Preparation of polyclonal antibody targeting Optic atrophy 1 (OPA1) of chicken and the effect of naringenin on OPA1 expression in the liver of broilers under heat stress

Abstract The mitochondrial protein optic atrophy 1 (OPA1) belongs to the dynamin-related GTPase family and plays a crucial role in maintaining mitochondrial morphology and function. Heat stress disrupts mitochondrial function, thereby impairing cellular viability and causing damage to multiple organs. Naringenin, a flavanone, has been reported to exert protective effects against various stressors. This study aimed to investigate whether naringenin is associated with the reversal of heat stress-induced downregulation of OPA1 in chicken liver and to explore its potential correlation with improved biochemical markers of liver injury and oxidative stress. However, direct studies examining the relationship between naringenin and heat stress remain limited, and the association between OPA1 and heat stress has been scarcely investigated. In this study,144 Ningdu Yellow chickens (28 days old) were randomly divided into four groups ( n = 36 per group) Control, Narimgenin, Heat stress (HS)and HS+Naringenin. Given the lack of commercially available antibodies specific to chicken OPA1, we first developed a rabbit anti-chicken OPA1 polyclonal antibody as a detection tool. To generate this antibody, a recombinant expression plasmid, pET32a-OPA1, was constructed using prokaryotic expression technology to express the exogenous gene. The recombinant target protein was produced through an induced expression method. Subsequently, rabbit anti-chicken OPA1 serum was generated by immunizing rabbits with purified and concentrated recombinant OPA1 protein as the antigen. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the antibody titer exceeded 1:51,200; antibody specificity was further validated by Western blotting showing a single band at the expected molecular weight.Western blotting combined with immunofluorescence analysis revealed that heat stress significantly reduced OPA1 protein levels in liver tissue ( p < 0.01), demonstrating a significant association between OPA1 and the heat stress response. Furthermore, naringenin treatment up-regulated OPA1 mRNA expression ( p < 0.05), fluorescence intensity ( p < 0.05), and protein levels( p < 0.01). Serum biochemical analysis showed that heat stress significantly increased AST and ALT levels, while naringenin treatment significantly reduced them (AST: p < 0.01) (ALT: p < 0.05). Additionally, hepatic MDA levels, a marker of oxidative stress, were significantly elevated in heat-stressed chickens and reduced by naringenin treatment ( p < 0.01). These findings provide preliminary evidence for the regulatory effects of naringenin on OPA1 expression under heat stress conditions and suggest a potential protective role that warrants further investigation into mitochondrial functional parameters.