Age-related differences in hypoxia-associated genes and cytokine profile in male Wistar rats

Abstract

Hypoxia tolerance of the organism depends on many factors, including age. High newborn organisms tolerance and high level of oxidative stress throughout aging were demonstrated by many studies. However, there is lack of investigations reflecting the expression of key hypoxia-inducible factor HIF in different age organisms in correlation to levels of pro-inflammatory and anti-inflammatory cytokines. Liver is a sensitive to hypoxia organ, and is an important organ in providing an acute reaction to infections - it synthesizes acute inflammation phase proteins, in particular, C-reactive protein. The aim of study was to determine relationship between age-related tolerance to hypoxia and HIF-1 and PHD2 (prolyl hydroxylase domain protein) expression levels in the liver and the production of cytokines in the spleen in newborn, prepubertal and adult Wistar rats. Newborn rats are characterized by high mRNA Hif-1α expression level in the liver, accompanied by a low content of HIF-1 protein and high level of PHD2. The growth in HIF-1α protein level throughout age is accompanied by the growth of pro-inflammatory cytokines level. Prepubertal animals are the least hypoxia resistant and their HIF-1α mRNA expression level was higher than in adult animals. The PHD2 activity in prepubertal animals was significantly reduced in comparison to newborn rats, and the HIF-1α protein level did not change. Further studies require the identification of additional mechanisms, determining the regulation of the HIF-1α level in prepubertal animals.

Keywords: Age-related differences; HIF-1; Hypoxia tolerance; Newborn; PHD2; Prepubertal age.

Figure 1

Evaluation of gasping time for newborn, prepubertal and adult Wistar rats in the decompression chamber (Me; 25%–75%). In all groups there were 6 observations. p – statistically significant differences, Kruskal–Wallis method with Dunn's post-hoc test. Newborn and adult rats are more tolerant to hypoxia than prepubertal animals. Not less important is the high variability of the parameter in adult rats.

Figure 2

The expression level of mRNA Hif-1α in the liver of newborn, prepubertal and adult Wistar rats (Me; 25%–75%). In all groups there were 6 observations. p – statistically significant differences, Kruskal–Wallis method with Dunn's post-hoc test. The Hif-1α expression level in the liver of newborn and prepubertal rats was significantly higher in comparison to adult animals, that means this indicator decreased throughout aging.

Figure 3

(a) – Relative protein levels of HIF-1α and PHD2 in the liver of newborn, prepubertal and adult Wistar rats, normalized on GAPDH level, representative Western blot images are shown. Full-length blots are presented in Supplementary Figure S1 (a-c). (b, c) – The expression level of HIF-1α (b) and PHD2 (c) proteins in the liver of newborn, prepubertal and adult Wistar rats (Me; 25%–75%). In all groups there were 6 observations. p – statistically significant differences, Kruskal–Wallis method with Dunn's post-hoc test. HIF-1α protein level was minimal in newborn and prepubertal rats and increased towards puberty. It should be stated that there is a high variability in the HIF-1α protein level in adult rats. PHD2 protein level in the liver of newborn animals was significantly higher than in prepubertal and adult rats.

Figure 4

The corticosterone concentration in blood serum of newborn, prepubertal and adult Wistar rats (Me; 25%–75%). In all groups there were 6 observations. p – statistically significant differences, Kruskal–Wallis method with Dunn's post-hoc test. The blood serum corticosterone concentration was the highest in prepubertal and the lowest in newborn rats.

Figure 5

Summary results of the study on the differences in the gasping time in the decompression chamber and the features of the expression of HIF-1α and PHD2 in newborn, prepubertal and adult Wistar rats. Newborn: high mRNA Hif-1α expression level, accompanied by a low content of HIF-1 protein and high PHD2 level. Prepubertal: the level of Hif-1α mRNA expression was higher than in adult animals. PHD2 activity in prepubertal animals was significantly reduced in comparison with newborn rats, but the HIF-1α protein level was not changed. Prepubertal animals are the least tolerant to hypoxia and are characterized by the shortest gasping time at the critical altitude. Adult: the relatively low level of Hif-1α mRNA expression in comparison to newborn and prepubertal rats during high levels of HIF-1α protein is in accordance with the low level of PHD2 activity. High HIF-1α protein level in adult rats is observed likely due to high ROS levels.