Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1β Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages
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Abstract
Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1β. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1β by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1β production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.
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SciScore for 10.1101/2021.07.25.453717: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: This study was approved by the Experimental Animal Ethics Committee in Kyorin
Euthanasia Agents: After the mice were euthanized by cervical dislocation, peritoneal exudate cells were harvested by sterile lavage of the peritoneal cavity with ice -cold Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan).Sex as a biological variable Animal care and use: Adult (8–12-week-old) male C57BL/6J mice (Sankyo Labo Service, Tokyo, Japan) were housed at a temperature of 23–25 °C and humidity of 50%–60% with a fixed light/dark cycle (light, 7:00–19:00; dark, 19:00–7:00). Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Re… SciScore for 10.1101/2021.07.25.453717: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: This study was approved by the Experimental Animal Ethics Committee in Kyorin
Euthanasia Agents: After the mice were euthanized by cervical dislocation, peritoneal exudate cells were harvested by sterile lavage of the peritoneal cavity with ice -cold Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan).Sex as a biological variable Animal care and use: Adult (8–12-week-old) male C57BL/6J mice (Sankyo Labo Service, Tokyo, Japan) were housed at a temperature of 23–25 °C and humidity of 50%–60% with a fixed light/dark cycle (light, 7:00–19:00; dark, 19:00–7:00). Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources After blocking with 1% bovine serum albumin, the primary antibody against p65 (8242S; Cell Signaling Technology) was applied at a 1:400 dilution for 1 h. p65suggested: (Cell Signaling Technology Cat# 8242, RRID:AB_10859369)Experimental Models: Organisms/Strains Sentences Resources Animal care and use: Adult (8–12-week-old) male C57BL/6J mice (Sankyo Labo Service, Tokyo, Japan) were housed at a temperature of 23–25 °C and humidity of 50%–60% with a fixed light/dark cycle (light, 7:00–19:00; dark, 19:00–7:00). C57BL/6Jsuggested: NoneSoftware and Algorithms Sentences Resources The density of each protein band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) ImageJsuggested: (ImageJ, RRID:SCR_003070)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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