Virlaza™ Inhibits Sars-COV-2-induced Inflammatory Response of Bronchial Epithelial Cells and Pulmonary Fibroblast

  1. Maysa Alves Rodrigues Brandao-Rangel
  2. Dobroslav Melamed
  3. Anamei Silva-Reis
  4. Boris Brill
  5. Lucas dos Santos Zamarioli
  6. Carlos Rocha Oliveira
  7. Rodolfo P Vieira

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Abstract

Coronavirus disease 2019 (COVID-19), which is currently a global public health emergency and beyond vaccines as a prophylactic treatment, no specific and effective therapeutical treatments are available. COVID-19 induces a massive release of proinflammatory cytokines, which drives COVID-19 progression, severity, and mortality. In addition, bronchial epithelial cells are the first pulmonary cells activated by coronavirus-2 (SARS-Cov-2) leading to massive cytokine release, which can hyperactivate lung fibroblasts, resulting in pulmonary fibrosis, a phenomenon observed even in moderate COVID-19 survivors. This in vitro study tested the hypothesis that Virlaza ™ , a herbal medicine, could inhibit the hyperactivation of human bronchial epithelial cells (BEAS-2B) and pulmonary fibroblasts (MRC-5) induced by SARS-Cov-2. BEAS-2B (5×10 4 /mL/well) and MRC-5 (5×10 4 /mL/well) cells were co-cultivated with 1ml of blood of a Sars-Cov-2 infected patient for 4 hours and Virlaza ™ (1ug/mL) was added in the first minute of the co-culture. After 4 hours, the cells were recovered and used for analysis of cytotoxicity by MTT and for mRNA expression of P2X7 receptor E iNOS. The supernatant was used to measure ATP and cytokines. Sars-Cov-2 incubation resulted in increased release of ATP, IL-1beta, IL-6, IL-8, and TNF-alpha by BEAS-2B and MRC-5 cells (p<0.001). Treatment with Virlaza ™ resulted in reduction of ATP, IL-1beta, IL-6, IL-8, and TNF-alpha release (p<0.001). In addition, Sars-Cov-2 incubation resulted in increased expression of P2X7 receptor and iNOS (p<0.001), which has been reversed by Virlaza ™ (p<0.001). In conclusion, Virlaza ™ presents important anti-inflammatory effects in the context of Sars-Cov-2 infection.

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    SciScore for 10.1101/2021.07.17.21260123: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: This study and all experimental procedures were analyzed and approved by ethical committee of School of Medicine of Anhembi Morumbi University (4.637.625 in 08 of April of 2021) and were carried out in accordance with Declaration of Helsinki for ethical principles for medical research involving human subjects.
    Consent: The written consent inform was obtained from the patient volunteer for this study.
    Sex as a biological variableBEAS-2B and MRC-5 cells were obtained from Rio de Janeiro Cell Bank and co-cultured in DMEM high glucose medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum in 500ul of whole blood of Sars-Cov-2 infected male (35-40) years old patient (original Wuhan Sars-Cov-2), presenting an estimated high viral load based on the cycle threshold (CT = 15).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    BEAS-2B and MRC-5 cells were obtained from Rio de Janeiro Cell Bank and co-cultured in DMEM high glucose medium (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum in 500ul of whole blood of Sars-Cov-2 infected male (35-40) years old patient (original Wuhan Sars-Cov-2), presenting an estimated high viral load based on the cycle threshold (CT = 15).
    MRC-5
    suggested: None
    Both BEAS-2B and MRC-5 cells were co-cultured at a concentration of 5×104/mL/well in 48 wells plate, at a humidified atmosphere in a CO2 incubator (5% CO2, 37°C) [14].
    BEAS-2B
    suggested: None

    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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  2. Version published to 10.1101/2021.07.17.21260123v1 on medRxiv