A Modified Chinese Herbal Decoction (Kai-Xin-San) Promotes NGF-Induced Neuronal Differentiation in PC12 Cells via Up-Regulating Trk A Signaling

Kai-Xin-San (KXS), a Chinese herbal decoction, has been applied to medical care of depression for thousands of years. It is composed of two functional paired-herbs: Ginseng Radix et Rhizoma (GR)-Polygalae Radix (PR) and Acori Tatarinowii Rhizoma (ATR)-Poria (PO). The compatibility of the paired-herbs has been frequently changed to meet the criteria of syndrome differentiation and treatment variation. Currently, a modified KXS (namely KXS2012) was prepared by optimizing the combinations of GR-PR and ATR-PO: the new herbal formula was shown to be very effective in animal studies. However, the cellular mechanism of KXS2012 against depression has not been fully investigated. Here, the study on KXS2012-induced neuronal differentiation in cultured PC12 cells was analyzed. In PC12 cultures, single application of KXS2012 showed no effect on the neuronal differentiation, but which showed robust effects in potentiating nerve growth factor (NGF)-induced neurite outgrowth and neurofilament expression. The potentiating effect of KXS2012 was mediated through NGF receptor, tropomyosin receptor kinase (Trk) A: because the receptor expression and activity was markedly up-regulated in the presence of KXS2012, and the potentiating effect was blocked by k252a, an inhibitor of Trk A. Our current results in cell cultures fully support the therapeutic efficacy of KXS2012 against depression.


INTRODUCTION
Depression, namely major depressive disorder, is a serious mental illness characterized by constant feeling of low mood, low self-esteem, and loss of interest or pleasure (Barlow and Durand, 2011;Ferrari and Villa, 2016). The major pathological alteration of depression includes neuron reduction, disorder in neurotransmitter system and loss of neurotrophic factors; these changes are more robust in hippocampus and cortex (Krishnan and Nestler, 2008;Masi and Brovedani, 2011). The dysfunction in neurotransmitter system has been widely recognized and considered as one of the most severe pathogenesis. However, anti-depressants targeted on regulating neurotransmitter levels are not able to exert an effect to all the patients, indicating the complex pathogenesis during depression (Pehrson and Sanchez, 2014;Duman and Aghajanian, 2015). In the brain of depressed patients, nerve reduction resulted from retarded neurogenesis deposits that depression is largely caused by an impairment of the brain's ability to maintain neurogenesis. The disorder is reversible when neurogenesis is improved. During neurogenesis, neuronal differentiation having the neurite outgrowth is of great importance for brain function and low mood regulation. Thus, the induction of neuronal differentiation predicts a novel and effective therapeutic target for development of anti-depressants (Qin et al., 2008).
Kai-Xin-San (KXS) from Beiji Qianjin Yaofang <Thousand Formulae for Emergency> by  in China has been applied to medical care of depression for thousands of years (Sun, 1997;Yasunori, 2011). KXS composes two functional paired-herbs, i.e., Ginseng Radix et Rhizoma (GR; root and rhizome of Panax ginseng C. A. Mey.; Araliaceae family)-Polygalae Radix (PR; root of Polygala tenuifolia Wild.; Polygalaceae family) and Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott; Acoraceae family)-Poria [PO; sclerotium of Poria cocos (Schw.) Wolf; Polyporaceae family]. The make-up of the paired-herbs contributes to antidepressant functions of KXS. The administration of KXS alleviated depressive symptoms in animal studies with the mechanism of restoring the levels of neurotransmitters and neurotrophic factors in the brain (Dang et al., 2009;Zhu et al., 2012), as well as in cultured astrocytes and neurons (Zhu et al., 2013;Hu et al., 2014). However, the combination of GR-PR and ATR-PO has been frequently changed to meet the criteria of syndrome differentiation and treatment variation, which hinders the development of anti-depressant. Thus, KXS 2012 (GR-PR: ATR-PO = 1:5) was prepared by optimizing the compatibility of GR-PR and ATR-PO (Yan et al., 2015). This new formulation was able to significantly alleviate depression-like symptoms and to regulate neurotransmitter and neurotrophic factor levels in animal study . However, the cellular mechanism of KXS 2012 against depression has not been fully illustrated.
Here, we evaluated the function of KXS 2012 against depression via promoting neuronal differentiation, an important stage of neurogenesis. Among different cell models for neuronal differentiation, cultured PC12 cell is usually selected for detection of neuronal differentiation in responding to various stimuli, e.g., NGF (Lortie et al., 2005;Marín-Vicente et al., 2011). The status of differentiated PC12 cells was determined morphological through measuring neurite outgrowth and biochemically through analyzing the expression of neurofilaments, i.e., neurofilament is the major structural components of differentiated neurons (Schimmelpfeng et al., 2004;Xu et al., 2012). In neurons, NGF activates its receptor, Trk A, to promote neuronal differentiation. Thus, the Trk A signaling was studied to predict the potential cellular mechanism against depression. Our results could accelerate the development of new therapy for anti-depression.

Preparation of Herbal Decoction
The plant materials, purchased from Qinping Market in Guangzhou China, were morphologically authenticated by one of the authors, Dr. Tina T. Dong. The corresponding voucher specimens were deposited in Center for Chinese Medicine of The Hong Kong University of Science and Technology. The herbs were tested to be qualified according to the requirements of Chinese Pharmacopeia (2015 Edition) and Hong Kong Materia Medica Standards.
According to Yi Xin Fang (Yasunori, 2011), GR-PR and ATR-PO were prepared according to the weight ratio of 1:1 and 1:2, respectively. In preparing the herbal extracts, 20 g of herb mixture was boiled in 160 mL of water for 2 h and extracted twice. The extracts were combined, dried under vacuum and stored at −80 • C. KXS 2012 was obtained by mixing the water extracts of GR-PR and ATR-PO together in 1:5 weight ratio. The quality control of herbal extracts was described in previous study (Yan et al., 2015). The extracts were solubilized in dimethylsulfoxide (DMSO) to give stock solution at a series of concentration from 15 to 50 mg/mL and stored at −20 • C.

Cell Culture
PC12 cells, originated from rat adrenal medulla, were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 6% fetal bovine serum (FBS) and horse serum (HS), 100 units/mL penicillin and 100 µg/mL streptomycin in a humidified CO 2 (7.5%) incubator at 37 • C. Fresh medium was applied every other day. All culture reagents were purchased from Life Technologies (Grand Island, NY).

Herbal Treatment
Cultured PC12 cells, after serum starvation for 3 h in DMEM having 1% FBS, HS, penicillin, and streptomycin, were treated with KXS 2012 for 48 h for western blotting, or 24 h for realtime quantitative PCR. The cell viability assay was performed to determine a safe concentration range (0-100 µg/mL) of each extract, at which all extracts did not induce cell proliferation or death (Yan et al., 2015). In the co-treatment, cultured PC12 cells were treated with NGF (1.5 ng/mL) and KXS 2012 (15 µg/mL) for 24 h for western blotting assay. In the pretreatment, PC12 cells were treated with herbal extract for 48 h and washed by 1x PBS twice before NGF (1.5 ng/mL) application for 24 h. In phosphorylation study, the cultures were starved for 5 h in DMEM before NGF (1.5 ng/mL) or herbal application.

Polymerase Chain Reaction
Total RNA was isolated from cell cultures by RNAzol RT reagent (Molecular Research Center, Cincinnati, OH) according to the manufacture's instruction. The amounts of RNAs were detected by UV absorbance at 260 nm. The total RNA was used to do the reverse transcription with moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies), according to the protocol provided by the manufacturer. Real-time PCR was performed by using FastStart SYBR Green Master (Roche, Indianapolis, IN), according to the manufacturer's instruction. The SYBR green signal was detected by Mx3000P TM muitiplex quantitative PCR machine (BD Biosciences Clontech, San Jose, CA).

Neurite Outgrowth Assay
A light microscope (Diagnostic Instruments, Sterling Heights, MI) equipped with a phase-contrast condenser, 10x objective lens and a digital camera (Diagnostic Instruments) was used to capture the images with the manual setting. For analyzing the number and length of neurite, ∼100 cells were counted from at least 10 randomly chosen visual fields for each culture. Using the photoshop software, the cells were then analyzed for the number and length of neurite. The cells were scored as differentiated if one or more neuritis was longer than diameter of the cell body, and they were classified to different groups according to the length of neurite that it possessed, i.e., <15, 15-30, and >30 µm (Xu et al., 2012). The number of neurite per cell was counting only those neurite that was longer than cell body.

Statistical Analysis
All data were analyzed using one-way ANOVA followed by the Students t-test. Statistical significance were classed as * P < 0.05; * * P < 0.01; * * * P < 0.001.

KXS 2012 Promotes NGF-Induced Neuronal Differentiation
The cellular study of KXS 2012 on PC12 cells was employed to investigate the function of herbal extracts in neuronal differentiation. The neuronal differentiation of PC12 cells could be determined morphologically in measuring the length of neurite: NGF induced the neurite outgrowth in a dose-dependent manner (Supplementary Figure 1). Three mammalian neurofilament subunits, NF68 (∼68 kDa), NF160 (∼160 kDa), and NF200 (∼200 kDa), form hetero-dimers in making the structural domain of neurite (Schimmelpfeng et al., 2004;Xiong et al., 2016). Application of NGF in cultured PC12 cells also induced the expressions of neurofilaments in dosedependent manners (Supplementary Figure 2). In cultured PC 12 cells, application of KXS 2012 caused up-regulation of neurofilaments, NF68, NF160, and NF200, in dose-dependent manners (Figure 2A). The treatment of KXS 2012 at 25 µg/mL slightly induced neurite outgrowth in PC12 cells. As compared to the control group, the effect possessed no significant difference neither on the length of neurite nor the number of neurite ( Figure 2B). Thus, the herbal extract was not able to promote neuronal differentiation as single treatment.
In depressed mammalian brain, the level of NGF is reduced, and NGF-induced neuronal differentiation is down-regulated. Thus, the effect of KXS 2012 in the presence of low level of NGF in neuronal differentiation was determined here. KXS 2012 at 15 µg/mL, a concentration just barely showed induction of neurofilament expressions, was applied together with a small amount of NGF onto cultured PC12 cells by the methods of preand co-treatments. A suitable concentration of NGF was selected: Frontiers in Cell and Developmental Biology | www.frontiersin.org this concentration should have no or little effect on induction of neurite outgrowth or neurofilament expressions. NGF at 1.5 ng/mL was selected in pre-treatment and co-treatment studies (see Supplementary Figures 1, 2): this concentration was to ensure the responses from a low amount of KXS 2012 could be revealed. In both pre-and co-treatments, the inductions of NF68, NF160, and NF200, induced by KXS 2012 plus NGF, were significantly higher at 3-to 4-folds (Figure 3). However, the effect triggered by pre-treatment was more robust than that of co-treatment. In neurite outgrowth assay, the number of differentiated cells by pre-treatment of KXS 2012 and NGF was reached to ∼30% (Figure 4A), and ∼20% increase was revealed in the co-treatment ( Figure 4B). By counting the number of neurite per cell, the pre-and co-treatment of KXS 2012 plus low dose of NGF caused an increase, significantly, by about 10% of cells having neurite (Figure 4). In addition, the differentiated cells, induced by KXS 2012 plus low amount of NGF, contained longer neurite, e.g., >15 and >30 µm (Figure 4). Compared to pre-treatment study, the inductive role of KXS 2012 showed less robust effect in promoting NGF-induced neurite outgrowth by co-treatment (Figure 4).

The Effect of KXS 2012 on Neuronal Differentiation via Up-Regulating Trk A Signaling
NGF achieves its function by binding to and activating Trk A in PC12 cells. The NGF-activated Trk A stimulates downstream signaling pathways leading to neuronal differentiation and promoting cell survival (Zhang et al., 2000;Vaudry et al., 2002). The role of KXS 2012 in phosphorylating Trk A was tested here. NGF induced Trk A phosphorylation in a dosedependent manner, and NGF at 1.5 ng/mL was selected in phosphorylation study, which barely showed phosphorylation capability (Supplementary Figure 3). KXS 2012 or co-treatment procedure did not effectively phosphorylate Trk A; however, the pre-treatment of KXS 2012 together with low level of NGF showed much better responsiveness ( Figure 5A).
In addition to Trk A, the downstream effectors of CREB and Erk1/2 were also determined here by pre-and co-treatment of KXS 2012 with low amount of NGF. In cultured PC12 cells, NGF induced CREB phosphorylation (∼40 kDa) at ∼5-folds; however, co-treatment with KXS 2012 did not show significant difference, as compared to NGF single treatment ( Figure 5B). In contrast, the phosphorylation of CREB was significantly induced by ∼7folds in the scenario of pre-treatment of KXS 2012 plus NGF (1.5 ng/mL; Figure 5B). Similarly, the Erk1/2 phosphorylation (∼42/44 kDa) was also being markedly up-regulated with pretreatment of KXS 2012 , while no significant difference, compared to NGF group, was observed in the co-treatment (Figure 5C).
To explore the role of KXS 2012 in NGF signaling, the expression of Trk A in cultured PC12 cells were analyzed. The expression of Trk A at ∼140 kDa, recognized by its specific antibody, was increased by 160% in KXS 2012 (15 µg/mL)treated cultures (Figure 6A). NF68 served as a positive control. The mRNA encoding Trk A was analyzed by real-time PCR. Application of KXS 2012 in PC12 cultures induced Trk A mRNA in a dose-dependent manner: the maximal induction of over 150% increase was revealed at ∼25 µg/mL (Figure 6B). This was in consistent with the phosphorylation study of KXS 2012 that the upregulation of Trk A expression could be an outcome of increasing sensitivity to low level of NGF during differentiation in PC12 cells.
Moreover, K252a, an inhibitor of tyrosine phosphorylation of Trk A, was applied onto cultured PC12 cells for 3 h before the preand co-treatment procedures. In the presence of K252a (0.1 µM), the protein expression of NF68 was totally decreased to that of control in KXS 2012 -treated cultures (Figure 7), which suggested FIGURE 5 | KXS 2012 promotes NGF-induced phosphorylation. In pre-treatment, KXS 2012 (15 µg/mL) was applied onto cultured PC12 cells for 48 h before serum starvation for 5 h, with or without application of NGF (1.5 ng/mL), for different time. In co-treatment, cultured PC12 cells, serum starvation for 5 h, were co-treated with NGF (1.5 ng/mL), KXS 2012 (15 µg/mL) and KXS 2012 (15 µg/mL) + NGF (1.5 ng/mL) for different time. (A) Total Trk A and phosphorylated Trk A were revealed by using specific antibodies. (B) Total CREB and phosphorylated CREB were revealed by using specific antibodies. (C) Total Erk1/2 and phosphorylated Erk1/2 were revealed by using specific antibodies. Quantification plot of the phosphorylation level in treatment of 5 (A) and 10 (B,C) min was shown. Values are expressed × Basal, where control value is set as 1, Mean ± SEM, n = 4. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control. that KXS 2012 -promoted neuronal differentiation was mediated by Trk A. These findings suggested that KXS 2012 promoted neuronal differentiation by up-regulating Trk A expression, which could increase the cellular response to low level of NGF.

DISCUSSION AND CONCLUSION
Depression is a common mental disorder, which shows severe neuron loss and dysfunction, mood disorder and behavioral disturbance (Barlow and Durand, 2011;Masi and Brovedani, 2011). In addition to neurodegenerative symptoms, depression is more likely to progress to suicide and other concurrent metabolic disorders, e.g., diabetes and cardiovascular diseases (Compare et al., 2014;Koponen et al., 2015). However, current anti-depressants are not able to deal with all the depressionrelated injuries, which make efficient drug development for antidepression a must. KXS, a classical Chinese herbal formula is expected to develop as a new anti-depressant. KXS is composed of two functional paired-herbs, i.e., GR-PR and ATR-PO. According to the theory of syndrome differentiation and Values are expressed as percentage of increase, where control value is set as 0, Mean ± SEM, n = 4. *p < 0.05 and **p < 0.01 compared to the control. treatment variation, the varied combination of paired-herbs in KXS has been recorded in Chinese medical books, which directs possible development of anti-depressants (Sun, 1997;Yasunori, 2011). Thus, a re-formulated KXS 2012 was prepared by optimizing the pairing of GR-PR and ATR-PO: the new formula presented robust anti-depressant action in animal study. It could also induce the expressions of neurotrophic factors as well as their receptors in rat cortical and hippocampal neurons . However, the underlying mechanism of KXS 2012 against depression has not been fully investigated. In the study, we investigated the cellular mechanism of KXS 2012 against depression by using PC12 cell as an in vitro model.
It is suggested that depression is largely caused by an impairment of neurogenesis in the brain. During neurogenesis, neuronal differentiation, in presence of neurite outgrowth, plays a critical role in synapse formation, which predicts a promising target of anti-depression (Hattiangady and Shetty, 2010;Snyder et al., 2011). Here, the potential drug targets and cellular mechanism on neuronal differentiation were addressed. The cultured PC12 cells are probably the most valid cell model for the detection of neuronal differentiation. The status of differentiated PC12 cells was determined morphologically through measuring neurite outgrowth and biochemically through analyzing the expression of neurofilaments (Schimmelpfeng et al., 2004;Xu et al., 2012). Our results indicated that KXS 2012 significantly potentiated NGF-induced neuronal differentiation. The potentiating effect was mediated through NGF receptor, Trk A. KXS 2012 greatly up-regulated Trk A signaling in the cultures, and the potentiating effect was blocked by k252a, an inhibitor of Trk A. Interestingly, the promoting mechanism of KXS 2012 in Trk A phosphorylation did not show great effect on the co-treatment situation. This discrepancy could be accounted by KXS 2012 -induced Trk A expression in cultured PC12 cells, which required longer time for Trk A activation. Moreover, the phosphorylations of Erk 1/2 and CREB, the downstream activators of Trk A, were explored here. The co-treatment of KXS 2012 and NGF did not show marked induction on Erk 1/2 and CREB phosphorylations, as compared to NGF group, which was in accord with our hypothesis that the promoting effect of KXS 2012 might require longer time of activation. By up-regulating Trk A expression and activation, KXS 2012 could increase the cellular response to low level of NGF, as to improve neuron adaptation and depression (Welberg, 2008;Peña et al., 2014). In line to this hypothesis, the study on siRNA knockdown of Trk A expression in cultured PC12 cells will be carried out as to further illustrate the function of KXS 2012 via Trk A signaling.
Considering the major ingredients in KXS 2012 responsible for anti-depression, different chemicals abundant in the herbal extract have been studied. GR-derived ginsenosides were reported to attenuate depression-like symptom by balancing neuronal cell proliferation and apoptosis and up-regulating neurotransmitter system (Xue et al., 2006;Li et al., 2014;Zhang et al., 2017). PR-derived 3,6 ′ -disinapoyl sucrose reversed mental disorder by improving hippocampal neuron plasticity and neurotrophic signaling pathway in stressed rats (Hu et al.,FIGURE 7 | K252a inhibits KXS 2012 -promoted neurofilament expression. (A) K252a (0.1 µM) was applied onto cultured PC12 cells, 3 h before KXS 2012 (15 µg/mL) treatment. After 48 h, PC12 cells were washed by PBS twice and treated with NGF (1.5 ng/mL) for 24 h. (B) K252a (0.1 µM) was applied onto cultured PC12 cells 3 h before KXS 2012 (15 µg/mL) treatment together with NGF (1.5 ng/mL) for 24 h. The cell lysates were collected to determine the expression of NF68. GAPDH served as a loading control. Quantification plot was shown in low panel. Values are expressed as × Basal, where control value is set as 1, Mean ± SEM, n = 4. *p < 0.05 and **p < 0.01 compared to the control. 2010). Asarones, derived from ATR, were more likely to pass the blood brain barrier, which may help improve distributions of other ingredients in the brain (Lu et al., 2014). The finding was consistent with our preliminary results that GR-derived ginsenoside Rb 1 and Rg 1 , PR-derived 3,6 ′ -disinapoyl sucrose, ATR-derived α-asarone and β-asarone were able to promote neuronal differentiation in neuronal cells. Therefore, it is reasonable to use KXS 2012 as a new regimen for anti-depression for its robust effect on neuronal differentiation and Trk A signaling.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2017. 00118/full#supplementary-material Supplementary Figure 1 | NGF induces neurite outgrowth. Cultured PC12 cells were treated with NGF (0.5-50 ng/mL) for 24 h (A) or 48 h (B). Cells were fixed with ice-cold 4% paraformaldehyde. Bar = 10 µm. The % of differentiated cell and length of neurite were counted as described in the Materials and Methods section. Values are expressed as % of total cells in 100 counted cells, Mean ± SEM, n = 4. * p < 0.05 and * * p < 0.01 compared to the control.
Supplementary Figure 3 | NGF increases Trk A phosphorylation in a dose-dependent manner. Cultured PC12 cells, serum starvation for 5 h, were treated with NGF (0.5-15 ng/mL) for 10 min. Total Trk A and phosphorylated Trk A were revealed by using specific antibodies. n = 4.