# In Vitro Evidence of Stating' Protective Role against COVID-19 Hallmarks _Comptes_ [PERSON]
## 3 Results
### Hudrophilic and lipophilic Staths Differentially Affect ACE2 Receptor Localization in Lipid Rafts  
Our data demonstrated that the statin-mediated upregulation of ACE2 receptor is accompanied by the reduction of many cytokines and chemokines strongly involved in COVID-19, in agreement with their anti-inflammatory effect. The key used by SARS-CoV-2 to enter through the host cell membrane is ACE2 receptor, and its statin-mediated upregulation has raised considerable concern about the potential use of these compounds in COVID-19 infection. Aiming to dissect the dynamic of ACE2 receptor after treatment with statins, we isolated plasma membrane-derived lipid rafts, after treatment for 24 h or 48 h with ATS 5 \(\upmu\)M or PVS 20 \(\upmu\)M. We previously showed that in A549-hACE2 cells, at  
Figure 4: IL6 secretion in culture medium from A549-hACE2 cells treated with ATS and PVS alone or after stimulation with LPS, measured by ELISA (**A**). Effect of ATS and PVS on LPS-induced cytokines secretion in culture media from A549-hACE2 cells. Histograms represent densitometric analyses of the membrane spots on the antibody array (**B–E**). Data are expressed as mean \(\pm\) SD of at least three independent experiments. * \(p\) < 0.05, **_p_ < 0.01, ***_p_ < 0.005 vs. control. $_p_ < 0.005, $_p_ < 0.001, $_p_ < 0.005 vs. LPS. these concentrations, both ATS and PVS significantly increase ACE2 receptor expression (Figure 1). Nevertheless, as shown in Figure 5, the general increase of ACE2 receptor induced by statins after 24 h and 48 h of treatment is not verified in isolated lipid rafts. In overexpressing A549-hACE2 cells, ATS 5 uM does not induce expression of ACE2 receptor in the lipid rafts fraction. Rather, after 24 h of treatment we noticed a slight decrease in its lipid rafts localization, which coincides with a slight reduction of Flotillin-1 in fractionated lipid rafts. Instead, after 24 h of PVS 20 uM, the amount of ACE2 receptor also increases in the lipid rafts compartment, parallel with the increase of Flotillin-1 amount, but tends to decrease after 48 h (Figure 5A). Plasma membrane-derived lipid rafts have been also isolated from CaCO2 cells, where the lipophilic ATS produces the same effect observed in overexpressing cells, with the reduction of both ACE2 receptor and Flotillin-1 amounts in the lipid rafts fraction after 24 h of treatment. The reduction of Flotillin-1 also appears persistent and more evident after 48 h. However, accordingly to what is observed in A549-hACE2 cells, in CaCO2 the hydrophilic PVS increases in time-dependent manner the amount of ACE2 receptor in lipid rafts, accompanied with a slight increase in Flotillin-1 (Figure 5B). These data confirm the role of some statins in affecting lipid rafts composition but, on the other hand, prompt us to hypothesize that lipophilicity could affect this ability, since only ATS is able to produce lipid rafts perturbation and to affect ACE2 receptor localization within them. Confocal microscopy analysis (Figures 6 and 7) substantially confirmed the increase in total ACE2 receptor expression in both cell lines analyzed, showing that its localization remains mainly intra-cytoplasm. The expression of total Flotillin-1 is coherent with the amount observed in the non-lipid rafts fraction showed in Figure 5.  
Figure 5: Representative Western blot analyses of ACE2 receptor and Flotillin-1 amount in lipid rafts and the complementary non-lipid rafts fractions, isolated from (**A**) A549-hACE2 and (**B**) CaCO2 cells, treated with ATS or PVS as indicated. Histograms on the right represent relative densitometric analyses of ACE2 receptor and Flotillin-1 in lipid raft fraction. GAPDH was used as loading control for the non-lipid rafts fraction. Data are expressed as mean \(\pm\) SD of at least three independent experiments. * \(p<0.05\), **\(p<0.01\) vs. control. Figure 6: Representative confocal microscopy images of Flotillin-1 (red) and ACE2 receptor (green) in A549-hACE2 cells treated with ATS or PVS at indicated doses and time points.