Protective effects of flunarizine and ATP against pembrolizumab-associated gastric injury in rats

Abstract Pembrolizumab, an immune checkpoint inhibitor targeting pro-grammed cell death protein-1 (PD-1), is widely used in cancer therapy. However, its potential gastrotoxic effects and underlying molecular mechanisms remain poorly understood. Flunarizine has antioxidant, calcium channel-blocking, and mitochondria-protective properties, whereas ATP plays a central role in maintaining cellular energy metabolism. Therefore, the present study investigated the possible protective effects of flunarizine, ATP, and their combination against pembrolizumab-associated gastric injury in rats. To our knowledge, the combined protective effects of flunarizine and ATP against pembrolizumab-associated gastric injury have not previously been investigated. Thirty male Wistar rats were randomly divided into five groups: healthy control (HG), pembrolizumab (PZB), flunarizine + pembrolizumab (FPZB), ATP + pembrolizumab (APZB), and flunarizine + ATP + pembrolizumab (FAPZB). Pembrolizumab (5 mg/kg, i.p.) was administered daily for one week. Flunarizine (5 mg/kg, oral gavage), ATP (5 mg/kg, i.p.), or their combination was administered 1 h before pembrolizumab. Gastric tissues were evaluated macroscopically, biochemically, histopathologically, and by double immunofluorescence analysis. Gastric tissue ATP, malondialdehyde (MDA), and total glutathione (tGSH) levels, together with cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression, were assessed. Pembrolizumab administration was associated with marked gastric mucosal injury characterized by ulcerative lesions, hyperemia, edema, decreased gastric tissue ATP and tGSH levels, increased MDA levels, increased COX-2 expression, and suppressed COX-1 expression compared with the healthy group. Flunarizine and ATP treatments significantly attenuated these alterations. Notably, ATP levels were significantly higher in the APZB group than in the FPZB group, whereas combined flunarizine and ATP treatment restored tissue ATP levels to values comparable to those of the healthy group. Among the treatment groups, FAPZB showed the most prominent gastroprotective effect by markedly improving biochemical, histopathological, and immunofluorescence findings. The findings indicate that pembrolizumab-associated gastric injury is accompanied by reduced gastric tissue ATP levels, oxidative stress, and inflammatory and structural alterations. Flunarizine and ATP attenuated these changes, while their combined administration produced the most pronounced overall protective effect and restored tissue ATP levels to values comparable to those of the healthy group. These findings support an association between preservation of tissue energy status and gastric mucosal protection; however, the underlying Ca²⁺- and mitochondrial-dependent mechanisms require further investigation.