Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis

Abstract

Severe coronavirus disease (COVID-19) is accompanied by acute respiratory distress syndrome and pulmonary pathology, and is presented mostly with an inflammatory cytokine release, a dysregulated immune response, a skewed neutrophil/lymphocyte ratio, and a hypercoagulable state. Though vaccinations have proved effective in reducing the COVID-19-related mortality, the limitation of the use of vaccine against immunocompromised individuals, those with comorbidity, and emerging variants remains a concern. In the current study, we investigate for the first time the efficacy of the Glycyrrhiza glabra (GG) extract, a potent immunomodulator, against SARS-CoV-2 infection in hamsters. Prophylactic treatment with GG showed protection against loss in body weight and a 35%–40% decrease in lung viral load along with reduced lung pathology in the hamster model. Remarkably, GG reduced the mRNA expression of pro-inflammatory cytokines and plasminogen activator inhibitor-1 (PAI-1). In vitro , GG acted as a potent immunomodulator by reducing Th2 and Th17 differentiation and IL-4 and IL-17A cytokine production. In addition, GG also showed robust potential to suppress ROS, mtROS, and NET generation in a concentration-dependent manner in both human polymorphonuclear neutrophils (PMNs) and murine bone marrow-derived neutrophils (BMDNs). Taken together, we provide evidence for the protective efficacy of GG against COVID-19 and its putative mechanistic insight through its immunomodulatory properties. Our study provides the proof of concept for GG efficacy against SARS-CoV-2 using a hamster model and opens the path for further studies aimed at identifying the active ingredients of GG and its efficacy in COVID-19 clinical cases.

SciScore for 10.1101/2022.05.16.492112: (What is this?)

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

Table 1: Rigor

Ethics	Euthanasia Agents: Intranasal infection of live SARS-CoV2 (SARS-Related Coronavirus 2, Isolate USA-WA1/2020)105PFU/ 100μl or with DMEM mock control was established with the help of catheter under mild anesthetized by using ketamine (150mg/kg) and xylazine (10mg/kg) intraperitoneal injection inside ABSL3 facility. IACUC: All the experimental protocols involving the handling of virus culture and animal infection were approved by RCGM, institutional biosafety and IAEC (IAEC/THSTI/105) animal ethics committee.
Sex as a biological variable	Isolation of murine BMDNs and human peripheral neutrophils: Murine bone marrow-derived neutrophils were isolated from femur and tibia bones of C57BL/6 wild-type male mice (20–25 g, 12–16 weeks) using the method described previously (71).
Randomization	not detected.
Blinding	Assessment for the histological score was carried out through blind scoring for each sample by a professional histologist on a scale of 0-5 (where 0 indicated absence of histological feature while 5 indicated highest score).
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
After centrifugation at 1700 x g for 30 min with acceleration 5 m/s2 and deceleration 4 m/s2, band between 81% and 62% were harvested and assessed for their viability by Trypan blue and purity by anti-Ly6G and anti-CD11b antibodies.	anti-Ly6G suggested: None anti-CD11b suggested: None
In a parallel experiment, immunofluorescence staining of BMDNs and PMNs was carried out using mouse anti-MPO and rabbit anti-H4Cit3 antibodies.	anti-MPO suggested: None anti-H4Cit3 suggested: None
After fixation and blocking, samples were incubated overnight with 1:100 dilution of primary antibodies and were visualized after incubation with the secondary antibodies (1:200, anti-mice AF488 and anti-rabbit AF594) using the confocal microscope (Olympus FV3000) at 100X resolution.	anti-mice suggested: (AgriSera Cat# AS04 040, RRID:AB_2226396) anti-rabbit suggested: None
Experimental Models: Cell Lines
Sentences	Resources
titration: SARS-Related Coronavirus 2, Isolate USA-WA1/2020 virus was grown and titrated in Vero E6 cell line cultured in Dulbecco’s Modified Eagle Medium (DMEM) complete media containing 4.5 g/L D-glucose, 100,000 U/L Penicillin-Streptomycin, 100 mg/L sodium pyruvate, 25mM HEPES and 2% FBS.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Isolation of murine BMDNs and human peripheral neutrophils: Murine bone marrow-derived neutrophils were isolated from femur and tibia bones of C57BL/6 wild-type male mice (20–25 g, 12–16 weeks) using the method described previously (71).	C57BL/6 suggested: None
Software and Algorithms
Sentences	Resources
The cells were then taken for flow cytometry using BD FACSCantoII and data was analysed with FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Multiple group comparisons have been performed using one-way ANOVA followed by the Bonferroni test using GraphPad Prism 8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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: We found the following clinical trial numbers in your paper:

Identifier	Status	Title
NCT04553705	Recruiting	Omega-3, Nigella Sativa, Indian Costus, Quinine, Anise Seed,…

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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Prophylactic treatment of Glycyrrhiza glabra mitigates COVID-19 pathology through inhibition of pro-inflammatory cytokines in the hamster model and NETosis

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Neeraj Kumar Sethiya

Robbert Boudewijns

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