Luteolin-rich fraction from Perilla frutescens seed meal inhibits spike glycoprotein S1 of SARS-CoV-2-induced NLRP3 inflammasome lung cell inflammation via regulation of JAK1/STAT3 pathway: A potential anti-inflammatory compound against inflammation-induced long-COVID

Abstract

Objective: The multi-systemic inflammation as a result of COVID-19 can persevere long after the initial symptoms of the illness have subsided. These effects are referred to as Long-COVID. Our research focused on the contribution of the Spike protein S1 subunit of SARS-CoV-2 (Spike S1) on the lung inflammation mediated by NLRP3 inflammasome machinery and the cytokine releases, interleukin 6 (IL-6), IL-1beta, and IL-18, in lung epithelial cells. This study has attempted to identify the naturally- occurring agents that act against inflammation-related long-COVID. The seed meal of Perilla frutescens (P. frutescens), which contains two major dietary polyphenols (rosmarinic acid and luteolin), has been reported to exhibit anti-inflammation activities. Therefore, we have established the ethyl acetate fraction of P. frutescens seed meal (PFEA) and determined its anti-inflammatory effects on Spike S1 exposure in A549 lung cells.

Methods: PFEA was established using solvent-partitioned extraction. Rosmarinic acid (Ra) and luteolin (Lu) in PFEA were identified using the HPLC technique. The inhibitory effects of PFEA and its active compounds against Spike S1-induced inflammatory response in A549 cells were determined by RT-PCR and ELISA. The mechanistic study of anti-inflammatory properties of PFEA and Lu were determined using western blot technique.

Results: PFEA was found to contain Ra (388.70 ± 11.12 mg/g extract) and Lu (248.82 ± 12.34 mg/g extract) as its major polyphenols. Accordingly, A549 lung cells were pre-treated with PFEA (12.5-100 μg/mL) and its two major compounds (2.5-20 μg/mL) prior to the Spike S1 exposure at 100 ng/mL. PFEA dose-dependently exhibited anti-inflammatory properties upon Spike S1-exposed A549 cells through IL-6, IL-1β, IL-18, and NLRP3 gene suppressions, as well as IL-6, IL-1β, and IL-18 cytokine releases with statistical significance (p < 0.05). Importantly, Lu possesses superior anti-inflammatory properties when compared with Ra (p < 0.01). Mechanistically, PFEA and Lu effectively attenuated a Spike S1-induced inflammatory response through downregulation of the JAK1/STAT3-inflammasome-dependent inflammatory pathway as evidenced by the downregulation of NLRP3, ASC, and cleaved-caspase-1 of the NLRP3 inflammasome components and by modulating the phosphorylation of JAK1 and STAT3 proteins (p < 0.05).

Conclusion: The findings suggested that luteolin and PFEA can modulate the signaling cascades that regulate Spike S1-induced lung inflammation during the incidence of Long-COVID. Consequently, luteolin and P. frutescens may be introduced as potential candidates in the preventive therapeutic strategy for inflammation-related post-acute sequelae of COVID-19.

Keywords: JAK1/STAT3 pathway; NLRP3 inflammasome pathway; Perilla frutescens extract; anti-inflammation; long-COVID; lung inflammation; luteolin (Lu); spike glycoprotein S1.

FIGURE 1

Cytotoxicity of PFEA and its active compounds on A549 cells. The cells were treated with PFEA (A), luteolin (B), and rosmarinic acid (C) for 24 and 48 h. Cell survival was determined using the MTT assay. Data are presented as mean ± S.D. values of three independent experiments. Data are presented as mean ± S.D. values of three independent experiments.

FIGURE 2

Anti-inflammatory properties of PFEA and its active compounds on cytokines releases upon Spike S1-induced inflammation in A549 cells. A549 cells were pre-treated with PFEA (A–C), at the concentration of 0–100 μg/mL or active compounds (D–F), rosmarinic acid and luteolin, at the concentration of 0–20 μg/mL for 24 h. The cells were then exposed to Spike S1 (100 ng/mL) for 3 h. The IL-6, IL-1beta, and IL-18 releases into the culture supernatant were examined by ELISA. The Spike S1-exposed A549 cells are presented as 100%. Data are presented as mean ± S.D. values of three independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the Spike-exposed control group. ^ap < 0.05 vs. rosmarinic acid at the same concentration.

FIGURE 3

Anti-inflammatory properties of PFEA and its active compounds on IL-6, IL-1β, IL-18, and NLRP3 gene expressions upon Spike S1-exposed A549 cells. A549 cells were pre-treated with PFEA (A–D) at concentration of 0–100 μg/mL and or active compounds (E–H), rosmarinic acid and luteolin, at concentration of 0–20 μg/mL for 24 h. The cells were then exposed to Spike S1 (100 ng/mL) for 3 h. The mRNA expressions were determined using RT-qPCR. Data are presented as mean ± S.D. values of three independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the Spike S1-exposed A549 cells. ^ap < 0.05 vs. rosmarinic acid at the same concentration.

FIGURE 4

Effects of PFEA and luteolin on the NLRP3 inflammasome pathway inhibition in Spike S1-exposed A549 lung cells. A549 lung cells were pre-treated with the PFEA at concentration of 0–100 μg/mL or luteolin at concentration of 0–20 μg/mL for 24 h, and then exposed to Spike S1 (100 ng/mL) for 3 h. The inhibitory effects of PFEA (A) and luteolin (B) on the expression of NLRP3, ASC, caspase-1 (p50), and the cleaved caspase-1 (p20) in the cell lysate of A549 cells were determined by western blot. The band density was measured using Image J software. Spike S1-exposed A549 cells are presented as 100% of control. Data are presented as mean ± S.D. values of three independent experiments, *p < 0.05, **p < 0.0,1 and ***p < 0.001 vs. the Spike S1-exposed A549 cells.

FIGURE 5

PFEA and luteolin inactivated the JAK1/STAT3 signaling pathway in Spike-S1-exposed A549 cells. A549 lung cells were pre-treated with the PFEA at concentration of 0–50 μg/mL or luteolin at concentration of 0–10 μg/mL for 24 h, and then exposed to Spike S1 for 3 h. The inhibitory effects of PFEA (A) and luteolin (B) on the phosphorylation of the JAK1, and STAT3 proteins in A549 cells were displayed in western blot and band density measurements. The Spike S1-exposed A549 cells are presented as 100% of the control. Data are presented as mean ± S.D. values of three independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the Spike S1-exposed A549 cells.

FIGURE 6

Schematic conclude mechanism of luteolin-enriched fraction from P. frutescens seed meal (PFEA) attenuated Spike S1-induced NLRP3 inflammasome inflammation through the inactivation of the JAK1/STAT3 signaling pathway in A549 cells.