# In Vitro Evidence of Stating' Protective Role against COVID-19 Hallmarks _Comptes_ [PERSON]
## 3 Results
### Statins Counteract the Effect of Induced Inflammation  
To investigate the dynamic of statins during an inflammatory state, such as those induced by SARS-CoV-2 infection, we analyzed the expression of the key players involved in COVID-19, after LPS-induced inflammation, in vitro. In A549-hACE2 cells, as expected, LPS stimulus decreases the expression of ACE2 receptor. Pre-treatment for 48 h with ATS and PVS reverts the effect of LPS stimulus, inducing the re-expression of ACE2 receptor in a dose-dependent manner (Figure 2A,B). As previously described, prolonged treatment with statins downregulated both the full length and cleaved domain of TMPRSS2 in a dose-dependent manner. Despite LPS treatment reducing the full-length TMPRSS2, its cleaved domain results strongly increased. Interestingly, both ATS and PVS revert the LPS-induced upregulation of TMPRSS2 in the cleaved form, but also the expression of the full-length protein, compared to LPS and statins alone, with a better effect at the highest concentration (ATS 5 uM and PVS 20 uM) (Figure 2A,C). One of the first events after SARS-CoV-2 infection is the activation of NfKB pathway [34]. We then investigatedif statins regulate NfKB activation, alone or after induced inflammation. Surprisingly, in A549-hACE2, only low concentrations of ATS (1 \(\upmu\)M) and PVS (10 \(\upmu\)M) significantly inhibited NfKB phosphorylation. However, after LPS stimulus, only the high concentrations (ATS 5 \(\upmu\)M and PVS 20 \(\upmu\)M) were able to revert the LPS-induced NfKB phosphorylation (Figure 2A,D). To expand our analysis, we investigated the effects of statins in different in vitro models, expressing different levels of ACE2 receptor, using the concentrations found to be most efficient in the A549-hACE2 cells. In CaCO2 cells expressing normal levels of ACE2 receptor, after 24 h of treatment, PVS 20 \(\upmu\)M and slightly ATS 5 \(\upmu\)M induce protein expression of ACE2 receptor, but the effect is persistent after 48 h of treatment only with PVS. However, both the compounds seem to be able to revert the LPS-mediated reduction of ACE2 receptor, increasing its expression (Figure 3A,B). Surprisingly, neither ATS nor PVS used alone, at the highest concentrations, were able to reduce the levels of phosphorylated NfKB. However, as in A549-hACE2 cells, at the same concentrations ATS (5 \(\upmu\)M) and PVS (20 \(\upmu\)M) strongly revert the LPS- induced NfKB activation (Figure 3A,C). Of note, in CaCO2 cells, we unable to detect the TMPRSS2 cleaved band, corresponding to the cleaved serine protease domain. In line with other reports, it seems that CaCO2 cells express mostly the full-length protein and very low levels of autoclavage [35]. In this study, in CaCO2 cells both ATS and PVS were able to reduce TMPRSS2 at full length only after 48 h of treatment, but they are ineffective in reducing the protease expression in cells treated with a proinflammatory stimulus such as LPS (Figure 3A,D). Similar to what was observed in CaCO2, in MRC5 cells only PVS significantly increases the protein expression of ACE2 receptor, while ATS seemed to decrease it. Moreover, both of the compounds  
Figure 1: Representative Western blot and densitometric analyses of (**A,B**) ACE2 receptor and (**A,C**) TMPRSS2 (total and cleaved forms) expression in A549-hACE2 cells treated with ATS or PVS as indicated. GAPDH was used as loading control. Data are expressed as mean \(\pm\) SD of at least three independent experiments. * \(p<0.05\),** \(p<0.01\),*** \(p<0.005\) vs. control. produced a significant up-regulation of ACE2 receptor, when combined with the LPS inflammatory stimulus. The expression of TMPRSS2 full length protein in MRC5 cells were not affected by the treatment with the two statins. However, the levels of the cleaved protein were almost completely abrogated after treatment with PVS, alone or with LPS (Supplementary Figure S2).