Hesperetin from Root Extract of Clerodendrum petasites S. Moore Inhibits SARS-CoV-2 Spike Protein S1 Subunit-Induced NLRP3 Inflammasome in A549 Lung Cells via Modulation of the Akt/MAPK/AP-1 Pathway

Abstract

Inhibition of inflammatory responses from the spike glycoprotein of SARS-CoV-2 (Spike) by targeting NLRP3 inflammasome has recently been developed as an alternative form of supportive therapy besides the traditional anti-viral approaches. Clerodendrum petasites S. Moore (C. petasites) is a Thai traditional medicinal plant possessing antipyretic and anti-inflammatory activities. In this study, C. petasites ethanolic root extract (CpEE) underwent solvent-partitioned extraction to obtain the ethyl acetate fraction of C. petasites (CpEA). Subsequently, C. petasites extracts were determined for the flavonoid contents and anti-inflammatory properties against spike induction in the A549 lung cells. According to the HPLC results, CpEA significantly contained higher amounts of hesperidin and hesperetin flavonoids than CpEE (p < 0.05). A549 cells were then pre-treated with either C. petasites extracts or its active flavonoids and were primed with 100 ng/mL of spike S1 subunit (Spike S1) and determined for the anti-inflammatory properties. The results indicate that CpEA (compared with CpEE) and hesperetin (compared with hesperidin) exhibited greater anti-inflammatory properties upon Spike S1 induction through a significant reduction in IL-6, IL-1β, and IL-18 cytokine releases in A549 cells culture supernatant (p < 0.05). Additionally, CpEA and hesperetin significantly inhibited the Spike S1-induced inflammatory gene expressions (NLRP3, IL-1β, and IL-18, p < 0.05). Mechanistically, CpEA and hesperetin attenuated inflammasome machinery protein expressions (NLRP3, ASC, and Caspase-1), as well as inactivated the Akt/MAPK/AP-1 pathway. Overall, our findings could provide scientific-based evidence to support the use of C. petasites and hesperetin in the development of supportive therapies for the prevention of COVID-19-related chronic inflammation.

Keywords: COVID-19; Clerodendrum petasites; NLRP3 inflammasome; anti-inflammation; chronic inflammation; hesperetin; spike glycoprotein S1.

Figure 1

Effects of C. petasites extracts and its active compounds on A549 lung cell viability. Cells were treated with CpEE (A), CpDM (B), CpEA (C), Cp-H₂O (D), hesperidin (E), hesperetin (F) and hispidulin (G) for 24 and 48 h. Cell survival was determined using SRB assay. Data are presented as mean ± S.D. values of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control.

Figure 2

Effects of ethyl acetate fraction of C. petasites (CpEA) and hesperetin on normal cell viability. Cells were treated with CpEA and hesperetin for 24 and 48 h. Cell survival was determined using SRB assay for human dermal fibroblasts (A,B) and THP-1 macrophages (C,D). Effects of CpEA (E) and hesperetin (F) on red blood cells (RBCs) were determined using RBC hemolysis assay, TritonX-100 was used as positive control. Data are presented as mean ± S.D. values of three independent experiments. *** p < 0.001 vs. negative 0.9% normal saline (NSS) control.

Figure 3

Inhibitory effects of C. petasites extracts and its active compounds on the pro-inflammatory cytokine release in Spike S1-induced A549 cells. A549 cells were pre-treated with C. petasites extracts (A), CpEE and CpEA, at a concentration of 0–200 μg/mL or active compounds (B), hesperidin or hesperetin, at a concentration of 0–20 μg/mL for 24 h. Then, the cells were 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-induced 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 S1-induced control group. ^a p < 0.05 vs. CpEE at the same concentration. ^b p < 0.05 vs. hesperidin at the same concentration.

Figure 4

Inhibitory effects of C. petasites extracts and hesperetin on the NLRP3, IL-1β and IL-18 gene expression in Spike S1-induced A549 cells. A549 cells were pre-treated with CpEE (A), CpEA (B) at a concentration of 0–200 μg/mL and hesperetin (C) at a concentration of 0–40 μg/mL for 24 h. Then, the cells were 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-induced A549 cells.

Figure 5

CpEA and hesperetin inhibited the NLRP3 inflammasome pathway in Spike S1-induced A549 lung cells. A549 lung cells were pre-treated with CpEA at a concentration of 0–200 μg/mL or hesperetin at a concentration of 0–40 μg/mL for 24 h, and then exposed to Spike S1 (100 ng/mL) for 3 h. The inhibitory effects of CpEA (A) and hesperetin (B) on the expression of NLRP3, ASC, and caspase-1 proteins in A549 cells. The inhibitory effects of CpEA and hesperetin on the expressions of cleaved caspase-1 in culture supernatant and cell lysate from A549 cells (C). The data is displayed in Western blot and band density measurements. The Spike S1-induced A549 cells are presented as 100% of control. Data are presented as mean ± S.D. values of three independent experiments, * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the Spike S1-induced A549 cells.

Figure 6

CpEA and hesperetin inactivated the Akt/Erk/c-Jun signaling pathway in Spike-S1-induced A549 cells. A549 lung cells were pre-treated with CpEA at a concentration of 0–200 μg/mL or hesperetin at a concentration of 0–40 μg/mL for 24 h, and then exposed to Spike S1 for 3 h. The inhibitory effects of CpEA (A) and hesperetin (B) on the phosphorylation of Akt, Erk1/2, and c-Jun proteins in A549 cells were displayed in Western blot and band density measurements. The Spike S1-induced A549 cells are presented as 100% of control. Data are presented as mean ± S.D. values of three independent experiments, ** p < 0.01 and *** p < 0.001 vs. the Spike S1-induced A549 cells.

Figure 7

Schematic concluding mechanism of hesperetin-enriched fraction from Clerodendrum petasites S. Moore (CpEA) and hesperetin-attenuated Spike S1-induced NLRP3 inflammasome inflammation through the inactivating of Akt/Erk/c-Jun signaling pathway in A549 cells.