A Novel Role of Lassbio-788 in Inhibiting Nf-&[Kappa]B Mediated Signaling in Platelet of Hypercholesterolemic Rats

Introduction Atherosclerosis is described as a chronic process closely related to inflammatory and proliferative responses of the endothelium after injury. Circulating platelets become hyperactive under hypercholesterolemic conditions, leading to the development and progression of atherosclerosis. LASSBio‐788 is a thienylacylhydrazone derivative with anti‐atherogenic properties in an animal model of hypercholesterolemia. However, the molecular mechanism underlying its antiplatelet effect remains unclear. The aim of this study was to investigate the signaling pathway involved in the antiplatelet effects of LASSBio‐788. Methods The animal protocols were approved by the Ethics Committee for Experimental Research of the Federal Fluminense University (CEPA/UFF 287/12). Adult male Wistar rats (150–200g) were randomly divided into four groups (n= 10, for each group): control group (C) and positive control (C+788) fed standard chow diet, hypercholesterolemic diet group (HC) and diet group + compound LASSBio‐788 (HC+788) fed a hypercholesterolemic diet. At 31° diet day, was performed the chronic treatment with LASSBio‐788 once daily, totalizing 15 days of treatment. The animals were euthanized by cervical dislocation and decapitated under anesthesia. Blood samples were collected from each animal and the platelets were isolated to biochemical and molecular analysis. Data were analyzed using one‐way ANOVA followed by a post‐hoc Bonferroni Multiple Comparison Test, p<0.05. Results The hypercholesterolemic diet increased serum lipids levels and reduces HDL levels in the HC group. LASSBio‐788 reduced serum lipids and increased HDL levels (p≤ 0.05). The HC group showed an increase of malondialdehyde levels in platelets (63.90 ± 5.50 nmol/mg protein) when compared with C group (17.40 ± 1.60 nmol/mg protein). Furthermore, the hypercholesterolemic diet increased TXA 2 (HC: 360.20 ± 9.18 × C: 259.30 ± 20.44 pg/1.2 × 10 8 platelets) and a decrease cAMP (HC: 85.05 ± 3.19 × C: 95.43 ± 1.49 pmol/1.2 × 10 8 ); cGMP (HC: 20.34 ± 3.41 × C: 120.00 ± 9.73 pmol/1.2 × 10 8 ) levels. LASSBio‐788 inhibited these effects promoted by diet: TXA 2 (294.50 ± 3.27 pg/1.2 × 10 8 platelets); cAMP (103.50±1.51 × 10 8 platelets); cGMP (94.56±4.42 pmol/1.2 × 10 8 platelets). NF‐kB signaling events, including PKC‐α, iNOS, P‐selectin and CD40L increased protein expression, IκB‐α degradation, eNOS, PKA and PKG decreased protein expression were observed in hypercholesterolemic rats. These signaling events were attenuated by chronic treatment with LASSBio‐788 (100μmol/kg) p<0.05. Conclusion The most important findings of this study demonstrate for the first time that LASSBio‐788 possesses potent antiplatelet activity, which may involve activation of the eNOS/NO/GC/cGMP/PKG pathway, resulting in inhibition of the NF‐κB/PLC/PKC/TXA 2 cascade, and, finally, inhibition of platelet aggregation. Our results indicate the LASSBio‐788 compound as a potential drug for the treatment of cardiovascular disorders such as atherosclerosis. Support or Funding Information CNPq, CAPES, PROPPI‐UFF, FAPERJ