# Protective Effects Induced by a Hydroalcoholic _Allium sativum_ Extract in Isolated Mouse Heart
## 3 Results and Discussion
### Toxicological and Pharmacological Studies
The inhibitory effects induced by the garlic hydroalcoholic extract on 8-iso-PGF\({}_{2\alpha}\) levels could be related to its phenolic acid and flavonoid content, with particular regard to the levels of protocatechic acid and gallic acid [41; 51; 52; 53; 54]. In this context, polyphenol compounds were found to be able to exert cardioprotective effects through the inhibition of oxidative stress and inflammation [55; 56; 57; 58; 59]. In particular, it has been demonstrated that protocatechic acid, one of the main secondary metabolites of garlic, exhibits antioxidant and anti-inflammatory effects [60]. Moreover, it was shown to inhibit the inflammation response, platelet aggregation, and cardiomyocytesocytes apoptosis on myocardial ischemia/reperfusion injury [61]. In addition, both antihypertensive and antioxidant effects of protocatechic acid have been described in hypertensive rats [62]. Gallic acid has also been hypothesized to protect the heart through the inhibition of lipid peroxidation, due to its scavenging effects on superoxide and hydroxyl radicals [63]. Furthermore, gallic acid pretreatment inhibited the serum levels of cardiac marker enzymes, such as cardiac troponin T, which is probably related to the decrease in myocardial damage [64]. We also showed that garlic hydroalcoholic extract inhibited LPS-induced PGE\({}_{2}\) levels in heart specimens (Figure 8), supporting its cardioprotective effects. Pro-inflammatory PGE\({}_{2}\) is produced by COX-2, in both neoplastic and inflamed tissue. The expression of the inducible enzyme COX-2 is known to be stimulated by mitogenic and inflammatory stimuli, such as cytokines and LPS [65]. In turn, LPS stimulated the macrophage production of pro-inflammatory cytokines, such as TNF-\(\alpha\), IL-1\(\beta\), and IL-6, as well as mediators, including PGE\({}_{2}\)[66; 67]. The mRNA levels of TNF-\(\alpha\), IL-6, NF-\(\kappa\)B, and COX-2 were evaluated, as well. In this context, we observed that garlic hydroalcoholic extract (1, 10 and 100 \(\mu\)g/mL) was able to reduce the mRNA levels of COX-2, without affecting _TNF-\(\alpha\)_ gene expression, in mouse heart specimens challenged with LPS inflammatory stimulus (Figures 9 and 10). Figure 8: Effects of garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\mu\)g/mL) on 8-iso-PGE\({}_{2}\) levels, in mouse heart specimens. Data are reported as means \(\pm\) SEM. ANOVA, \(p<0.0001\); *** \(p<0.001\) vs. LPS group. Figure 10: Relative quantification of _TNF-a_ gene expression in mouse heart specimens treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\upmu\)g/mL), ex vivo. Data are reported as means \(\pm\) SEM. ANOVA, \(p\) < 0.05; \({}^{\star}p\) < 0.05 vs. LPS group. Figure 9: Relative quantification of _COX-2_ gene expression in mouse heart specimens treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\upmu\)g/mL), ex vivo. Data are reported as means \(\pm\) SEM. ANOVA, \(p\) < 0.05; \({}^{\star\star}p\) < 0.01; \({}^{\star}p\) < 0.05 vs. LPS group. induced IL-6 mRNA levels (Figure 11), whereas only the highest concentration (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) suppressed NF-kB mRNA induced by LPS treatment in heart specimens (Figure 12). Figure 11: Relative quantification of _IL-6_ gene expression in mouse heart specimens treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), ex vivo. Data are reported as means \(\pm\) SEM. ANOVA, \(p<0.01\); ***\(p<0.001\) vs. LPS group. Figure 12: Relative quantification of _NF-kB_ gene expression in mouse heart specimens treated with garlic hydroalcoholic extract (GHE) (1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), ex vivo. Data are reported as means \(\pm\) SEM. ANOVA, \(p<0.01\); ***\(p<0.001\); ***\(p<0.001\) vs. LPS group. Accordingly, [PERSON] et al.