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. 2024 Jun 24;14(1):14545.
doi: 10.1038/s41598-024-65447-w.

Effect of apigetrin in pseudo-SARS-CoV-2-induced inflammatory and pulmonary fibrosis in vitro model

Hengmin Han #  1 , Jung-Eun Kim #  2 , Hyo-Jeong Lee  3   4
Affiliations

Effect of apigetrin in pseudo-SARS-CoV-2-induced inflammatory and pulmonary fibrosis in vitro model

Hengmin Han et al. Sci Rep. .

Abstract

SARS-CoV-2 has become a global public health problem. Acute respiratory distress syndrome (ARDS) is the leading cause of death due to the SARS-CoV-2 infection. Pulmonary fibrosis (PF) is a severe and frequently reported COVID-19 sequela. In this study, an in vitro model of ARDS and PF caused by SARS-CoV-2 was established in MH-S, THP-1, and MRC-5 cells using pseudo-SARS-CoV-2 (PSCV). Expression of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and HIF-1α was increased in PSCV-infected MH-S and THP-1 cells, ARDS model, consistent with other profiling data in SARS-CoV-2-infected patients have been reported. Hypoxia-inducible factor-1 alpha (HIF-1α) siRNA and cobalt chloride were tested using this in vitro model. HIF-1α knockdown reduces inflammation caused by PSCV infection in MH-S and THP-1 cells and lowers elevated levels of CTGF, COLA1, and α-SMA in MRC-5 cells exposed to CPMSCV. Furthermore, apigetrin, a glycoside bioactive dietary flavonoid derived from several plants, including Crataegus pinnatifida, which is reported to be a HIF-1α inhibitor, was tested in this in vitro model. Apigetrin significantly reduced the increased inflammatory cytokine (IL-6, IL-1β, and TNF-α) expression and secretion by PSCV in MH-S and THP-1 cells. Apigetrin inhibited the binding of the SARS-CoV-2 spike protein RBD to the ACE2 protein. An in vitro model of PF induced by SARS-CoV-2 was produced using a conditioned medium of THP-1 and MH-S cells that were PSCV-infected (CMPSCV) into MRC-5 cells. In a PF model, CMPSCV treatment of THP-1 and MH-S cells increased cell growth, migration, and collagen synthesis in MRC-5 cells. In contrast, apigetrin suppressed the increase in cell growth, migration, and collagen synthesis induced by CMPSCV in THP-1 and MH-S MRC-5 cells. Also, compared to control, fibrosis-related proteins (CTGF, COLA1, α-SMA, and HIF-1α) levels were over two-fold higher in CMPSV-treated MRC-5 cells. Apigetrin decreased protein levels in CMPSCV-treated MRC-5 cells. Thus, our data suggest that hypoxia-inducible factor-1 alpha (HIF-1α) might be a novel target for SARS-CoV-2 sequela therapies and apigetrin, representative of HIF-1alpha inhibitor, exerts anti-inflammatory and PF effects in PSCV-treated MH-S, THP-1, and CMPVSC-treated MRC-5 cells. These findings indicate that HIF-1α inhibition and apigetrin would have a potential value in controlling SARS-CoV-2-related diseases.

Keywords: Acute respiratory distress syndrome (ARDS); Apigetrin; Hypoxia-inducible factor-1α (HIF-1α); Pulmonary fibrosis (PF); SARS-CoV-2.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Evaluation of in vitro ARDS model. MH-S (A) and THP-1 cells (B) were infected with PSCV for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the expression of Spike1, HIF-1α, IL-6, IL-1β, TNF-α, and β-actin. The results are presented as the mean ± SD of three independent experiments. **p < 0.01, and ***p < 0.001 (compared to normal control).
Figure 2
Figure 2
Evaluation of in vitro PF model. MRC-5 cells were treated with CMPSCV from MH-S (A) or THP-1 (B) cells for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the CTGF, COLA1, α-SMA, HIF-1α, and β-actin expressions. The results are presented as the mean ± SD of three independent experiments. **p < 0.01, and ***p < 0.001 (compared to normal control).
Figure 3
Figure 3
Effect of HIF-1α siRNA and CoCl2 in PSCV-infected MH-S or THP-1 cells and CPMSCV-exposure MRC-5 cells. MH-S (A) and THP-1 cells (B) were transfected with HIF-1α siRNA for 48 h and were incubated in the presence or absence of PSCV for 24 h. PSCV-infected MH-S (C) and THP-1 cells (D) were treated with CoCl2 for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the Spike1, HIF-1α, IL-6, IL-1β, TNF-α, and β-actin expressions. The results are presented as the mean ± SD of three independent experiments. ##p < 0.01, ###p < 0.001 (compared to normal control), **p < 0.01, and ***p < 0.001 (compared to PSCV-infected control). MRC-5 cells were transfected with HIF-1α siRNA for 48 h and were incubated in the presence or absence of PSCV-treated MH-S (E) or THP-1 cells (F) CMPSCV for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the CTGF, COLA1, α-SMA, HIF-1α, and β-actin expressions. The results are presented as the mean ± SD of three independent experiments. ##p < 0.01, ###p < 0.001 (compared to normal control), **p < 0.01, and ***p < 0.001 (compared to CMPSCV-infected control).
Figure 4
Figure 4
Evaluation of apigetrin on spike protein expression and binding to S protein RBD in PSCV-infected MH-S and THP-1 cells. (A) The SARS-CoV-2 inhibitor screening kit was used to test the ability of apigetrin to bind to SARS-CoV-2 S protein RBD. The results are presented as the mean ± SD of three independent experiments. ***p < 0.001 (compared to normal control). MH-S (B) and THP-1 cells (C) were treated with PSCV and apigetrin for 24 h. The cells were fixed in 100% methanol, exposed to the diluted Spike 1 antibody, and incubated with the goat anti-rabbit IgG. The reacted cells were incubated with VECTASHIELD antifade mounting medium with DAPI, and the image was captured using a fluorescence microscope. a–c means in a row by different superscripts are significantly different by LSD (least significant difference).
Figure 5
Figure 5
Anti-inflammatory effect of apigetrin in PSCV-infected MH-S and THP-1 cells. MH-S (A) and THP-1 cells (B) were infected with PSCV and apigetrin for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the Spike1, HIF-1α, IL-6, IL-1β, TNF-α, and β-actin expressions. IL-6, IL-1β, and TNF-α concentrations in the serum samples of MH-S (C–E), and THP-1 (F–H) cells were determined by ELISA. The results are presented as the mean ± SD of three independent experiments. ##p < 0.01, ###p < 0.001 (compared to normal control), **p < 0.01, and ***p < 0.001 (compared to PSCV-infected control).
Figure 6
Figure 6
Effect of apigetrin on pulmonary fibrosis in CMPSCV-treated MRC-5 cell model. MRC-5 cells were treated with apigetrin in MH-S (A,B) or THP-1 cells (D,E) CMPSCV for 24 h. CELLOMAX kit was used to measure cell proliferation. a–c means in a row by different superscripts are significantly different by LSD (least significant difference). After treatment the cells were resolved in 3.5% formaldehyde, permeabilized by methanol, and stained by crystal violet. Picro-Sirius Red Stain Kit was used to stain collagen fibers and the image was captured using a light microscope (B,E). a–d means in a row by different superscripts are significantly different by LSD (least significant difference). MRC-5 cells were treated with apigetrin in MH-S (C) or THP-1 cells (F) CMPSCV for 24 h. Cell lysates were prepared and subjected to western blotting to analyze the CTGF, COLA1, α-SMA, HIF-1α, and β-actin expressions. The results are presented as the mean ± SD of three independent experiments at p < 0.05. ##p < 0.01, ###p < 0.001 (compared to normal control), **p < 0.01, and ***p < 0.001 (compared to CMPSCV-infected control).
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