Characterization of Keap1a and Keap1b in grass carp (Ctenopharyngodon idella) and Keap1-Nrf2 pathway-related transcriptional responses under heat stress.

The Keap1-Nrf2 pathway is a critical pathway for cellular antioxidant defense, with Keap1 acting as its central regulator. Unlike mammals, which possess only a single Keap1 gene, fish have two keap1 paralogs, keap1a and keap1b, which may have functionally diverged. In this study, we cloned and characterized keap1a and keap1b genes in grass carp (Ctenopharyngodon idella) and investigated their responses to heat stress through both in vivo and in vitro experiments. Our results showed that keap1a was downregulated and nrf2 was upregulated in both the spleen and liver during heat stress. In contrast, keap1b exhibited tissue-specific transcriptional patterns, with downregulation in the spleen at 6 and 24 h but upregulation in the liver at 24 and 48 h. Functional studies further revealed that knockdown of either keap1a or keap1b significantly enhanced cell viability of grass carp hepatocytes under heat stress. Interestingly, keap1b knockdown increased the mRNA levels of nrf2, gsh-px, and ho-1, whereas keap1a knockdown reduced the transcriptional responses of several Keap1-Nrf2 pathway-related genes, suggesting differential roles of Keap1a and Keap1b in the transcriptional response to heat stress. In summary, this study supports potential functional divergence between grass carp Keap1a and Keap1b during heat stress and suggests that they may have distinct regulatory roles, providing new insights into the molecular responses of fish to heat stress.