Gongjin-Dan Enhances Neurite Outgrowth of Cortical Neuron by Ameliorating H2O2-Induced Oxidative Damage via Sirtuin1 Signaling Pathway

Gongjin-dan (GJD) is a multiherbal formula produced from 10 medicinal herbs and has been traditonally used as an oriental medicine to treat cardiovascular diseases, alcoholic hepatitis, mild dementia, and anemia. Additionally, increasing evidence suggests that GJD exerts neuroprotective effects by suppressing inflammation and oxidative stress-induced events to prevent neurological diseases. However, the mechanism by which GJD prevents oxidative stress-induced neuronal injury in a mature neuron remains unknown. Here, we examined the preventive effect and mechanism of GJD on primary cortical neurons exposed to hydrogen peroxide (H2O2). In the neuroprotection signaling pathway, Sirtuin1 is involved in neuroprotective action as a therapeutic target for neurological diseases. After pre-treatment with GJD at three concentrations (10, 25, and 50 µg/mL) and stimulation by H2O2 (30 µM) for 24 h, the influence of GJD on Sirtuin1 activation was assessed using immunocytochemistry, real-time PCR, western blotting, and flow cytometry. GJD effectively ameliorated H2O2-induced neuronal death against oxidative damage through Sirtuin1 activation. In addition, GJD-induced Sirtuin1 activation accelerated elongation of new axons and formation of synapses via increased expression of nerve growth factor and brain-derived neurotrophic factor, as well as regeneration-related genes. Thus, GJD shows potential for preventing neurological diseases via Sirtuin1 activation.


Introduction
Oxidative stress is a pathological hallmark of regenerative failure resulting from an oxidative imbalance between cellular oxidant and antioxidant defense in the nervous system [1,2]. One of the major reasons for being worsened with diseases progression is antioxidant system depletion, which induces the degradation of almost all cell contents including the lipid membrane, protein, and DNA [3]. Accumulation of ROS, such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), and hydroxyl radical (HO·), that exceeds the capacity of the antioxidant system results in neuronal death and functional impairment [4]. Increasing evidence suggests a strong relationship between oxidative stress and neurological diseases, such as Alzheimer's disease and Parkinson's disease [5,6]. In addition, oxidative stress contributes to the pathogeneses of secondary damage after traumatic injuries to the spinal cord and brain [7,8]. Therefore, the regulation of oxidative stress has been suggested for resolving neurological diseases. In recent years, natural substances that have been empirically verified as safe and effective have been widely studied.
The seeded cells were cultured in neuron medium for 1 day, and then the medium was replaced with 10, 25, or 50 µg/mL GJD-containing medium. After 30 min of incubation to allow for the preventive effect of GJD, H2O2 was added to the medium at a concentration of 30 µM. The cells were then incubated for 24 h in the presence of both GJD and H2O2. The timeline of the experiment is shown in Scheme 1.

Neuronal Viability Assays
First, the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used for confirmation of the cell viability after GJD pretreatment at various doses of 1, 10, 25, 50, and 100 µg/mL with or without H2O2 exposure. CCK-8 reagent (10% of total volume) was treated and incubated for 4 h at 37 °C, and then absorbance measurement at 450 nm was analyzed using a microplate reader (Epoch, BioteK, Winooski, VT, USA). Live and dead cells were confirmed by a live/dead cell imaging kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The cells were treated with live dead assay staining solution (100 µL) for 15 min at 37 °C. To quantify the number of live and dead cell, 10 images per group were randomly captured at 10× objective using a confocal microscope (Eclipse C2 Plus, Minato, Tokyo, Nikon, Japan).

Neuronal Viability Assays
First, the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used for confirmation of the cell viability after GJD pretreatment at various doses of 1, 10, 25, 50, and 100 µg/mL with or without H 2 O 2 exposure. CCK-8 reagent (10% of total volume) was treated and incubated for 4 h at 37 • C, and then absorbance measurement at 450 nm was analyzed using a microplate reader (Epoch, BioteK, Winooski, VT, USA). Live and dead cells were confirmed by a live/dead cell imaging kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The cells were treated with live dead assay staining solution (100 µL) for 15 min at 37 • C. To quantify the number of live and dead cell, 10 images per group were randomly captured at 10× objective using a confocal microscope (Eclipse C2 Plus, Minato, Tokyo, Nikon, Japan).

Flow Cytometry
The cell-permeable fluorogenic probe, 2 , 7 -dichlorodihydrofluorescein diacetate (DCFDA; Sigma-Aldrich), which is a cell permeant reagent produced by oxidation of DCF by ROS within the cell, was used to evaluate the production of intracellular ROS. The 9.3 mg DCFDA powder was dissolved in high-quality 3.8 mL anhydrous dimethyl sulfoxide (Sigma-Aldrich), and then the pellet was suspended with 1 mL of 10 µM DCFDA solution. After further incubation at 37 • C for 30 min, a spectrofluorometer was used for measuring DCFDA fluorescence at 484 and 530 nm. The mean percentages of DCFDA-positive cells were determined relative to the control group.

DNA Dot-Blotting
A DNA dot-blotting was performed using extracted genomic DNA from a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). The DNA concentration were measured by a microplate reader (Epoch, BioteK, Winooski, VT, USA) at 260 nm/280 nm absorbance. Purified DNA were loaded onto 0.2 µM nitrocellulose membrane and hybridized for 2 h at 80 • C. The membrane was then blocked with 5% skim milk in 1X Tris-buffered saline (Bio-Rad) with 0.1% Tween 20 (TBST; Sigma-Aldrich) at room temperature for 1 h. After incubation with dsDNA (1:2000, Abcam) and 8-OHdG (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 • C for overnight, membrane was washed in three changes of TBST for 15 min and then incubated with an anti-mouse or anti-rabbit horseradish-peroxidaseconjugated antibody (1:2500, Abcam) at room temperature for 2 h. Dot blots were visualized using electrochemiluminescence (Bio-Rad, Hercules, CA, USA) and evaluated with an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden).

Real-Time Polymerase Chain Reaction (PCR)
The changes in mRNA levels of Sirt1, Sirt2, BDNF, NGF, GAP43, and NF200 were analyzed in each group using real-time PCR. Total RNA was isolated using TRIzol reagent (Ambion, Austin, TX, USA). cDNA was synthesized using oligo(dT)20 primer and Accupower RT PreMix (Bioneer, Daejeon, Korea). All primers are listed in Table 1. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). The cycling conditions were 40 cycles at 95 • C for 3 min, 95 • C for 15 s, and 60 • C for 30 s. All real-time PCR samples were evaluated in at least triplicate. The gene expression was normalized to that of GAPDH and is shown as the fold-change relative to the control group.

GJD Exerted a Neuroprotective Effect on H 2 O 2 -Treated Cortical Neurons
We preliminarily determined the optimal doses of GJD to achieve therapeutic effects in H 2 O 2 -injured neuron using the CCK-8 assay. GJD was not cytotoxic to cortical neurons at doses of 1-100 µg/mL and significantly increased cell proliferation from 10 to 50 µg/mL ( Figure 1A). Moreover, pretreatment with 10-50 µg/mL GJD effectively blocked cytotoxic activity and specifically exerted a significant neuroprotective effect on cortical neurons following H 2 O 2 exposure ( Figure 1B). We confirmed these optimal ranges of GJD for neuroprotective effect using imaging assays for live and dead cells. The number of green-stained living cells was significantly reduced after H 2 O 2 treatment compared with the blank group. The number of live cells was significantly increased following GJD pretreatment, demonstrating that the cells were dose-dependently protected from H 2 O 2 stress ( Figure 1C,D). Based on the cell viability results, GJD treatment at up to 50 µg/mL was considered as safe in primary cortical neurons. blank group. The number of live cells was significantly increased followin pretreatment, demonstrating that the cells were dose-dependently protected fro stress ( Figure 1C,D). Based on the cell viability results, GJD treatment at up to 50 was considered as safe in primary cortical neurons. showing live (green) and dead (red) cells in the blank, control, and GJD groups. White sc 200 µm, yellow scale bar = 100 µm. Data are expressed as the mean ± SEM. Significant dif indicated as #### p < 0.0001 vs. blank group; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs group were analyzed via ordinary one-way analysis of variance (ANOVA) with Tukey's analysis.

GJD Suppressed H2O2-Induced ROS Production by Activating Nrf2/HO-1 Expressio Cortical Neurons
The major consequence of oxidative stress is the induction of DNA damag with increased intracellular ROS. To determine whether the GJD could preven damage caused by H2O2-induced oxidative stress, we used three methods to evalu and 8-OHdG expression. First, we measured intracellular ROS levels using H2D as a good indicator of cellular ROS by flow-cytometry. The ROS level was dram increased following exposure of the neurons to H2O2 but was dose-dependently de following pretreatment with GJD ( Figure 2A). Changes in cellular ROS leve Data are expressed as the mean ± SEM. Significant differences indicated as #### p < 0.0001 vs. blank group; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group were analyzed via ordinary one-way analysis of variance (ANOVA) with Tukey's post hoc analysis.

GJD Suppressed H 2 O 2 -Induced ROS Production by Activating Nrf2/HO-1 Expression in Cortical Neurons
The major consequence of oxidative stress is the induction of DNA damage along with increased intracellular ROS. To determine whether the GJD could prevent DNA damage caused by H 2 O 2 -induced oxidative stress, we used three methods to evaluate ROS and 8-OHdG expression. First, we measured intracellular ROS levels using H2DCF-DA as a good indicator of cellular ROS by flow-cytometry. The ROS level was dramatically increased following exposure of the neurons to H 2 O 2 but was dose-dependently decreased following pretreatment with GJD ( Figure 2A). Changes in cellular ROS levels were precisely measured by quantifying the percentage of H2DCF-DA positivity. The cells were detected in approximately 23% of 10,000 single cells after H 2 O 2 treatment. Additionally, H2DCF-DA-positive cells showed a dose-dependent decrease after GJD pretreatment, and a significant decrease compared with the control group ( Figure 2B). We next investigated iNOS, HO-1, and Nrf2 expression to confirm whether GJD inhibits oxidative stress-related iNOS activity by modulating Nrf2/HO-1 activation. The iNOS level increased with H 2 O 2 treatment, whereas this level gradually decreased in the 25 and 50 µg/mL GJD groups ( Figure 2C,D). In accordance with previous studies, activating the antioxidant protein HO-1 and nuclear factor erythroid derived 2-related factor 2 (Nrf2) play crucial regulatory roles in the cellular stress response and neuroprotection [26]. Thus, we also performed Western blotting and immunochemical staining to confirm whether GJD could induce the expression of Nrf2/HO-1 in cortical neurons against H 2 O 2 -induced neurotoxicity. After H 2 O 2 treatment, the level of HO-1 protein was not increased in control group; however, the HO-1 protein level showed a tendency to increase in a concentration-dependent manner with GJD pretreatment, as shown in Figure 2C. Quantitative analysis revealed significant increase in 50 µg/mL GJD group compared to control group ( Figure 2E). We also analyzed the protein expression of Nrf2 and confirmed the antioxidant activity and prevention of oxidative damage by GJD. As shown in Figure 2C,F, the Western blot assay revealed that 25 and 50 µg/mL GJD groups significantly increased Nrf2 level compared to control group. Confocal images also showed that the Nrf2 level significantly decreased after H 2 O 2 treatment compared to that in the blank group, with GJD affecting Nrf2 expression in a dose-dependent manner, supporting its antioxidant action ( Figure 2G,H).
play crucial regulatory roles in the cellular stress response and neuroprotection [26]. we also performed Western blotting and immunochemical staining to confirm wh GJD could induce the expression of Nrf2/HO-1 in cortical neurons against H2O2-ind neurotoxicity. After H2O2 treatment, the level of HO-1 protein was not increased in co group; however, the HO-1 protein level showed a tendency to increase in a concentra dependent manner with GJD pretreatment, as shown in Figure 2C. Quantitative an revealed significant increase in 50 µg/mL GJD group compared to control group (F 2E). We also analyzed the protein expression of Nrf2 and confirmed the antiox activity and prevention of oxidative damage by GJD. As shown in Figure 2C,F Western blot assay revealed that 25 and 50 µg/mL GJD groups significantly increased level compared to control group. Confocal images also showed that the Nrf2 significantly decreased after H2O2 treatment compared to that in the blank group, GJD affecting Nrf2 expression in a dose-dependent manner, supporting its antiox action ( Figure 2G,H).  . Data are expressed as the mean ± SEM. Significant differences indicated as ### p < 0.001, #### p < 0.0001 vs. blank group; * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. control group were analyzed via one-way ANOVA with Tukey's post hoc analysis.

GJD Prevented Oxidative Damage to DNA in H 2 O 2 -Treated Cortical Neurons
Next, we confirmed the dose-dependent preventive effect of GJD against DNA damage. 8-OHdG is used widely as a reliable marker of oxidative DNA damage and stress [27]. The 8-OHdG level was first determined using immunochemical staining and a dot-blot assay. GJD pretreatment reduced the 8-OHdG expression increased by H 2 O 2 ( Figure 3A). There was significantly greater difference between the control and GJD groups with respect to the 8-OHdG intensity. H 2 O 2 treatment induced positively stained cells with 8-OHdG; however, the intensity of 8-OHdG-positive neurons significantly decreased in a dose-dependent manner by GJD pretreatment (Figure 3B). We also assessed the 8-OHdG level in genomic DNA using a dot blot assay ( Figure 3C). The 8-OHdG level in genomic DNA was markedly decreased in the GJD groups compared to the control group. These findings indicate that GJD inhibits H 2 O 2 -induced DNA damage in cortical neurons.

GJD Prevented Oxidative Damage to DNA in H2O2-Treated Cortical Neur
Next, we confirmed the dose-dependent preventive effect of GJ damage. 8-OHdG is used widely as a reliable marker of oxidative DNA da [27]. The 8-OHdG level was first determined using immunochemical sta blot assay. GJD pretreatment reduced the 8-OHdG expression increased 3A). There was significantly greater difference between the control and G respect to the 8-OHdG intensity. H2O2 treatment induced positively stai OHdG; however, the intensity of 8-OHdG-positive neurons significantl dose-dependent manner by GJD pretreatment ( Figure 3B). We also asses level in genomic DNA using a dot blot assay ( Figure 3C). The 8-OHdG DNA was markedly decreased in the GJD groups compared to the cont findings indicate that GJD inhibits H2O2-induced DNA damage in cortica

GJD Activated Sirt1 Expression for Axonal Outgrowth but Did Not Affect Sirt2 Expression in H 2 O 2 -Induced Cortical Neurons
We further assessed the promoting effect of GJD on axonal outgrowth in H 2 O 2 -treated neurons. Sirt1 was previously reported to be involved in the processes of neuron survival, outgrowth, and synaptic plasticity [28]. Thus, we examined the effect of GJD on the promotion of neurite outgrowth by activating the Sirt1 signaling pathway. GJD pretreatment not only protected cortical neuron against H 2 O 2 damage, but also dose-dependently increased neurite outgrowth. A significant increase in MAP2-positive neurite was observed in GJD-pretreated neurons ( Figure 4A). When axon outgrowth was quantified by three parameters, the results revealed a significant dose-response effect in neurons pretreated with GJD against H 2 O 2 -induced injury ( Figure 4B-D). Additionally, F-actin assembly was observed at the leading edge to the axons. We next examined whether GJD affected Sirtuin expression to accelerate axonal growth in H 2 O 2 -treated neurons. Almost all neurons strongly expressed Sirt1 before H 2 O 2 exposure, whereas Sirt1 was rarely expressed following H 2 O 2 treatment. Thus, Sirt1 was activated by GJD pretreatment to promote axonal growth ( Figure 4E), and its intensity was significantly elevated in a dose-dependent manner compared to that in the control group ( Figure 4F). In addition, the mRNA and protein expression of Sirt1 was investigated using real-time PCR and Western blot. GJD pretreatment induced a significant dose-dependent increase in Sirt1 expression, which showed a dose-related increasing trend in GJD-pretreated neurons as directly observed in the confocal images ( Figure 4G,H). Quantifying the Western blot exhibited a significant increase of Sirt1 level in 25 and 50 µg/mL GJD groups compared to the control group ( Figure 4I). We also analyzed the Sirt2 level using the same methods as in Sirt1 analysis. Sirt2 expression was also decreased by H 2 O 2 treatment, but GJD did not affect Sir2 expression in H 2 O 2 -treated neurons. Confocal images revealed that Sirt2 expression was decreased after H 2 O 2 treatment, but GJD did not stimulate a significant increase in H 2 O 2 -treated neurons (Supplementary Figure S1A). Sirt2 intensity was decreased by H 2 O 2 exposure, but there was no significant difference between the control and GJD groups (Supplementary Figure S1B). This trend was also observed in the real-time PCR and Western blotting results (Supplementary Figure S1C,D). Quantifying data generated by Western blot revealed significant reduction in control group, compared to blank group. However, GJD pretreatment does not induce increased expression of Sirt2 level (Supplementary Figure S1E). Based on these findings, GJD can stimulate axonal outgrowth by increasing Sirt1 expression but does not affect Sirt2 expression in H 2 O 2 -injured neurons.    4). Data are expressed as the mean ± SEM. Significant differences indicated as #### p < 0.0001 compared vs. blank group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group were analyzed via ordinary one-way ANOVA with Tukey's post hoc analysis.

GJD Did Not Induce Axonal Growth in Sirt1-Silenced Neurons
Additionally, we tested whether GJD can induce axon growth when the Sirt1 pathway was inhibited by the specific Sirt1 inhibitor EX-527 to better understand the relevance of Sirt1 activation more clearly. In our work, we confirmed that Sirt1 expression decreased after H 2 O 2 treatment, while increased with axonal elongation in GJD-pretreated conditions ( Figure 5A), indicating that Sirt1 may be related to the neuroprotective and axon growthpromoting effect of GJD pretreatment. Surprisingly, after adding a Sirt1 inhibitor EX-527, we found that neuronal death was increased and cell viability was decreased, confirming that Sirt1 was involved in neuronal survival. Next, when adding both GJD and EX-527 in H 2 O 2 -injured neuron, axonal outgrowth was not induced even if GJD was pretreated in the neuron before H 2 O 2 exposure. All dose-dependent effects of GJD pretreatment were abrogated by the Sirt1 inhibitor EX-527 ( Figure 5B). Therefore, we demonstrated that Sirt1 may be closely related to the neuroprotective effect of GJD, suggesting its dependency on the Sirt1 signaling pathway. Moreover, axon growth showed no obvious difference either with or without GJD under the Sirt1-inhibited condition ( Figure 5C-E), indicating its great potential pathway for the preventive effect of GJD. In addition, the Sirt1 intensity did not show any difference between GJD and control groups under the Sirt1-inhibited condition ( Figure 5F). These Sirt1 expression tend to have similar patterns at the RNA and protein levels. Under condition of EX-527 exposure, there was no significant overall difference between GJD and control groups ( Figure 5G-I).

GJD Influenced Neurotrophic Factor Upregulation in H 2 O 2 -Damaged Cortical Neurons
Based on the results highlighting the effect of GJD in mediating the Sirt1 level, we also investigated neurotrophic factor expression to better understand how GJD can accelerate axon growth by increasing Sirt1 expression. Previous studies demonstrated that Sirt1 acts as a neuroprotective regulator by activating BDNF and NGF expression, and neurotrophic factors are well-known to be essential for neuronal survival and growth by participating in synaptic function and plasticity [29,30]. Confocal microscopy images revealed that BDNF expression was decreased after H 2 O 2 treatment but dose-dependently increased in the GJD groups ( Figure 6A). Quantitative analysis of BDNF showed that the intensity was significantly higher in the GJD groups than in the control group ( Figure 6C). In addition, the BDNF mRNA level, analyzed by real-time PCR, was significantly upregulated in the 25 and 50 µg/mL GJD groups compared to that in the control group ( Figure 6D). A similar trend was observed in the confocal images of NGF, as BDNF increased from GJD pretreatment and promoted axonal growth in H 2 O 2 -injured neurons ( Figure 6B). The NGF intensity was significantly increased in 25 and 50 µg/mL GJD groups ( Figure 6E). The NGF mRNA level had significant upregulation in the 25 and 50 µg/mL GJD groups compared to that in the control group ( Figure 6F). These findings suggest that pretreatment with GJD can promote axonal growth by activating BDNF and NGF, which involves the Sirt1 signaling pathway, in H 2 O 2 -injured neurons.
GJD. In addition, the Sirt1 intensity did not show any difference between GJD and contro groups under the Sirt1-inhibited condition ( Figure 5F). These Sirt1 expression tend to hav similar patterns at the RNA and protein levels. Under condition of EX-527 exposure, ther was no significant overall difference between GJD and control groups ( Figure 5G-I).  . Data are expressed as the means ± SEM. Significant differences indicated as # p < 0.05, ### p < 0.001, and #### p < 0.0001 compared vs. blank group; * p < 0.05, ** p < 0.01 and **** p < 0.0001 vs. control group were analyzed via ordinary one-way ANOVA with Tukey's post hoc analysis.

GJD Enhanced Synapse Formation by Upregulating Synasin1 Expression in H2O2-Induced Cortical Neurons
Synapse contacts are typically formed between pre-and postsynaptic cells to ensure (D) Relative fold-changes in the level of BDNF mRNA in the blank, control, and GJD groups (n = 5). (E,F) Quantification of NGF intensity and mRNA in the blank, control, and GJD groups (n = 5). Data are expressed as the means ± SEM. Significant differences indicated as # p < 0.05, ### p < 0.001, and #### p < 0.0001 compared vs. blank group; * p < 0.05, ** p < 0.01 and **** p < 0.0001 vs. control group were analyzed via ordinary one-way ANOVA with Tukey's post hoc analysis.

GJD Enhanced Synapse Formation by Upregulating Synasin1 Expression in H 2 O 2 -Induced Cortical Neurons
Synapse contacts are typically formed between pre-and postsynaptic cells to ensure the normal function of neural networks. Remarkably, during injury to the central nervous system, synapse loss occurs almost simultaneously in billions of neurons, resulting in permanent disability. Thus, to determine whether GJD can lead to synapse formation in H 2 O 2 -injured neurons, Syn1 staining was performed to confirm the synaptic contacts induced by GJD pretreatment. The signal for Syn1 was bright between neurons within the cell soma and axons. Meanwhile, this signal was dramatically decreased after H 2 O 2 exposure. The GJD-pretreated condition induced an increase in the Syn1 signal in a dosedependent manner ( Figure 7A), with significant increases in the 25 and 50 µg/mL GJD and control groups ( Figure 7B). Furthermore, neurofilament 200-kDa (NF200) and growthassociated protein (GAP43) were enhanced by GJD pretreatment. Quantitatively, GJD induced a dose-dependent increase in NF200 expression, but only 50 µg/mL GJD showed a significant effect ( Figure 7C). GAP43 mRNA level was also dose-dependently increased and significantly different in the 25 and 50 µg/mL GJD groups relative to the control group ( Figure 7D).

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14 of 17 cell soma and axons. Meanwhile, this signal was dramatically decreased after H2O2 exposure. The GJD-pretreated condition induced an increase in the Syn1 signal in a dosedependent manner ( Figure 7A), with significant increases in the 25 and 50 µg/mL GJD and control groups ( Figure 7B). Furthermore, neurofilament 200-kDa (NF200) and growthassociated protein (GAP43) were enhanced by GJD pretreatment. Quantitatively, GJD induced a dose-dependent increase in NF200 expression, but only 50 µg/mL GJD showed a significant effect ( Figure 7C). GAP43 mRNA level was also dose-dependently increased and significantly different in the 25 and 50 µg/mL GJD groups relative to the control group ( Figure 7D).  (C,D) Relative fold-changes in the level of NF200 and GAP43 mRNA in the blank, control, and GJD groups (n = 5). Data are expressed as the mean ± SEM. Significant differences indicated as #### p < 0.0001 compared vs. blank group; * p < 0.05, *** p < 0.001 and **** p < 0.0001 vs. control group were analyzed via ordinary one-way ANOVA with Tukey's post-hoc analysis.

Discussion
Although GJD has been demonstrated to have neuroprotective effects, its mechanism in the repair and prevention of neurological damage has not been widely studied. Thus, we examined whether GJD can protect the neurons from H 2 O 2 -induced oxidative damage by activating the Sirt1 signaling pathway. Sirt1 is involved in numerous biological and pathophysiological processes and has recently been considered as an emerging neuronal therapeutic target for neurological diseases [31]. The GJD used in this study showed excellent in vitro activity against H 2 O 2 -induced oxidative stress; we demonstrated these specific effects directly in primary cortical neurons. GJD was highly effective for protecting against neuronal death caused by oxidative stress. Here, we found that 10-50 µg/mL GJD protected primary cortical neurons; these concentrations are recommended as the optimal therapeutic dose range for achieving antioxidant neuroprotective effects.
Previous studies also showed that GJD enhanced neurite outgrowth in neuronal-like cell lines, PC12 cells and increased NGF secretion in astrocytes treated at 250 µg/mL [17]. Although the therapeutic dose range of GJD was previously suggested by Moon et al., the authors did not directly demonstrate these effects in neural cells with the same morphological and biochemical characteristics as mature neurons. We also demonstrated that GJD has excellent antioxidant activity by reducing the ROS level following H 2 O 2 exposure in cortical neurons and specifically prevented oxidative DNA damage. One important feature of oxidative stress is the accumulation of oxidative damage to DNA, RNA, protein, and organelles [32]. GJD prevents oxidative stress-induced DNA damage and promotes neurite outgrowth under H 2 O 2 -treated conditions. Specifically, axon growth was accelerated through the Sirt1 signaling pathway following GJD treatment. Sirt1 was dose-dependently upregulated and peaked at 50 µg/mL of GJD, and concomitantly induced axon elongation from oxidative damage. In contrast, Sirt2 expression was not elevated by GJD treatment in H 2 O 2 -treated neurons.
Additionally, we examined whether GJD could induce axon growth when Sirt1 pathway was inhibited by inhibitor to confirm the relevance of Sirt1 activation. Sirt1 inhibition by EX-527 did not robustly induce axon growth in neurons, even following GJD treatment. This result indicates that the preventive neuroprotection effect of GJD is closely associated with the Sirt1 signaling pathway in neurons following oxidative injury. Cumulative evidence regarding the mechanisms involved in Sirt1-induced neuroprotection indicates that Sirt1 activation can directly control Forkhead box protein 1 to defense oxidative stress, NF-кB to anti-inflammation, and p53 to anti-apoptosis and providing effective neuroprotection against neurological diseases [33,34]. Therefore, the Sirt1 activation effect of GJD may represent a therapeutic target for future research on anti-aging and immune regulation.
One limitation of our study was that it was difficult to identify a single target responsible for the neuroprotective properties and mechanism of action of GJD, which is a complex multi-herbal formula. Another limitation was that interpreting functional recovery at the tissue level was difficult. Thus, studies are needed to investigate the preventive effect of GJD in an animal model and human-induced cell line. Our results reveal the antioxidant neuroprotective effects of GJD in H 2 O 2 -injured mature cortical neurons, and that the Sirt1 signaling pathway is involved in the mechanism of action of GJD.

Conclusions
GJD enhances the axonal outgrowth of cortical neurons through Sirt1 regulation following H 2 O 2 -induced oxidative injury.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.