Antidepressant Mechanism of Kaixinsan and Its Active Compounds Based on Upregulation of Antioxidant Thioredoxin

Objectives Kaixinsan (KXS), a traditional Chinese medicine formula, has been demonstrated to be effective in the treatment of depression. The present study applied a network pharmacology approach to dig out the new targets and mechanism of action of KXS and the active compounds in the treatment of depression. Methods A network pharmacology approach based on public databases including ADME (absorption, distribution, metabolism, and excretion) evaluation, targets prediction, construction of networks, and molecule docking was used and validated the predicted new antioxidant targets and mechanisms in vitro. Based on an in vitro experiment, we verified the AKT1/Nrf2 pathway related to the thioredoxin (Trx) antioxidant mechanism. Results The present study sorted 31 pharmacologically active components (kaempferol, ginsenoside rh2, ginsenoside rh4, stigmasterol, etc.) through the ADME algorithm from KXS. 136 potential molecular targets (AKT1, TNF, IL-1b, JUN, ESR1, NOS3, etc.) were predicted, of which there were 69 targets clearly related to depression. By compound-depression targets (C-DTs) network constructed, and protein-protein interaction networks (PPI) and KEGG pathway enrichment analyzed, we identified active compounds mediating depression-related targets to exert synergism on the predictive AKT1/Nrf2 pathway related to thioredoxin (Trx) antioxidant mechanism and other inflammation-related signaling pathways as well as neurotransmitter related signaling pathways. In the H2O2 induced SH-SY5Y cell damage model, this showed kaempferol and ginsenoside rh2 could enhance the activity of the Trx system by upregulation of AKT1 to activate Nrf2 in vitro. Conclusions Taken together, by comprehensive systems pharmacology approach analysis, we found that KXS and its active compounds might exhibit antioxidant effects by stimulating the AKT1/Nrf2 pathway in the treatment of depression, which might shed new light on innovative therapeutic tactics for the new aspects for depression in traditional Chinese medicine in future studies.


Introduction
Depression has huge social risks of high incidence, disability, and suicide. At present, the main clinical antidepressants are selective 5-HT reuptake inhibitors, tricyclic or tetracyclic antidepressants, and monoamine oxidase inhibitors. Chronic treatment with antidepressants is only 60 to 70 percent effective with significant side effects [1].
With a long history of thousands of years, traditional Chinese medicine (TCM) plays important roles in the treatment of mental illness. Natural products, such as plants, animals, and minerals, are typically prescribed in TCM, in which herbal or botanical products are most widely used. Depression can be traced back to the traditional Chinese medicine book "Huang Di Nei Jing" more than 2000 years ago, which belongs to the category of "depressive syndrome-YuZheng" in TCM. TCM shows its advantage for containing a variety of chemical components, which targets through various channels, and significantly alleviating complications such as insomnia and anxiety while treating depression.
In fact, the serotonergic, dopaminergic, and noradrenergic systems are deeply involved in the mechanisms of TCMs' antidepression process. Among them, inhibition against monoamine oxidase reaction and increasing synaptic availability of monoamines are the main factors in TCMs' monoamine regulation mechanism. For example, Xiaoyaosan could improve the serotonergic metabolism by regulating the expression levels of tryptophan hydrogenase 2 (TPH2) and indoleamine 2,3-dioxygenase 1 (IDO1), thus exerting an antidepressant effect [2]. e antidepressant-like effect was also the main function in the Banxia-Houpu decoction by attenuating abnormalities in the serotonergic and dopaminergic system functions in animal models of depression [3]. e antidepressant effect of Kaixinsan (KXS) could be explained by modulation of the noradrenergic system and the 5-hydroxytryptamine (5-HT) system in mice as well [4]. Other than the neurotransmitter mechanism, depression is triggered by other complicated pathogenetic factors such as oxidative stress. Chronic SNRI\SSRI treatment in effective antidepressant doses was shown to protect against chronic stress-increased oxidative cellular or DNA damage and inhibited reactive oxygen species (ROS) and lipid peroxidation levels in the hippocampus [5], and this mechanism might rely on the AKT /Nrf2/HO-1 pathway [6].
ese results indicated that inhibition of oxidative stress played a vital role in the pathogenesis of depression. erefore, there might be a novel way to evaluate the therapeutic effects and mechanisms of TCM and its active compounds.
KXS is a classic ancient prescription, first recorded the "Qianjin Yaofang" written by Sun Simiao in the Tang Dynasty. It is composed of Ginseng Radix (Panax ginseng C. A. Mey.), Polygalae Radix (Polygala tenuifolia Wild.), Poria (Poria cocos (Schw.) Wolf ), and Acori Tatarinowii Rhizoma (Acorus tatarinowii Schott), with different ratio. As our previous clinical trials showed that KXS (3 : 3 : 2 : 2 ratios) ameliorated clinical symptoms of patients with minor and modest depression [7]. Pharmacological studies indicated that KXS significantly reduced depressive-like behavior on the forced swim test, the open-field test, in the CUMS animal models. It mainly focused on the mechanism of neurotransmitter regulation, or alleviating the hypothalamic-pituitary-adrenal (HPA) axis dysfunctions, lightening the impairment of neuroplasticity, and inflammatory regulation [8]. Although some studies have confirmed that some of the active compounds from the formula plants like ginsenoside rg1, ginsenoside rk1, polygalaxanthone III, essential oil, and polysaccharides from Ginseng Radix [9,10], Polygalae Radix [11], Acori Tatarinowii Rhizoma [12] and Poria [13] have an obvious antioxidant effect. However, the rather complicated multicomponent compounds of KXS and its antioxidant molecular mechanisms are yet to be revealed.
Herein, we screened the potential active compounds of the four herbs contained in KXS with the computational ADME and selected depression-related targets, then combined the KEGG enrichment analysis and PPI were performed illustrate its possible mechanisms, especially those involved with oxidant stress, and finally performed to reveal the potential compounds in KXS on its specific antioxidant pathway. e work scheme of this research is shown in Figure 1.

Protein-Protein Interaction Networks of Common Targets between KXS and Depression.
e Disgenet (https://www. disgenet.org) [17] database selected the depression-related targets. e depression search terms were "mental depression" and "depression." e same targets of depression were obtained after KXS treatment with Venny 2.1.0 online software. e Uniprot (https://www.uniprot.org/) [18] database converted KXS targets related to depression into international common gene names, and Uniprot IDs were obtained. Using String 11.0 (https://string-db.org/), the obtained common targets of KXS in the treatment of depression were visualized, and the protein-protein interaction networks (PPI) were drawn.

KEGG Pathway Analysis.
e KEGG database (https:// www.kegg.jp/) [19] conducted a KEGG pathway analysis on the common targets of KXS in the treatment of depression. P < 0.05 was set, the species was limited to homo sapiens, the top 10 targets related to KEGG pathway enrichment were listed, and the antioxidant pathways were found.

Molecular Docking.
e Autodock Vina software calculated the free energy of molecular docking between the selected key targets and the corresponding compounds of KXS. e Pymol software was used for mapping, and the results of molecular docking were visualized to verify the network pharmacological analysis.

Cell Culture and Drug Treatment.
e human neuroblastoma SH-SY5Y cells were selected to construct the model of oxidant stress damage in vitro. e cell-complete medium was DMEM supplemented with 10% fetal bovine serum. SH-SY5Y cell suspension with a concentration of 5 × 10 5 /mL and a volume of 100 μL in the logarithmic growth phase was added to the 96-well plate. After screening the H 2 O 2 induced damage concentration, it is set on 100 μmol/L to induced injury model. en, the active compounds kaempferol and ginsenoside rh2 with different dosages were added to the cell for 24 h. Four groups were set up: untreated control group, H 2 O 2 induced injury model group, kaempferol treatment group, and ginsenoside rh2 treatment group.

Cell Viability.
e cell viability was measured with the Cell Counting Kit-8 (CCK8) Assay (Beyotime). e SH-SY5Y cells (1 × 10 5 ) were cultured in an incubator with 5% CO 2 at 37°C for 24 h under different treatments in the 96well plate. Totally 10 μL CCK8 solution was added into each well, and the cells were cultured for another 1 h. e absorbance was finally determined at 450 nm with a microplate reader.

Western Blot.
e SH-SY5Y cells were inoculated in the six-well plate after four ways of treatment, and 100 μL RIPA lysis buffer was inoculated in each well. e cells were blown evenly and shaken evenly in a 4°C shaker for 30 min.

Activity of ioredoxin Reductase (TrxR) Determination.
e activity of TrxR was measured by the Solarbio thioredoxin reductase activity Kit. We used 2-vinylpyridine to inhibit the original reduced glutathione in each sample. e activity of TrxR was calculated by measuring the absorbance at 412 nm. Evidence-Based Complementary and Alternative Medicine 2.9. Statistical Analysis. Statistical data were analyzed by GraphPad Prism 5 and compared with a one-way analysis of variance. e bilateral 95% confidence interval was used for all tests, and a value of P < 0.05 represented statistical significance.  (Table 1).

Protein-Protein Interaction Networks of Compound-Depression Targets (C-DTs).
Disgenet database screened depression targets and 1479 depression-related targets were obtained. According to the Venn diagram, there were 69 related targets of depression and KXS (Figure 3(a)). According to STRING 11.0 protein interactions, the network of 69 targets related to the treatment of depression by KXS was extensively connected with AKT1 as the core. A node represents one protein, and the degree value determines the size of the node. PPI also proved that AKT1 was much more likely to be a key target in this network. Among the targets, there were antioxidant-related targets, such as AKT1 (RACalpha serine/ threonine-protein kinase, degree � 43), NOS3 (Nitric-oxide synthase, endothelial, degree � 28), Nrf2 (Nuclear factor erythroid 2-related factor 2, degree � 18)) and KEAP1 (Kelch-like ECH-associated protein 1, degree � 18) with a high degree, which initially proved that antioxidant active the important role in KXS antidepressive treatment (Figure 3(b)).

Functional and Pathway Enrichment Analysis and Antioxidant Pathway Identification.
e KEGG pathway analysis of the related targets of KXS in the treatment of depression was performed by the KEGG database, and the tissue origin was limited to homo sapiens. e result showed that target genes were mapped into 227 pathways. e top 10 significantly enriched (adjusted P < 0.05) signaling pathways were preserved for further analysis. Multiple signaling pathways were involved in the treatment of depression with KXS, including pathways in cancer and metabolic pathways, and neurotransmitter-related pathways (Figure 4(a)). Based on the results of pathway analysis, besides the first two high-degree pathways, which were related to caner and the prime central nervous system function, it was found that the third pathway was closely related to the fluid shear stress and atherosclerosis pathway which included AKT1/Nrf2 pathways. Further analysis found that AKT1/Nrf2 pathways play an important role in antioxidant function, which could achieve antioxidant effects by influencing the activation of ioredoxin (Trx) (Figure 4(b)).

Molecular Docking Validation of Selected Compound-Target Interactions.
e molecular docking free energy of a ligand was associated with the number and strength of its binding forces with its receptor. rough molecular docking analysis, antioxidant targets as probes for validation of the compound-Target interactions of the formula were screened. At last, we used ligands and binding sites for molecular docking to verify whether the compound and target have good binding activity. e results showed that ginsenoside rh2 and AKT1, kaempferol, and AKT1 had larger free energy (Table 2). e molecular docking images of ginsenoside rh2 and AKT1, kaempferol, and AKT1 showed a high degree of fitting (Figure 5). According to the results, we speculated that AKT1 might be the key target of kaempferol and ginsenoside rh2, which is also the hub core gene in the PPI network. erefore, we predicted kaempferol and ginsenoside rh2 as potential active compounds of KXS involved in its antioxidant mechanism.

Experimental Validation of Active Compounds in KXS Protected Oxidative Stress Induced Damage in SY5Y Cells by
Increased the Antioxidant TrxR Activity. In Figure 6, both kaempferol and ginsenoside rh2 significantly inhibited 100 μmol/L H 2 O 2 induced SY5Y cell damage, at 25 μmol/L and 40 μmol/L, respectively. At the same time, H 2 O 2 (100 μmol/L) in 24 h induced a significant increase of TrxR activity, compared with the control group, and then decreased at 72 h, which might be a process of depletion. Kaempferol and ginsenoside rh2 kept the activity of TrxR from 24 h to 72 h, compared with H 2 O 2 induced damage group, indicating they might keep the Trx protein system maintained for stronger antioxidant capacity for a longer time. And the antioxidant effect of kaempferol was significantly better than that of ginsenoside rh2.

Keampferol and Ginsenoside rh2 Exerted Antioxidant
Functions rough AKT1/Nrf2 Pathwaysz. According to the retrieval of Trx protein-related pathways in the KEGG database, AKT1 and Nrf2 were the upstream proteins in the antioxidant pathway of Trx. Here, the results indicated that kaempferol and ginsenoside rh2 could increase the expression of Trx protein. Meanwhile, these two monomer compounds could also increase the expression of AKT1 and Nrf2, which boosted the targets in the AKT1/Nrf2/Trx pathways cascade working together. Similar to Trx activity, kaempferol works better than that of ginsenoside rh2 in the AKT1 /Nrf2/Trx pathways (Figure 7).

Discussion
According to our results, by systems pharmacology approach which contained ADME screening, targets prediction, network analysis, and pathway screening, we verified the "multicompound, multitarget, and multipathway" properties of the KXS formula. We identified depressionassociated targets of KXS compounds, among them AKT1 was identified as core antidepressive target of KXS. More specifically with its antioxidant mechanism, we found that KXS and its anti-oxidant monomer compounds might work on AKT1/Nrf2 pathways to activate the Trx antioxidant system. According to the results, the advantages of KXS for treating depression mainly concentrate on the following aspects.
Firstly, the active compounds of the KXS formula are large in number, which might greatly increase the multitarget effect and reduce the possibility of side effects and drug resistance. KXS takes flavonoids, saponins, oligosaccharides, polysaccharides, and volatile oil as the main active compounds [21], and its antidepressant pharmacology and compound studies have made certain progress [22,23]. By network pharmacology analysis, kaempferol and ginsenoside rh2 were recognized as key compounds of KXS.

Evidence-Based Complementary and Alternative Medicine
Ginsenoside rh2 belongs to compounds with a tetracyclic skeleton and kaempferol belongs to structural analogs of catecholamines. Kaempferol is a kind of flavonoid, which can inhibit oxidative stress and cell apoptosis. It has been shown to have protective effects on depression models. It exerts antidepressive effects, which might be mediated by enhanced antioxidant abilities and anti-inflammation effects in the prefrontal cortex of CSDS mice [24]. Ginsenoside rh2 is a secondary ginsenoside produced by the decomposition of some proto-ginsenosides by heat, resulting in the degradation of the sugar chain on their ligands. It was confirmed that ginsenoside rh2 can significantly down-regulate the levels of TNF-α and IL-6 in the serum of depressed mice, and up-regulate the activity of superoxide dismutase (SOD) in the hippocampus [25]. ese references suggest the potential of these two compounds in antioxidant studies. Secondly, many studies had shown that the mechanism of KXS in the treatment of depression was to improve the activity of neurotransmitters such as 5-HT [26] and acetylcholine [27], repair the secretion decline of neurotrophic factors such as BDNF [28] and NGF [29] or their related signaling pathway damage, reduce the level of central nervous inflammatory factors such as inhibiting the increase of IL-1β, IL-6, and TNF-α [30], and increase the activity of superoxide dismutase to reduce lipid peroxidation, protecting hippocampal nerve cells. Further review of the literature showed that KXS, as a traditional Chinese medicine prescription for the treatment of emotional diseases, was also effective in the treatment of Alzheimer's disease(AD) in addition to depression. KXS could reduce the deposition of amyloid β-protein (Aβ) [31], improve the plasticity of neurons [32], inhibit the activity of acetylcholinesterase, and significantly improve mitochondrial function by reducing ROS levels and increasing mitochondrial membrane potential [33].
ese studies suggest that the antioxidant mechanism of KXS plays an important role in the treatment       of emotional diseases, including depression. However, most studies on the antioxidant mechanism are relatively superficial and lack of in-depth explanation. So, we mainly explored the antioxidation-related mechanisms by KEGG pathway enrichment analysis. We found that antioxidantrelated targets AKT1, NOS3, Nrf2, and KEAP1 have high research potential and KXS could activate the AKT/Nrf2 pathway to up-regulate the activity of several antioxidant systems, such as glutathione (GSH) system [34], reactive oxygen species (ROS) [35], heme oxygenase(HO) system [36], and Trx system. ese antioxidant mechanisms may play an important role in the treatment of depression. irdly, most studies on the antioxidation of antidepressants focused on the GSH system, ROS, and HO systems. e mechanism of antioxidants was mainly on Trx antioxidant system, relatively less studied. e Trx system is a small molecule protein system associated with redox action in the body. is system is composed of Trx, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH). It can reduce the cysteine residues of oxidized Trx and further acts as an electron donor for ribonucleotide reduction, maintaining its antioxidant capacity [37], so that it is an important participant in antioxidant free radicals, antiapoptosis, and promoting cell growth in cells. Several reports have shown that Trx has an obvious antioxidant effect [38,39], and the serum content of Trx is decreased in patients with depression. As the preclinical systematic review study found, KXS, most treated for Alzheimer's Disease and Depression, has an effect on antioxidant activity by decreased ROS and MDA levels via increased GSH and SOD levels. Here, in a combined analysis of the KXS antidepressive related targets identifying and targets pathway, we illustrated the potential AKT/Nrf2/Trx pathway as a novel antioxidant mechanism of KXS therapy. Furthermore, found that kaempferol and ginsenoside rh2 as effective compounds of KXS protected H 2 O 2 oxidative damage of SH-SY5Y cells in vitro, and instantly keeping enhanced the activity of Trx by increasing its upstream proteins AKT1 and Nrf2. While, the antioxidant effect of kaempferol is better than that of ginsenoside rh2, which makes kaempferol become a valuable active compound with new targets for treating depression. Of course, there are some deficiencies in this study. For example, the components and effective targets of KXS after screening by ADME are relatively few, and only in vitro model studies have been carried out. ere is still a lot of work to be made up in the following studies.
In conclusion, for the first time, we identified the AKT/ Nrf2 pathway and targets of KXS compounds using an integrative systems pharmacology approach. Furthermore, we validated the mechanisms by which KXS and its active compound ameliorated depression as shown in Figure 8.