Ginsenoside Rc Protects Neurons From Oxygen-glucose Deprivation Injuries and Its Mechanistic Investigation via a Network Pharmacology-based Analysis

Background: Ginsenoside Rc (Rc) is one of the major active components of Panax ginseng Meyer. Studies have shown that Rc has remarkable effect in protection of nervous system. However, the potential molecular mechanism of its neuroprotective effect remains unclear. Our study aim to investigate the neuroprotective effect of Rc on neuron damage and explore the potential mechanism on its regulation of TNF-α and DRP-1. Methods: Oxygen-glucose deprivation reperfusion (OGD/R) cell neuron damage modle was induced by Na 2 S 2 O 4 and EBSS solution. After preventive administration, cell viability and cell toxicity were detected to evaluate the putative neuroprotective properties of Rc. Network pharmacology and molecular docking simulation studies were performed to predict the potential targets and pharmacological mechanism. Furthermore, the prediction was validated via western blot assay and specic antagonist. Results: In OGD/R injured cells, Rc signicantly improved cell viability (Rc middle dose vs. OGD/R model: 67.3±2.33% vs. 55.7±1.14%, P<0.05) and obviously decreased cell toxicity (Rc middle dose vs. OGD/R model: 147±39.7% vs. 232±29.4%, P<0.01). Analysis of network pharmacology and molecular docking indicated that the key targets of Rc are TNF-α and DRP-1. Subsequently molecular biological studies showed a signicant increase on expression of TNF-α and DRP-1 in model group. Conversely, administration of Rc reversed the alteration signicantly and presented a dose dependence. By adding antagonist, we validated that Rc had an indirect regulation on TNF-α and DRP-1. Conclusions: Rc possess protective properties against OGD-induced neuron damage by regulating the expression of TNF-α and DRP-1.


Background
Stroke is a brain injured disease with high incidence, death, and disability rates. The global burden of disease in 2016 showed that stroke is the leading cause of life years loss in China [1,2]. In clinic, ischemic stroke (IS) accounts more than 85% of stroke. For the past two decades, the mainstay of acute ischemic stroke (AIS) management has been attempted reperfusion of ischemic tissue with intravenous thrombolysis [3]. Until now, alteplase, one of the recombinant tissue plasminogen activator, is the only brinolytic agent with Food and Drug Administration (FDA)-approved for AIS treatment. However, there were less than 3% patients who can bene t from intravenous thrombolysis because of the strict therapeutic time window limit and fatal side effects [4]. Besides, brinolytic therapy may cause ischemiareperfusion (I/R) injury, which results in high morbidity and high mortality [5]. Currently, neuroprotective therapies have shown the potential to prolong the therapeutic window for reperfusion prior to endovascular interventions [6]. However, neuroprotection for stroke has shown great promise but has had little translational success. Therefore, the development of a clinically effective and safe neuroprotective drug for the treatment of ischemic stroke remains an urgent unmet need.
Ginsenoside Rc (Rc) is a major natural product isolated from Panax ginseng Meyer, which is widely used as both a preventive and therapeutic treatment against various diseases. Rc has exhibited effects on protection of central nervous system [7,8], prevention of diabetes [9], anti-in ammatory [10], antioxidation [11] and anti-adipogenesis [12]. In addition, Rc can be absorbed into blood and brain tissue rapidly after administration [13]. Researches have shown that ginsenosides are effective in treating cerebral I/R and other nervous system diseases [14]. Resently, some ginsenosides are regarded as neuroprotective agents to attenuate IS damages [15,16]. However, although the pharmacological activities of Rc have been well studied, it remains not clear whether RC has neuroprotective effect in IS. Moreover, the speci c mechanisms have not been fully elucidated. From a therapeutic perspective, it is important to understand the functional mechanisms of RC to be developed into clinically effective and safe neuroprotective drug.
Network pharmacology is a prospective strategy to explore the multi-targets regulation networks of chemicals with combining systematic methods [17]. Until now, it has been reported in lots of researches on exploring the molecular mechanisms of effective components from TCM herbs [18]. Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (TCMIP) is an intelligent data mining platform which has been extensively used to explain the functional mechanisms of TCM [19,20].
Besides, molecular docking is a computational method for predicting the placement of ligands in the binding sites of their receptors. It has been widely used in drug discovery [21]. Oxygen-glucose deprivation(OGD/R) cell modelhas been well recognized for stroke in vitro. Therefore, this study aimed to investigate the neuroprotective potential of Rc and explore the mechanism for treatment on IS via methods of network pharmacology, including target prediction of chemical, topological feature analysis, and molecular docking. Then the putative potential targets of Rc in OGD/R injury will be validated by molecular biology approach.
Cell treatment and OGD/R model Cells were seeded in 96-well plate at a density of 6×10 3 cells/well and incubated for 24 hours. Thereafter, Nimodipine (positive drug) group was treated with 5μmol/L Nimodipine. Treatment groups were treated with 1000, 500, and 100μmol/L Rc, respectively. While control group and model group were just cultured in normal medium. After culturing for 24 hours, Earle's Balanced Salts (EBSS) with 5 mmol/L Na 2 S 2 O 4 was added into groups except control group. After culturing for 80 minutes, the medium was replaced by normal DMEM with glucose and cells were cultured for 24 hours for reoxygenation under normoxic condition.

Preparation of cell lysates
Adherent cells were harvested using a cell scraper, washed twice with PBS and maintained on ice for 5 min. Following centrifugation at 14, 000 rpm for 15 min at 4℃, the cell pellets were dissolved in cell lysis buffer containing 1µL PMSF and Cocktail (Sigma-Aldrich; Merck KGaA), and maintained on ice for 30 min. Following centrifugation at 10,000 rpm for 5 min at 4˚C, the supernatants were collected. Protein concentration in cell lysates was determined by the BCA kit assay according to the manufacturer's instructions.
Cell viability and toxicity assay.
In order to investigate the putative protective properties of Rc in OGD on PC12 cells, cell viability was evaluated using a CCK-8 assay, and cell toxicity was determined by LDH assay. After treatment, CCK-8 assay and LDH assay were performed according to the manufacturer's instructions as described previously [22].

Target prediction
Identifcation of targets of Rc is a key step in understanding its mechanisms. In this study, the methods,includingTCMIP [19] (http://www.tcmip.cn/TCMIP/index.php/Home/Login/login.html),was used to derive molecular target information for target prediction. TCMIP is a new powerful platform for predicting targets of actual bioactive ingredients that based on similarity ensemble analysis.
Functional analysis of the putative targets by STRING.
The STRING database (https://string-db.org/), including data on interacting proteins or genes in humans, was used to determine protein-protein interactions (PPI). Interactions among the putative targets were identi ed using a threshold score of 0.7. To mine the critical targets related to ischemic stroke, a targetfunction network of ischemic stroke was constructed, with the target-function relation based on the network topological analysis by Cytoscape 3.7.1.
Molecular docking of Rc.
Molecular docking was analyzed using the SYBYL-X 2.1.1 software (Certara, L. P.). The scoring function total-Score equal to 5 was used as a threshold to evaluate the interaction between ingredients and targets. The structure of disease targets employed in the analysis of docking was obtained from the Protein Data Bank (PDB, http://www.rcsb.org). The co-crystalized ligand and water molecules were removed from the structure, while H atoms were added and side chains were xed during protein preparation. The Sur ex-Dock (SFXC) docking mode was used, and the procedure was conducted as previously described. Total Sur ex-Dock scores represent binding a nities.
Western blot analysis.
Cellular lysates (protein content, 20 µg/lane) were separated using SDS PAGE on a 10% (w/v) gel and electrotransferred to a polyvinylidene uoride membrane (Millipore; USA). The membranes, after blocking with 5% nonfat milk (Sigma-Aldrich; Merck KGaA) for 1 hour at room temperature, were probed with the primary antibodies at 4˚C overnight, and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (TNF-α Rabbit Antibody, ab11564; DRP-1 Rabbit Antibody, ab184247; Abcam, UK) at room temperature for 2 hours. Protein bands were visualized by using the ECL western detection reagent. GAPDH was used as the loading control. Densities of the protein bands were determined using ImageJ2x software.

Statistical analysis.
Data are expressed as the mean ± standard deviation. The statistical signifcance of the differences between groups was assessed using One-way analysis of variance. Graphpad Prism version 8.0.1 for Windows was used to perform the statistical analyses. P<0.05 was considered to indicate a statistically signifcant difference.

Results
Effects of ginsenoside Rc on cell viability and toxicity in OGD/R injured cells.
To evaluate the putative neuroprotective properties of Rc in OGD/R injured cells, the cell viability was detected by CCK-8 assay and the cytotoxicity was determined by LDH leakage. As shown in Fig.1A , Eighteen potential targets of Rc were predited by TCMIP database. Then protein-protein interactions of these targets were determined by The STRING database as shown in Fig.2B. As revealed in Fig.2C, the enrichment of GO was performed by STRING. The enrichment of functional pathway was found out that the top 10 of pathway, such as TNF signaling pathway, is illustrated in Fig.2D. The network topological analysis by Cytoscape demonstrated that targets with the top ve scores of degree were TNF. The key pathway of Rc maybe the TNF signal pathway.
The targets were analysed in Cytoscape and ranked by degree as shown in Table 1. The top 4 were TNF/CASPA3/NFKB1/IL6. To identify the key targets of Rc, the targets in TNF pathway related to ischemic stroke were imported into SYBYL to nd out key target. The results of molecular docking are shown in Fig.3A. As depicted in Fig.3A, RC is bound to residues outside the active pocket of TNF (key residues including ARG201/ASN200/ASP254/ASN182/ARG143/ASP294/ARG295)by hydrogen bonds. The overall spatial structure indicates that the TNF/Rc complexes are stable, indicating the interactions of Rc with their targets may have an active functional role.
Referring to literature on ischemic stroke, TNF signal pathway (As shown in Fig.3B Effects on cell survival rate induced by ginsnoside Rc and EvP4593/Mdivi-1 To further determine the correlation between Rc and TNF-α/DRP-1, cells were treated by EvP4593 (antagonist of TNF-α) and Mdivi-1 (antagonist of DRP-1), respectively. Cell survival rate was detected by CCK-8 assay. The result was shown in Fig.4D. Compared with Rc group (65.87±9.67%), the survival rate of Rc+EvP4593 (57.42±5.22%) was tended to decrease. Compared with model group, the survival rate of EvP4593 group showed no signi cant difference. The result indicated that Rc may had an indirect effect on TNF-α. Besides, the survival rate of Rc+Mdivi-1 showed no signi cant difference with Rc group. There was also no signi cant difference between model group and Mdivi-1 group, which indicated that Rc may also had an indirect effect on DRP-1.

Discussion
Ischemic stroke is a complex multifactorial disease caused by infarction and result in the loss of neurologic function. For decades, the most effective way to treat cerebral infarction is brinolytic therapy [23]. However, there isn't any effective treatment for stroke caused by neuronal damage and death.
In this study, we investigated the putative neuroprotective properties of Rc and the molecular machanism, which showed a positive signi cance for development of neuroprotective drugs for the treatment of cerebral I/R injury to reduce safety concerns caused by antithrombotic drugs in IS.
In our study, we found that pretreatment of Rc enhanced the cell viability and reduced the cell toxicity on OGD/R injured cells (Fig.1), which provided evidence for Rc as one of the active components of P. ginseng.Then, network pharmacology and molecular docking were applied to predict the related targets and corresponding mechanisms of Rc in treatment of IS. A protein-protein interaction network based on Rc was constructed. Bynetwork topological analysis, we revealed and highlighted that Rc were involved in TNF signal pathway (Fig.2). Analysis of molecular docking and literature on IS indicated that TNF signal pathway (Fig.3), including TNF, DRP-1, play important role in IS. The correlation between Rc and predicted targets was explored via western blots. Results showed that Rc reversed the expression of TNF-α and DRP-1 on OGD/R injured cells when compared with model group (Fig.4). To further explore whether Rc possess neuroprotective effect by directly targeting TNF-α and DRP-1, the antagonists were added as interfering agent respectively. The result showed that Rc had an indirect effect on TNF-α and DRP-1 (Fig.4). In terms of experimental method, network pharmacology and molecular docking are not enough to judge the targets of Rc in IS. So cell experiments were combined in this study for illustration. This work may has practical signi cance for rapidly discovery on related targets and corresponding mechanisms of monomer drugs.
The tumor necrosis factor (TNF) superfamily of cytokines activate signaling pathways plays an important role in cell survival, death, and differentiation. A network pharmacology research had indicated that the treatment of compounds on stroke may related to TNF signaling pathway and TNF-α may be one of the molecular markers for stroke [24]. TNF-α is a pleiotropic in ammatory cytokine cytokine with various biological functions, which is mainly produced by macrophages and monocyte. TNF-α participates in several immunity disease and in ammatory disease [25], and it is a crucial determinant of in ammatory reaction in stroke [26]. Studies have shown that the expression of TNF-α after cerebral I/R has neurotoxic effect on nervous system [27]. Moreover, it was suggested that TNF-α may play a neurorotective role in stroke by downregulating apoptosis [28]. In addition, we found that stroke may be related to NFκB1 and CASP3 (table 1), which is consistant with the existing research nding [29]. It is well known that TNF-α plays a critical role on the activation of NFκB and caspase-3 [30,31], which also provided evidence to illustrate TNF-α as a key target.
Dynamin-related protein-1 (DRP-1) is the dynamin of mitochondrial ssion [32]. The imbalance of mitochondrial fusion/division has great in uence on I/R injuries [33]. Previous studies had shown that TNF-α and DRP-1 played a regulatory role in amelioration of cerebral ischemic injury and neuroin ammation [34]. Moreover, TNF-α is the predominant inducer of DRP1 S616 phosphorylation during sepsis [35]. DRP-1 had neuroprotective effect in OGD-induced hippocampal neurons [36]. The overexpression of DRP-1 in nerve cells will eventually lead to cell apoptosis and mitochondrial lysis through activation of caspase system and damage of important proteins and organelles in mitochondria [37]. Our study document and validation suggested that Rc ameliorates neuron damage on oxygen-glucose deprivation associated with regulating TNF-α and DRP-1. The neuroprotective effects of Rc may be related to in ammatory response and necroptosis during nerve cell injury.

Conclusions
This study identi ed the neuroprotective activity of ginsenoside Rc mediated by its regulation on TNF-α and DRP-1. Rc may be involved in the pathological process of cell necroptosis and in ammatory. This work provide a step forward in the understanding of the neuroprotective effect and underlying mechanism of Rc on OGD induced neuron damage. However, the current study is performed based on in vitro experiments and the conclusions remain to beconfrmed by in vivo experiments.

Consent for publication
This manuscript is approved by all authors for publication.

Availability of data and materials
The datasets generated for this study are available on request to the corresponding author.
Competing interests