Effects of Flunarizine and Lutein on Atezolizumab-Associated Ovarian Alterations in Rats

Immune checkpoint inhibitors (ICIs), including atezolizumab, enhance antitumor immune responses by interfering with inhibitory immune checkpoint signaling. Atezolizumab targets programmed death-ligand 1 (PD-L1), thereby disrupting its interaction with programmed death-1 (PD-1). Although ICIs have improved cancer treatment outcomes, their potential associations with ovarian alterations remain insufficiently understood. This study aimed to compare ovarian biochemical, hormonal, histopathological, and immunofluorescence outcomes following the co-administration of flunarizine or lutein with atezolizumab in rats. Twenty-four female Wistar rats were randomly assigned to four groups (n = 6 per group): healthy control (HG), atezolizumab (ATZ), flunarizine plus atezolizumab (FUAZ), and lutein plus atezolizumab (LTAZ). Ovarian oxidative stress markers (MDA, tGSH, SOD, and CAT), inflammatory cytokines (TNF-α and IL-1β), serum anti-Müllerian hormone (AMH) and prolactin levels, histopathological alterations, and immunofluorescence intensities of COX-1, COX-2, IL-1β, and IL-6 were evaluated. Compared with HG, the ATZ group exhibited increased MDA, TNF-α, and IL-1β levels and decreased tGSH, SOD, CAT, and serum AMH levels (p < 0.05). Histopathological examination revealed more pronounced follicular degeneration, parenchymal edema, and hyperemia in the ATZ group, whereas these alterations were milder in the FUAZ group and moderate in the LTAZ group. Immunofluorescence analysis demonstrated reduced COX-1 and increased COX-2, IL-1β, and IL-6 intensities in the ATZ group compared with HG. Both co-administration groups showed higher COX-1 and lower COX-2, IL-1β, and IL-6 intensities than the ATZ group. Several biochemical, histopathological, and immunofluorescence outcomes in the FUAZ group were closer to HG values than those in the LTAZ group. Serum prolactin levels differed significantly only between the ATZ and FUAZ groups in pairwise comparisons. Atezolizumab administration was associated with ovarian oxidative, inflammatory, and histopathological alterations. Co-administration of flunarizine or lutein was associated with differences in several evaluated parameters, with more favorable findings observed in the FUAZ group at the selected doses. However, the absence of appropriate antibody controls and adjunct-only groups limits causal attribution and precludes conclusions regarding independent protective efficacy.