Riboflavin deficiency (RD) induces liver damage, abnormal embryonic development, and high mortality. We hypothesized that the phenotype could be rescued by inhibiting ER stress. The objectives of the present study were to investigate the underlying molecular mechanisms of RD-induced embryonic defects using in vitro and in vivo models. Primary duck embryonic hepatocytes were treated with an ER stress inhibitor (4-PBA) or transfected with CHOP siRNA, and cultured in RD medium and riboflavin-sufficient (CON) medium for 8 days. Laying ducks (n = 20 cages/diet, 1 bird/cage) were fed an RD diet or CON diet for 14 wk, and the eggs were collected for hatching. At day 7 of incubation, the fertilized RD eggs were injected with or without 4-PBA into the yolk. RD decreased cell number and cell viability compared to the CON group, induced oxidative stress and apoptosis in primary duck embryonic hepatocytes. However, after being treated with an ER stress inhibitor (4-PBA) or transfected with CHOP siRNA, the apoptosis rate in RD hepatocytes decreased by 60.6 % and 86.1 %, respectively, being equal to the CON. These results indicated that RD-induced hepatocyte apoptosis is mediated by ER stress and the CHOP pathway. In vivo, RD embryos showed low hatchability, abnormal development, liver damage, ER stress, and apoptosis compared to the CON group. However, 4-PBA administration, as a model of ER stress inhibition, substantially restored embryonic development and alleviated liver damage in the RD group, including ER stress and apoptosis. Notably, hatchability in the RD group increased from 21.7 % to 72.7 % after 4-PBA treatment, though it remained less than the CON group (87.7 %). These results implicated ER stress-CHOP-apoptosis pathway as molecular mechanisms underlying RD-induced abnormal embryonic development and death, this target with potential for therapy or intervention.
Keywords: Apoptosis; CHOP; Deficiency; ER stress; Embryonic development; Riboflavin.
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