With the increasing number of stress factors in today’s society, depression has become one of the main causes of disability in the world, and the recurrence rate is high [1, 2]. Drug treatment often brings a series of adverse reactions. The search for safe and effective new drugs has always been a hot topic. The reserpine model is a commonly used and effective depression model which can be used for behavioral tests such as forced swimming tests, tail suspension tests, and open field tests . With the merits of simple operation, animal-friendly, and high efficiency, this model played an important role in the evaluation and development of antidepressant drugs . This experiment analyzed the chemical composition of CREOs and explored its role in the depression model induced by reserpine.
Weight loss is an important indicator of depression performance , and the immobility of forced swimming test and tail suspension test is a behavioral desperate performance, which can be used to evaluate the depressive behavior of mice . Our experiments show that the weight of mice treated with reserpine has decreased significantly. What’s more, the immobility time of FST and TST in the experiment has been significantly prolonged. These results indicated that the reserpine depression model is successful and effective, which is in line with previous studies [28, 29, 38]. Fluoxetine is a clinically recognized and effective antidepressant which has a significant antidepressant effect according to previous studies . In this experiment, it is used as a positive control to compare and verify the antidepressant effect of tangerine. By analyzing the body weight changes and behavioral tests of each group, it can be seen that CREOs treatment can prevent weight loss and reduce the immobility time of FST and TST in reserpine-treated mice. Among them, the therapeutic effect of CREOs-H optimal. In general, CREOs can at least improve depression-like behaviors in mouse depression models induced by reserpine.
Nissl bodies are composed of rough endoplasmic reticulum and free ribosomes interspersed in it. Basophilic particles are presented and the shape and number of Nissl bodies are different in different neurons . They are related to the synthesis of structural proteins needed to renew organelles, the enzymes needed to synthesize neurotransmitters, and the neuromodulators of peptides. Previous research has shown that neuron damage could cause the sensitivity of Nissl body to decrease, dissolve or disappear . Therefore, the shape and number of Nissl body are often used to identify neurons and their pathophysiological changes. Study has found that the total number of neurons in the hippocampus of patients with depression was reduced by 20–35% compared with the control group , and injection of reserpine could cause neuronal damage . Our research shows that the treatment of CREOs can reverse the nerve damage caused by reserpine treatment. The number of neurons in the CREOs treatment group is close to that of the control group, indicating that CREOs treatment cause less damage to neurons. In addition, the results demonstrate that CREOs treatment can protect the morphology of brain tissue neurons, and have relatively good treatment safety.
In depression-related patients and some animal experiments, it has been usually observed that symptoms of depression were accompanied by disorders of glucocorticoid secretion. Therefore, the hypothalamic-pituitary-adrenal (HPA) axis dysfunction has been studied by evaluating GR and MR levels in the past work . Hypothalamic-pituitary-adrenal axis (HPAA) disorder is an important finding in the pathophysiology of depression. This disorder is thought to be due to the central glucocorticoid receptor (GR) level and the chronic glucocorticoid (GC) release or changes in function, leading to the receptor positively or negatively regulate the expression of glucocorticoid-responsive genes . Excessive stress stimulus may activate GR through cortisol, so that GR stimulates the hippocampus to issue negative feedback commands, resulting in HPA axis imbalance, showing excitement . As shown before, antidepressants could improve the GR-mediated inhibition of corticosteroids by increasing the expression of GR on the HPA axis, thereby reducing cortisol levels in different regions . The results of this study found that reserpine caused a significant reduction in GR expression in the brain, indicating the negative feedback regulation of the HPA axis was impaired and the HPA axis was hyperactive, which is in line with previous studies . In addition, our research shows that treatment with CREOs and fluoxetine could increase the expression of GR, indicating that CREOs had antidepressant effects by regulating neuroendocrine. The HPA axis may be a mechanism of CREOs, but the difference in our data is not significant and need to be further explored.
The monoamine hypothesis was considered by many researchers as the mechanism of action of depressive drugs. The hypothesis believes that depression is caused by insufficient activity of monoaminergic neurons . Previous study has shown that patients with major depressive episodes had lower serotonin transporter binding potential in the midbrain and amygdala, compared with non-depressed individuals . Although the complexity of the emotional state cannot be attributed to the imbalance of a single neurotransmitter, it is recognized that 5-HT is significantly involved in depression [4, 5, 50], and 5HT-1A receptors are considered to treat mental illness, Especially a potential target for depression [50, 51].Studies have found that the antidepressant effect of lemon oil whose main component is limonene was closely related to the 5-HT energy pathway, especially through the 5-HT 1A receptor pathway . In this study, immunohistochemical methods were used to detect the content of 5-HT 1A in different areas of the brain. It is found that fluoxetine and CREOs can increase the content of 5-HT 1A in the brains of reserpine-induced depression mice, but there are differences in dosage and location differences. Insignificant effects are shown in the treatment of low-concentration essential oils and in the treatment of hippocampus and hypothalamus. Studies have found that excessive activity of the HPA axis might damage hippocampal monoaminergic neurons, resulting in a decrease in monoamines , and 5-HT1A receptor agonist suppressed stress-induced activation of the HPA axis as measured . It is speculated that CREOs may increase 5-HT1A in the cerebral cortex through the action of the HPA axis, thereby increasing neuronal activity.
BDNF is a neurotrophic factor that can stimulate neurogenesis and regulate synaptic plasticity . Widespread in the brain, especially in the hippocampus and cerebral cortex, BDNF could promote the survival of dopaminergic, GABAergic and serotonergic neurons . It is also involved in regulating the activities of HPA axis [55, 56]. Antidepressant drugs can increase the expression of BDNF in the brain of mice, such as selective serotonin reuptake inhibitors (SSRI) and norepinephrine reuptake inhibitors (NARI) . Of course, there are also antidepressants that have different effects on the mRNA and protein levels of BDNF . The regulating effect of these drugs on depression might be related to neurotrophic activity and benefited from long-term chronic regulation . Study has shown that the loss of BDNF in the hippocampus could induce neuronal apoptosis and ultimately led to depression . This experimental data supports the study. WB staining shows that the expression of BDNF protein in reserpine-depression mice is decreased. Further study on BDNF mRNA by RT-qPCR is found that BDNF mRNA level expression and protein level expression have an opposite expression trend, and there is a negative feedback relationship. The treatment of CREOs and fluoxetine can significantly increase the BDNF protein and reduce the expression of BDNF mRNA, which further confirms this negative feedback effect. Interestingly, the changes in the 5HT-1A protein in the brain homogenate detected by WB have different and opposite results from the changes in the 5HT-1A protein in different regions of the brain detected by immunohistochemistry. This may be related to the location of 5HT-1A receptors, which have opposite effects on depression regulation. For example, the post-synaptic 5HT-1A receptor is thought to cause anxiety and depression effects. On the contrary, it is believed that the activation of 5-HT1 autoreceptor will inhibit the activity of serotonergic neurons and release serotonin, thereby reducing the marginal zone and producing anti-anxiety and anti-depressant effects . Similarly, the use of RT-qPCR to detect the mRNA expression of 5HT-1A also showed a different trend from the expression of 5HT-1A protein. Therefore, we speculate that there is a brain-derived neurotrophin-neurotransmitter interaction in at least a certain area of the brain, which makes BDNF and 5HT-1A feedback regulation in protein and mRNA. The disorder of this mechanism can be regulated by CREOs, while fluoxetine is invalid for it.
Hyperactive HPA axis is one of the main abnormal phenomena found in depression, and the treatment of depression promotes the production of BDNF [61, 62]. Therefore, the decreased expression of BDNF in reserpine animals may indicate abnormal changes in HPA axis and monoaminergic circuit function. Previous studies also found that limonene also restored CUMS-induced depressive behavior, HPA axis hyperactivity, and decreased levels of monoamine neurotransmitters by down-regulating hippocampal BDNF and its signaling pathways . Our experimental data also meets this conclusion, which provides a reference for the antidepressant mechanism of CREOs, indicating that CREOs may improve the expression of GR, BDNF and 5HT-1A through the connection of the HPA axis.