COVID-19, caused by SARS-CoV-2, has resulted in millions of deaths globally. Severe cases involve a "cytokine storm," characterized by excessive proinflammatory cytokines like IL-6 and TNF-α. SARS-CoV-2 enters cells via ACE2 and TMPRSS2. While vaccines exist, effective treatments remain limited. Cannabis and its cannabinoids, including CBD, have shown potential anti-inflammatory and anti-cancer properties, although the mechanisms are not fully understood. Previous research indicated that cannabis extracts can downregulate ACE2, IL-6, and TNF-α. This study aims to investigate the mechanism by which high-CBD cannabis extracts inhibit ACE2 and TMPRSS2 expression, and attenuate inflammatory mediator expression in human normal cell lines, focusing on the potential anti-SARS-CoV-2 host entry and anti-cytokine release syndrome properties.

The literature review section highlights the urgent need for effective COVID-19 treatments given the severity of the disease and the limitations of current therapies. It emphasizes the long history of medicinal cannabis use and the various cannabinoids present in *Cannabis sativa*, including CBD and Δ-THC. The 'entourage effect' – the synergistic interaction of cannabinoids and terpenes – is also mentioned. The anti-inflammatory and anti-cancer potential of cannabinoids is noted, but the underlying mechanisms remain unclear. Prior research from the authors on the effects of cannabis extracts on ACE2, IL-6, and TNF-α in 3D tissue models is referenced, setting the stage for the current study focusing on cellular mechanisms.

The study utilized human lung fibroblast WI-38 cells and human normal foreskin fibroblasts (BJ-5ta) as model systems. Cells were treated with CBD, various high-CBD cannabis extracts (#1, #5, #7, #129, #169), and individual components (CBN, THC, and terpenes) at various concentrations. Western blot analysis was used to assess protein expression of ACE2, TMPRSS2, COX2, IL-6, IL-8, AKT, and p65. qRT-PCR measured mRNA levels of ACE2, TMPRSS2, IL-6, IL-8, and COX2, as well as miRNA levels (miR-200c-3p, let-7a-5p). Luciferase assays were performed to confirm miRNA targeting of ACE2 and TMPRSS2. The AKT pathway was inhibited using MK2206, and AKT1 was knocked down using CRISPR/Cas9. MTT assays assessed the effect of extracts on cell proliferation. Statistical analysis using two-tailed Student's t-test determined significance.

CBD and selected high-CBD cannabis extracts (#1, #5, #7, #129) inhibited ACE2 and TMPRSS2 expression, both transcriptionally and post-transcriptionally, via the AKT pathway. miR-200c-3p and let-7a-5p were identified as key miRNAs mediating this effect; their induction was blocked by AKT inhibition. Extracts #1, #5, #7, and #169 suppressed COX2 expression and attenuated TNFα/IFNγ-induced IL-6 and IL-8 expression, also through AKT activation. The most abundant components in extracts #1 and #7 individually and in combination modulated the expression of COX2, IL-6, and IL-8. CBD and terpene PTWT2.2 showed additive effects in downregulating ACE2 and TMPRSS2. The extracts did not affect the proliferation of normal fibroblasts.

The findings demonstrate a novel mechanism of action for high-CBD cannabis extracts in potentially combating COVID-19. The AKT pathway's role in regulating both ACE2/TMPRSS2 expression and the inflammatory response is a key discovery. The identification of miR-200c-3p and let-7a-5p as mediators further strengthens the understanding of this process. The synergistic effects of CBD and certain terpenes, as well as the differential effects of individual extract components highlight the complexity of the 'entourage effect'. The study provides substantial preclinical evidence for the potential of these extracts as anti-COVID-19 agents, focusing on both viral entry inhibition and cytokine storm mitigation. Further research is needed to explore the clinical significance and potential side effects.

This study reveals that high-CBD/low-THC cannabis extracts effectively suppress SARS-CoV-2 host entry proteins (ACE2 and TMPRSS2) and inflammatory mediators (COX2, IL-6, and IL-8) via AKT pathway activation, both transcriptionally and post-transcriptionally. These findings strongly suggest the potential of these extracts as anti-COVID-19 agents and warrant further investigation through clinical trials. Future research should focus on identifying the optimal ratios of individual components for clinical formulations and conducting thorough clinical studies to assess efficacy and safety.

The study was conducted using in vitro cell culture models which may not fully reflect the complexity of in vivo responses. The study focused on specific extracts and their components and may not generalize to all CBD-dominant cannabis extracts. The mechanisms underlying the interaction between certain components and the AKT pathway require further clarification. While the study shows no impact on normal fibroblast proliferation, in-vivo studies are needed to assess potential toxicity or side effects in a whole organism.