Depression is one of the most common mental illnesses that may remarkably affect one’s quality of life. Knowledge of the pathophysiology will assist in smart treatment of depression . In the clinical setting, elements of RAS are known as blood pressure regulators. In addition to its "classical" role, RAS exists locally in various tissues, including the brain . Binding of Ang II to its main receptors, AT1 and AT2, is accountable for most of RAS function in the brain [15, 36]. The potential harmful effects of Ang II in the brain are often due to its binding to the AT1 receptor. RAS plays an important role in balancing the inflammatory and anti-inflammatory pathways through its two receptors in the brain [37, 38]. Recent studies have also shown that increased RAS activity increases the risk of depression by increasing AT1 receptor response to angiotensin II [37, 39, 40]. This stimulation leads to the release of inflammatory mediators through various intracellular mechanisms in the CNS and ultimately causes oxidative stress [7, 41].
Increased levels of ROS and intracellular calcium in Ang II-stimulated Neuro-2A cells has been repeatedly reported [15, 21, 42, 43]. On the other hand, there is some evidence that ROS can also induce Ang-II activity. The mitogen-activated protein kinase (MAPK), an important regulator of signal transduction pathways, is independently activated by Ang-II and ROS. Increased MAPK leads to further ROS formation through NADPH oxidase activation. As a result of MAPK activation, nuclear transcriptional factors are activated that modulate the expression of tyrosine AT1 receptors. Indeed, Ang-II and ROS enhance each other's activities in a variety of ways. Finally, all of the pathways generally lead to an increase in the intracellular calcium concentration that causes neuronal death and an inflammatory response [15, 44-48].
It has been seen that most antihypertensive drugs improve the symptoms of depression as well as brain damage in cardiovascular patients [49-52]. The first evidence of a possible role for RAS in depression was reported following the improvement of depressive symptoms in captopril-treated hypertensive patients [53, 54]. In many experiments, captopril has a similar healing effect to imipramine on depressed rat model induced by foot shock . On the other hand, many findings show that some antidepressants can have some positive effects by inhibiting the brain RAS [55, 56].
In the present study, captopril-treated Neuro-2a cells in the presence of H2O2 showed a significant difference in viability comparing with the H2O2 and control groups. No cytotoxic effects were observed at any of the captopril concentrations. The result showed that exposure of Neuro-2a cells to H2O2 significantly decreased cell viability, and captopril can protect the cells from H2O2-induced cytotoxicity. Captopril also significantly reduced the accumulation of intracellular ROS, which may be the reason for the significant difference in cell viability compared to H2O2 -treated cells. The greatest effect was observed at a concentration of 100 μM. At all concentrations except for 10 μM, captopril significantly decreased ROS level in the H2O2-treated cells compared to the H2O2 alone but not in a dose-dependent manner. Due to the increased oxidative stress of the cells at a concentration of 50 μM, the activity of the SOD enzyme was expected to increase to prevent cell necrosis, which was consistent with our observations. However, the percentage of dead cells with high intracellular calcium in captopril-treated cells at all concentrations except 10 μM was significantly different from the H2O2-treated cells. However, cell viability has improved at this concentration of captopril. According to the improvement of cell viability, ROS generation and the percentage of dead cells with high intracellular calcium, especially in the concentration of 100 μM, the neuroprotective effects of captopril may be related to the inhibition of the RAS system in Neuro-2a cells. But at lower concentrations, the increased viability of H2O2-treated cells may be dependent on SOD activity. These results are in line with previous report that studied the neuroprotective effects of captopril in high glucose-induced toxicity in PC12 cells .
At different concentrations of losartan, we observed a good cell viability in H2O2-treated Neuro-2a cells. However, in none of the losartan concentrations there was a significant difference in SOD activity compared to the H2O2 group. Losartan along with imipramine were among the drugs that captured ROS the most compared to the H2O2-treated group. Percentage of dead cells with intracellular calcium indicated that higher concentrations of losartan was associated with death due to high intra cellular calcium and at the concentration of 1 μM it can prevent cell death due to high calcium. According to the results, we concluded that losartan at lower concentrations such as 1 μM, plays a neuroprotective role by reducing mitochondrial oxidative stress and thus reducing intracellular calcium levels. While at higher concentrations, losartan showed its neuroprotective effects by decreasing ROS activity through the inhibition of the RAS system. Various studies have shown that ARBs such as losartan have protective effects in some tissues following various injuries . Losartan has shown protective properties against the neuronal damage caused by oxidative stress [59-61]. Also, in line with the results of our study, losartan inhibits oxidative stress-induced neurotoxicity in animal models of Parkinson's disease and also in cell lines such as PC12 [57, 62].
Venlafaxine is known as a serotonin-norepinephrine reuptake inhibitor (SNRI) that is widely used in the treatment of MDD, anxiety, and neuropathy . Venlafaxine has neuroprotective effects due to its anti-inflammatory activities [64, 65]. Based on our results, the highest cell viability belonged to 100 μM of venlafaxine. The activity of the SOD enzyme in all concentrations of venlafaxine was significantly higher in H2O2-treated group. ROS levels were significantly reduced in venlafaxine-treated cells in different concentrations compared with the H2O2 group. Also, at the concentration of 50 μM, treatment with venlafaxine caused the lowest percentage of dead cells with high calcium compared to all other groups treated with other drugs and even the control group. According to the results, it appears that venlafaxine increases the activity of SOD enzyme and also prevents the accumulation of ROS and calcium and thus protects Neuro-2a cells against damage. Considering other neuronal cell lines, venlafaxine entail its neuroprotective effects in PC12 cells through an unclear mechanism . It also suppresses oxidative stress in human cerebrovascular microvascular endothelial cells (HBMECs) damaged by methylglyoxal by reducing intracellular ROS.
Imipramine, a tricyclic antidepressant (TCA) known as an effective drug in treating depressive disorders, neurodegenerative diseases, and other mental disorders . Treatment with antidepressants such as imipramine has shown to increase BDNF . Many results also confirm that imipramine reduces the expression of inflammatory cytokines and has neuroprotective and anti-inflammatory effects [70-74]. In our study, no cytotoxic effect was observed in any of the concentrations of imipramine. Imipramine and losartan showed the lowest levels of ROS at different concentrations compared to other drugs. The level of ROS in cells treated with concentrations of 40, 50, and 100 μM of imipramine was almost the same as the control group. Cell death due to incline in intracellular calcium was significantly lower in all concentrations of imipramine compared to both the control and the H2O2-treated groups. Considering the neuroprotective effects of imipramine in most studies on neurons [71, 73, 75, 76] and our results, it seems that imipramine is involved in the protection and survival of neurons by inhibiting the production and accumulation of ROS and associated downstream pathways such as MAPK/extracellular-regulated kinase (ERK) in neural stem cells (NSCs) . In SH-SY5Y cells, imipramine plays its neuroprotective effects against H2O2-induced cell death by increasing the expression of the anti-apoptotic protein, Bcl-2 . Additionally, imipramine inhibits LPS-induced toxicity in oligodendrocyte cell line, OLN-93 by reducing the levels of ROS.
In summary, the findings of the present study demonstrated that imipramine, venlafaxine, as well as captopril and losartan, reduce H2O2-induced oxidative stress in Neuro-2a cells and may exert their protective effects against neuronal cell death by inhibition RAS. Losartan and captopril at low concentrations protect cell death due to elevation of intracellular calcium.
Based on the results, we can conclude that losartan and captopril have the ability to produce neuroprotective effects. Therefore, if approved in animal models, it may be used in the future as an adjunct in psychiatric diseases such as depression.