Lactoferrin Induces Erythropoietin Synthesis via HIF Signaling and Improves Cognitive Functions in Rat Offspring Subjected to Prenatal Hypoxia

Introduction: Lactoferrin (LF) is a cationic transferrin found in milk, some other exocrine secretions, and in neutrophils. LF has bactericidal, antianemic, immunomodulatory, antitumor, and antiphlogistic effects. Almost 90% of LF in human milk is iron-free (apo-LF) and has an extreme affinity toward Fe(III), which makes it an efficient iron chelator. We showed previously that, due to its chelating activity, it stabilizes in vivo hypoxia-inducible factor (HIF)-1α and HIF-2α, the redox-sensitive multitargeted transcription factors. In the absence of an iron chelator, this effect is not observed at least at normoxia, since HIF-1α is continuously modified by iron-sensitive hydroxylases and undergoes ubiquitination and proteasomal degradation. In our experiments, various tissues of animals treated with recombinant human LF (rhLF) responded by expressing HIF-1α target genes. As a result, such proteins as erythropoietin (EPO), ceruloplasmin, etc., were synthesized in noticeable amounts.[1] [2] [3] These results allowed us to suggest that LF is an efficient natural antihypoxic factor. Various harmful factors during pregnancy, including hypoxia, cause postnatal motor and cognitive dysfunctions as shown in our previous studies.[4] [5] In this study, we analyzed the effects of LF on the cognitive function of the offspring of pregnant rats subjected to hypoxia.

Materials and Methods: rhLF purified from the milk of transgenic goats was obtained from the Belorussian State University and Scientific Practical Centre of Animal Breeding of the Belorussian National Academy of Sciences. The product is officially branded “CAPRABEL.” About 90% of such LF was iron-free (apo-LF). Pregnant Wistar rats (200 g) were subjected to hypoxia (7% O₂, 3 hours) on the 14th day of gestation in a special chamber as described in Zhuravin et al.[4] Half the pregnant rats were injected i.p. with 10 mg of CAPRABEL each on the 9, 12, 13, and 15th days of gestation or during nurturing (every day starting from P0 after delivery up to P15) and were killed 3 hours after the last injection. Organ homogenates of some females and suckling pups were analyzed by Western blotting or ELISA with anti-HIFs or anti-EPO. Another group of pups was allowed to grow and their short-term working memory was tested in a two-level radial maze. The “novel object recognition” (NOR) test was also used to assay both short-term and long-term memories.

Results: Western blotting detected HIF-1α, HIF-2α, and EPO in the brain, liver, heart, spleen, and placenta, but not in the embryos of rat dams subjected to prenatal hypoxia. HIFs or EPO were not detected in the organs of control pregnant rats. In young (P22) or adult (P90) rats born from the apo-LF-treated mothers subjected to hypoxia, a significant memory improvement was registered in comparison with the offspring of untreated rats, as judged by the radial maze and NOR tests. Injections of CAPRABEL to hypoxia-treated lactating dams caused the presence of human apo-LF in their milk during 4 to 24 hours after the treatment[6] and resulted in induction of HIF-1α, HIF-2α and EPO expression in the pup brain, liver, and spleen. CAPRABEL injections to hypoxia-treated lactating dams also resulted in significant improvement of short- and long-term memories of their offspring in the NOR test on P22 and P90.

Conclusion: Apo-LF applied to pregnant or lactating dams protects the developing brain both during prenatal and postnatal ontogeneses against the harmful effects of prenatal hypoxia, and this effect is most likely due to the ability of apo-LF to induce EPO biosynthesis in the brain and other tissues via a HIF-signaling mechanism. This observation is in line with our recent results showing that apo-LF significantly mitigated neurological symptoms in rotenone-treated rats (a model of Parkinson’s disease) and rescued rats with experimental allergic encephalomyelitis (a model of multiple sclerosis).[6] An i.p. injection of apo-LF to mice 1 hour after they were subjected to the occlusion of the medial cerebral artery significantly diminished the necrosis area in the brain.[6]

Taken together, the results of this study and previous data suggest that endogenous LF in the breast milk or as a pharmaceutical in the substitute formulas can have therapeutic value providing a neuroprotective effect.

Keywords: lactoferrin; erythropoietin; hypoxia; HIF; cognitive functions; neural lesions

Conflict of Interest: None declared.

Funding: This work received support from the Russian State budget (AAAA-A18–118012290373–7).

No conflict of interest has been declared by the author(s).

• References

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