates SARS-CoV-2-S1-induced pulmonary injury in K18-hACE2 mice model

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a challenge for public health globally since transmission of different variants of the virus does not seem to be effectively affected by the current treatments and vaccines. During COVID-19 the outbreak in Taiwan, the patients with mild symptoms were improved after the treatment with NRICM101, a traditional Chinese medicine formula developed by our institute. Here, we investigated the effect and mechanism of action of NRICM101 on improval of COVID-19-induced pulmonary injury using S1 subunit of the SARS-CoV-2 spike proteininduced diffuse alveolar damage (DAD) of hACE2 transgenic mice. The S1 protein induced significant pulmonary injury with the hallmarks of DAD (strong exudation, interstitial and intra-alveolar edema, hyaline membranes, abnormal pneumocyte apoptosis, strong leukocyte infiltration, and cytokine production). NRICM101 effectively reduced all of these hallmarks. We then used nextgeneration sequencing assays to identify 193 genes that were differentially expressed in the S1+NRICM101 group. Of these, three (Ddit4, Ikbke, Tnfaip3) were significantly represented in the top 30 enriched downregulated gene ontology (GO) terms in the S1+NRICM101 group versus the S1+saline group. These terms included the innate immune response, pattern recognition receptor (PRR), and Toll-like receptor signaling pathways. We found that NRICM101 disrupted the interaction of the spike protein of various SARS-CoV-2 variants with the human ACE2 receptor. It also suppressed the expression of cytokines IL-1 beta, IL-6, TNF-alpha, MIP-1 ss, IP-10, and MIP-1 alpha in alveolar macrophages activated by lipopolysaccharide. We conclude that NRICM101 effectively protects against SARS-CoV-2-S1-induced pulmonary injury via modulation of the innate immune response, pattern recognition receptor, and Toll-like receptor signaling pathways to ameliorate DAD.