**4. Discussion**

Given its long history usage in Asia, a reliable amount of clinical experience with TCM has been accumulated, forming a comprehensive and vital medical and cultural system. Chinese herbal medicine is the fundamental of TCM, which includes several medicinal herbs. The typical characteristics of TCM treatments are "multi-component, multi-channel, and multi-target", which is a requirement for treating complex diseases including depression or MDD [39].

Repeated CORT injection in rodents have been considered as a reliable animal model for evaluating chronic stress depressive disorder [27]. The TST test is one of the classic experiments used to evaluate the anti-depression pharmacology of drugs and has often been used to observe the anti-depression effect of drugs by causing behavioral despair in animals [40,41]. In the present study, compared with the control group, a significant prolonged immobility time in the TST test was observed in the CORT-treated group mice. As expected, the mice treated with PAPZ+CORT exhibited a significant decrease in immobility time as compared to the CORT group (Figure 1b). PAPZ showed minor side effects on locomotor activities at the indicated dose (Figure 1c,d). Depression is often accompanied by cognitive impairment, and especially obvious learning and memory impairment [42]. In this study, the NOR test and MWM test were used to evaluate the learning and memory ability of mice. A significant increase in the DI score for new objects and new locations was shown in the PAPZ+CORT-treated group compared with the CORT-only treated group (Figure 2a,b). From the MWM results (Figure 2c), we found that the latency period of escape increased in the model group even though there was no statistical difference, whereas treatment with PAPZ significantly decreased the latency. These results demonstrated that PAPZ slightly decreased the progression of the chronic treatment of CORT-induced mild stress-mediated depressive-like behaviors as observed by the TST, NOR, and MWM tests.

Studies have shown that various pathophysiological mechanisms are involved in depression and cognitive impairment. Synaptic dysfunction and structural damage have become the focus of research. BDNF functions as an endogenous growth factor within the central nervous system [43], playing a key role in processes such as neuronal differentiation and growth, synapse formation and synaptic plasticity, and higher cognitive functions [44,45]. BDNF has also been implicated in a number of psychiatric disorders, including schizophrenia, and the development of mood disorders such as MDD and its treatment [46]. Many experiments have shown that chronic stress can down-regulate the expression of BDNF protein in the hippocampus, and this phenomenon can be effectively reversed by antidepressant therapy [47–49]. Neto et al. [49] found that up-regulation of neurotrophic factor expression and its signaling pathway activity could be a common pathway of antidepressant action. Therefore, we speculated that PAPZ may promote neurogenesis or change neuroplasticity by increasing the level of BDNF protein, and thus play an antidepressant role in reversing hippocampal atrophy and cell damage. According to the results of the qPCR and Western blot tests, we found that the expression of hippocampal BDNF protein in the repeated CORT injection group significantly decreased when compared with the control group. Treatment with PAPZ showed significantly increased the expression of BDNF in the hippocampal tissue when compared to the CORT group (Figure 3). Therefore, we concluded that PAPZ might exert an antidepressant role by up-regulating the expression of BDNF.

The antioxidant defense system is comprised of a series of enzymatic and non-enzymatic components. SOD is a critical antioxidant enzyme that can remove oxygen free radicals produced by the body and protect tissues from damage. Maes et al. [50] revealed that the pathogenesis of MDD might primarily or secondarily be related to oxidative stress. Antidepressant drugs function by affecting the oxidative or antioxidative systems [51], and are partly connected with their effects on the immune system [52]. Many reports have shown that disturbances in SOD activity are generally dysregulated in depressed populations. For example, the SOD activity of a red blood cell was reported to be lower in MDD patients [53]. Freitas et al. [2] reported that acute restraint stress can increase SOD activity and this effect can be abrogated by treatment with agmatine; however, no changes were observed in the unstressed animals. These disagreements in the data on SOD activity could be explained by means of variations in different drugs or a different administration period.

The results of our study showed that the SOD activity of hippocampal tissue in the CORT group mice significantly decreased, indicating that the function of the oxygen free radical defense system of depressed mice was significantly reduced, and the oxygen free radicals could not be effectively removed. PAPZ administration could reverse the trend of SOD activity. The increased SOD activity was in line with a decreased MDA level (Figure 4). Therefore, we concluded that the role of PAPZ in anti-depression could be related to the involvement of SOD and MDA. The results (Figure 5) demonstrated that PAPZ significantly attenuated CORT-induced neuroexcitotoxicity in HT22 cells, which was indicated that PAPZ could protect HT22 cells in vitro. However, the complex mechanism of PAPZ are not yet completely clear, which need to be explored in further study. In future studies, we will pay more attention to the exploration of the relevant herb of PAPZ under the guidance of the theory and practice of TCM, and clarify the role of each herb and the relationship and influences among them.
