Background: Benign prostatic hyperplasia (BPH) is the most common urogenital condition in aging males, while inflammation and tissue proliferation constitute the main pathophysiological factors. The adverse effects of currently available BPH medications limit patient compliance. We tested the protective effect of aescin against the development of BPH in rats. Methods: A total of 18 male Wistar rats were divided into 3 groups: control (sesame oil 1 mL/kg, s.c.); BPH (testosterone oenanthate 3 mg/kg, s.c., in sesame oil), and BPH-aescin rats (testosterone oenanthate 3 mg/kg, s.c. + aescin 10 mg/kg/day, p.o.). All treatments continued for 4 weeks. Serum and prostatic samples were harvested for biochemical and histopathological examination. Results: Induction of BPH by testosterone increased the prostate weight and prostate weight index, serum testosterone, prostate expression of inflammatory (IL-1β, TNF-α, and COX-2), and proliferative markers (PCNA and TGF-β1). Concurrent treatment with aescin decreased the testosterone-induced increase in prostatic IL-1β, TNF-α, and COX-2 expression by 47.9%, 71.2%, and 64.4%, respectively. Moreover, aescin reduced the prostatic proliferation markers TGF-β1 and PCNA by 58.3% and 71.9%, respectively, and normalized the prostate weight. Conclusion: The results of this study showed, for the first time, that aescin protected against the development of experimental BPH in rats via its anti-inflammatory and antiproliferative effects. These findings warrant further studies to clinically repurpose aescin in the management of BPH.

The 8th-helix (H8) and carboxyl terminal tail (CT) of GPCRs are crucial for interactions with intracellular signaling molecules and regulatory proteins. We investigated how H8 and CT residues influence signaling in PAR4, a tethered-ligand activated GPCR. Our analysis revealed that PAR4 activation by thrombin or AYPGKF-NH2 stimulates β-arrestin-1/-2 recruitment and activates multiple Gα proteins (Gαq, Gα11, Gαz, Gα15, Gα12, Gα13, Gαi1-3, GαoA/B). Mutation of the H8 sequence (R ³⁵² AGLFQRS ³⁵⁹ ) significantly reduced G protein activation and β-arrestin recruitment, with residues Leu ³⁵⁵ -Glu ³⁵⁷ being particularly important. Mutations of specific lysine residues in H8 and CT also impaired signaling. Additionally, we identified a crucial TM7-H8 interaction and found that CT phosphorylation sites regulate β-arrestin recruitment. Finally, using AlphaFold3 we predicted interactions between PAR4, β-arrestins, and G proteins, revealing novel receptor regulatory sites in the intracellular loops, transmembrane domains, H8 and CT.