This study proved that the treatment with hPH produced significant decreases in the despair behavior in the FST compared to the control group. The hPH treated groups showed markedly reduced expression of the NADPH-d in the PVN compared to the control group. In addition, the repeated treatment of hPH showed an increasing trend of GPx levels in the hippocampus when compared to the control group. These results suggested that the effects of the repeated treatment of hPH are mediated by the regulation of a repeated stress-induced depressive behavior and the oxidative stress system in the brain.
In a pilot study, we compared the effects of various routes of administration on the immobility in FST using intravenous, intramuscular or subcutaneous injections of hPH (0.002, 0.02, 0.2, 1 and 2 ml/rat). It was found that the most effective route to reduce immobility was intravenous injection with ranges of 0.2 to 2 ml (data not shown). Therefore, we used intravenous injections of hPH at the doses of 0.02, 0.2 and 1 mL/rat in this study.
Chronic stress induces oxidative stress via hyper-activation of the hypothalamic pituitary adrenal (HPA) axis, leading to oversecretion of corticosterone [
31]. Moreover, the oversecretion of corticosterone induces depressive-like behaviors such as anxiety and despair. The present study showed that the repeated immobilization stress increased the despair behavior in the FST which was reduced by treatment with hPH or fluoxetine. Recently, some studies reported that repeated restraint stress induced not only depressive behavior but also oxidative stress in the brain region [
32‐
37]. The brain is more susceptible to oxidative stress due to its relatively high consumption of oxygen, high iron content, high fatty acid peroxidation, and low antioxidant capacity compared to other organs [
32‐
34,
36]. Nitric oxide (NO) is the major oxidative stress marker for cellular component’s damage [
35,
37‐
40] and repeated stress increases the NO levels in the brain [
9,
41,
42]. Our result also showed that repeatedly stressed rats have significantly increased levels of NADPH-d expression in the PVN. NADPH oxidase has been studied as a major source of reactive oxygen species (ROS) generation in mammalian cells. Sanchez et al. reported that the swim stress enhances the NADPH-d histochemical staining in the PVN of the hypothalamus [
43]. In mice, the repeated stress mediates depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and the treatment of NADPH oxidase inhibitor apocynin during the stress or post-stress period reduced stress induced responses [
8]. In the present study, hPH decreased the expression of NADPH-d in the PVN, suggesting that the inhibition of NADPH oxidase may provide beneficial anti-stress effects. Among important changes in the brain of stress-induced animal models, accumulation of oxidative stress has been noted [
8,
44‐
46]. GPx, an antioxidant enzyme, is a defense system against oxidative stress [
47‐
49], and many antioxidants and free radical scavenging enzyme systems exist in the cell to protect it against the damaging effects of free radicals produced as a part of normal cell respiration and other cellular processes [
50]. However, repeated stress affected the level of the antioxidant components as superoxide dismutase, GPx, catalase in the central and peripheral nervous systems [
51‐
54]. Consistent with previous studies, our results showed that the repeatedly stressed rats showed a decreasing trend of GPx levels in the brain regions. Moreover, the repeated treatment of hPH or fluoxetine showed a trend toward increased GPx levels in the hippocampus of the repeatedly stressed rats. Other studies also reported that fluoxetine, one of antidepressants, reduced the amounts of free oxygen radicals [
55], catalase levels, and lipid peroxidation [
56]. Some studies showed that the human placenta extract and their components have neuro-protective effects by regulating antioxidant actions [
57‐
60].
Taken together, repeated treatment of hPH has an antioxidant effect and HPA axis modulatory activity that could be the important therapeutic strategy for repeated stress-induced behavioral and biochemical changes. These results suggest that hPH may play an active role in the treatment of stress-related responses via regulation of oxidative stress.