Bioinformatics findings reveal the pharmacological properties of ferulic acid treating traumatic brain injury via targeting of ferroptosis

ABSTRACT Ferroptosis refers to a lytic cell death avenue that may be related to the development of neuropathological disorders. Traumatic brain injury (TBI) is the brain impairments and ferroptosis is found with the pathological role in TBI development. Our previous findings showed that ferulic acid exerts pharmacological benefited against TBI. However, the therapeutic efficacy and mechanism targeting of ferroptosis in ferulic acid against TBI is still unclear. In current report, an integrated approach by using network pharmacology and molecular docking analyses was subjected to identify the ferulic acid-anti-TBI mechanisms and targets associated with ferroptosis. The network pharmacology analysis had screened 184 ferulic acid-related targets, 1834 TBI-associated targets and 616 ferroptosis-linked targets, characterized with 14 overlapping genes among ferulic acid, TBI, and ferroptosis. All core targets in ferulic acid treating TBI via regulation of ferroptosis were identified through parametric determination, including PTGS2, TLR4, RELA, GSK3B, NFE2L2, EGFR, and MIF. Following with enrichment analysis, anti-TBI functions of ferulic acid against TBI targeting ferroptosis were revealed in multiple biological processes, including regulation of polymerase activities, transcription factor functions, ubiquitin protease binding capabilities. Pharmacological mechanisms were detailed in signaling pathways mainly involved in neuroprotection, microenvironmental restoration and neural regeneration were the key functional characteristics. Further in silico validation exhibited that PTGS2 was a potential pharmacological target associated with ferroptosis in ferulic acid against TBI owing to the potent binding energy. Collectively, current bioinformatics data provide a new preclinical insight of the pharmacological function and mechanism targeting of ferroptosis in ferulic acid treating TBI. Compared to other correlative researches, this study may effectively exhibit the preclinical perspective in new drug research and development on ferulic acid against TBI through revealing multiple targets and pathways.


Introduction
Traumatic brain injury (TBI), more known as brain trauma, is a severe neurocranial lesion stricken by external force, and its outcome is characterized with elevated disability and mortality. [1]Clinical treatment advances on TBI are achieved currently through targeted drugs for promoting patient rehabilitation. [2]he TBI cases diagnosed in China are greater than other countries owing to the hug population, and it can cause a major public health concerns. [3]Computerized tomography medical imaging is recommended as the prioritized screening for TBI prior to making personalized treatment regimen for the patients. [4]The histopathological onset of TBI is involved in astrocyte loss, neuronal necrosis, and cell death in the brain. [5]The occurrence of astrocyte swelling, brain edema, hypoxia-ischemia, inflammatory infiltration is the neuropathological features of TBI, in which these outcomes may be lethal if untreated. [6]t is reported that astrocytes are functionally related to brain plasticity, synaptic transmission, and neuronal metabolism. [7]Additionally, astrocytic dysfunction can cause adverse actions and it may result in the development of neurological disorders. [8]Therefore, post-brain injury recovery via melioration of neuronal functions may be a promising therapeutic approach against TBI. [9]In clinical treatment, antiinflammatory, antiepileptic, pro-growth medication can be used commonly for TBI, [10] however, some poor therapeutic effects may be observed.Thus, substitute pharmacotherapy for TBI still needs to be explored successively, especially screening novel pharmacological target.Ferroptosis, a nonapoptotic form of cell death, can exert the key action in degenerative brain disorders, including intracerebral hemorrhage, stroke. [11]Increasing studies have indicated that ferroptosis is involved in the pathophysiological process of TBI, and thus suppression of ferroptosis may achieve positive benefits for TBI. [12]As revealed in the underlying pathogenesis of ferroptosis in TBI, excessive iron deposition in brain tissue results in lipid peroxidation reaction, reactive oxygen species-induced oxidative injury, mitochondrial dysfunction and neuroinflammatory stress, eventually causing neuronal death. [13]Therefore, the screening of ferroptosisinterrelated biomarkers may help to development of promising therapeutics for TBI clinical applications. [14]Owing to shortage of clinical drugs that can effectively activate or inhibit ferroptosis, modulating ferroptosis is still difficult presently. [15]Thus, further exploration of potential bioactive compounds, especially natural ingredients, is needed imminently.
Pharmacologically bioactive compounds from Traditional Chinese medicine have potential benefits in relieving brain disorders, [16] including TBI.Ferula sinkiangensis K. M. Shen is an ethno-drug that may use for relieving human ailments. [17]Other pharmacological evidences indicate that some bioactive ingredients isolated from Ferula sinkiangensis K. M. Shen exhibit neuroprotection by inhibiting microglial activation and reducing the inflammatory response. [18]It is believed that Ferula sinkiangensis K. M. Shen may rich in potential phytochemicals that exert promising neuroprotective effects.Ferulic acid, one of the bioactive compounds found in Ferula sinkiangensis K. M. Shen, has various pharmacological activities, including antidiabetes, anti-cardiovascular action and preventing neurodegenerative diseases. [19]As a potent antioxidant, ferulic acid may be used to suppress the release of free radicals to mitigate oxidative damage. [20]nterestingly, other preclinical data show that ferulic acid exhibits potential therapeutic benefits against neurological disorders through regulating neuro-signaling pathways. [21]An experimental study demonstrates the neuroprotective effect of ferulic acid in traumatic brain impairment in vivo, and this pharmacological activity is involved in primarily reducing neuronal apoptosis. [22]However, molecular mechanisms of ferulic acid in the treatment of TBI remain unreported.Although ferroptosis is found as a potential mechanism against myocardia injury following ferulic acid treatment, [23] anti-TBI mechanism targeting of ferroptosis is still needs to be investigated.Network pharmacology is merging as a multidisciplinary methodology that may be used to determine potential curative targets and mechanisms of compounds or ingredients against human diseases. [24,25]Interestingly, molecular docking approach can be served as an advanced tool to reveal the protein structure-based drug design, including therapeutic targets. [26]In this study, we applied the network pharmacology approach and molecular docking analysis to identify the anti-TBI core targets of ferulic acid through modulation of ferroptosis.In addition, core target-enriched molecular mechanisms of ferulic acid against TBI were characterized by using the analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively.Thus, we aimed to propose a promising hypothesis that current preclinical evaluation methodology may be helpful to promote researchers' interest of exploring mechanisms of Chinese herb compounds against TBI, especially ferulic acid.

Acquisition of intersection targets in ferroptosis, TBI and ferulic acid
All ferroptosis, TBI, and ferulic acid targets were mapped using the online tool Venn diagram analysis that applied network propagation on gene-gene interaction network to correlate multiple targets, characterized by gene rankings in interaction diagram with additional analysis. [27]

Core target identification and network construction
The intersection targets were determined with the String (http://string-db.org)database to assess functional target protein interactions, in which the lowest reciprocal value was 0.09.The resultant data were then imported into Cytoscape (https://cytoscape.org)database to construct the ferulic acid anti-TBI-iron death target protein-protein interaction network in ferulic acid against TBI associated with ferroptosis.NetworkAnalyzer plug-in in Cytoscape tool was used to analyze topological parameters in interaction network, including median and maximum degrees of freedom.The core targets were screened and identified according to degree values, in which the upper limit of the screening range was the maximum degree value in the topological data and the lower limit was the median degrees of freedom.

GO and KEGG enrichment analysis and network visualization of core targets
The DAVID (http://www.david.niaid.nih.gov)database was used to obtain the interactions between the core targets and functional and mechanical findings, R language packages such as "GOplot" were used to visualize the GO-associated biological process and signaling pathway enrichment assayed the core targets, subsequently producing the resultant bubble chart and histogram.These data were then imported into an online tool (http://www.bioinformatics.com.cn) to obtain the Circos circle chart.Additionally, aggregating network diagram characterized with ferulic acid anti-ferroptosis-TBI actions was presented using the Cytoscape tool.

Core target molecular docking validation
Molecular docking is an emerging technique that may use to identify the interactions in ligand attached to target protein. [28]The chemical structure of the ferulic acid compound were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov).The crystalline structures of potential proteins were gained from RCSB Protein Data Bank (PDB, https://www.rcsb.org).The Chem Bio Office 2010 software was used to offer as a central repository for knowledge on three-dimensional structures of ferulic acid via force field optimization.Using AutoDock Vina software analysis, [29] the ligands contained the water molecules (represented by virtual atoms) that might or might not be involved in the intermolecular interaction.The modified AutoGrid distribution map was further used during docking to give favorable ratings when the water was well placed, in which water molecule overlapping receptor was ignored.Subsequently, the site finder from the GridBox setting was subjected to predict the binding pockets of target proteins, and MGLTools in AutoDock software [30] was applied to effectively docking between ferulic acid compound and PTGS2 protein.We identified the bound poses that had not more than 4 Å in root mean square deviation (RMSD) and binding free energy.The bound scores were applied as assessment criteria for screening of ferulic acid compound and PTGS2 target before exclusion of other potential target proteins.

Ferroptosis, TBI, and ferulic acid target data
The bioinformatics analysis showed that total of 616 genes related to ferroptosis were collected and 1,834 TBI-interrelated and 184 ferulic acid-acted targets were screened, respectively.After further analysis, other 14 shared targets in ferroptosis, TBI, and ferulic acid were obtained after correction by the UniProt database.The data connection is shown in Figure 1a.

Interaction network, topological parameters, and core targets
The protein-protein interaction data related to 14 shared targets of ferroptosis, TBI, and ferulic acid were constructed in a correlative network.The mapped intersecting targets were calculated for the topological parameters, and the median degree of freedom of core targets was 4, whereas the maximum degree of freedom was 9.The range of core targets screening standard was set from 4 to 9. The results showed that seven core targets were obtained, respectively: PTGS2, TLR4, RELA, GSK3B, NFE2L2, EGFR, and MIF (Figure 1b).

GO and KEGG enrichment data and network relationship visualization
Both GO and KEGG pathway enrichment analysis of the core intersection genes were characterized, respectively.The output data of ferulic acid against TBI targeting of ferroptosis were exhibited in the histogram (Figure 2a), and Circos circle plot (Figure 2b) in details.The KEGG signaling pathway enrichment results are shown in a bar chart (Figure 3a), and Circos circle chart (Figure 3b).The enrichment results elucidated that the biological processes associated with the action of ferulic acid against TBI targeting of ferroptosis were mainly involved in RNA polymerase II-specific DNA-binding transcription factor binding, DNA-binding transcription factor binding, heme binding, ubiquitin protein ligase binding, ubiquitin-like protein ligase binding, activating transcription factor binding, flavin adenine dinucleotide binding, DNA-binding transcription activator activity, RNA polymerase II-specific, DNA-binding transcription activator activity.The KEGG signaling pathways revealed that the core genes are notably enriched in the HIF-1 signaling pathway, Leishmaniasis, Alcoholic liver disease, PI3K-Akt signaling pathway, Alzheimer disease, Pertussis, Lipid and atherosclerosis, Chemical carcinogenesis reactive oxygen species, Human cytomegalovirus infection, PD-L1 expression, and PD-1 checkpoint pathway in cancer, IL-17 signaling pathway, and NF-kappa B signaling pathway.Furthermore, network visualization of ferulic acid-GO-KEGG-ferroptosis-TBI was visualized by using Cytoscape 3 (Figure 4).Finally, the core genes were predicted as the potential anti-TBI targets in ferulic acid and were identified for molecular docking assay.

Discussion
It is reported that targeted treatment of aquaporin, a transmembrane protein, to astrocytes and glial cells could be potentially used for brain edema following stroke or trauma. [31]Therefore, small-molecule aquaporin inhibitor for therapeutic development will provide new treatments for brain diseases, [32] including TBI.Thus, we speculate that exploring potential target may promote the identification of new bioactive compounds in the treatment of TBI.Traditionally, Chinese medicine compounds are effective therapeutics in the prevention and of neurodegenerative disorders. [33]Interestingly, ferulic acid is a bioactive small molecule from Ferula sinkiangensis K. M. Shen that may protect secondary TBI-related injury in a cell culture study . [34]However, the molecular mechanisms of ferulic acid against TBI remain unclear.Ferroptosis inhibitor is evolving as a potential therapeutic application in clinical neurodegenerative diseases. [35]Ferulic acid, a potent antioxidant, may target ferroptosis action to exert promising pharmacological activities, including anti-cancer, [36] anti-sepsis, [37] and beta-cell protection. [38]Based on these  reference reports, it is reasoned that the potential molecular mechanisms of ferulic acid against TBI may be associated with the regulation of ferroptosis action.In this study, we aimed to apply bioinformatic approach using network pharmacology and molecular docking for validating our proposed hypothesis.As results, all 14 shared target genes in ferulic acid-ferroptosis-TBI were selected and used for constructing correlative network.Among these shared genes, we determined PTGS2, TLR4, RELA, GSK3B, NFE2L2, EGFR, and MIF as core targets associated with ferroptosis in ferulic acid against TBI.The core target genes were reported and enriched in ferroptosis-related biological processes and functions, in which these gene regulations contributed to neuroprotection, microenvironmental restoration and neural regeneration were the key functional characteristics.In addition, enriched KEGG pathways were characterized in detail, including HIF-1 signaling pathway, PI3K-Akt signaling pathway, Alzheimer disease, and Chemical carcinogenesis-reactive oxygen species.Hypoxia is one of the main leading causes of TBI [39] and hypoxia reportedly acts as a regulatory component in regulation of ferroptosis action in brain heart injury . [40]Using the molecular docking approach, a few of the core target proteins were further identified in silico, including PTGS2.PTSG2, a proinflammatory cyclooxygenase gene, is highly related to the initiation and progression of inflammatory reactions. [41]In certain neurological disorders, over expression of PTSG2 is likely inducing inflammasome-activated neuroinflammation diseases . [42]In addition, abnormal activation of PTSG2 is positively associated with neuroinflammation development in TBI in vivo . [43]Other reports show that PTSG2 may be a potential molecule targeting ferroptosis in cerebral ischemia reperfusion lesions. [44]owever, a few evidences on PTSG2 targeting ferroptosis induced by ferulic acid against TBI were still unknown.Here, in silico findings indicated that ferulic acid may modulate PTSG2 protein activity through targeting ferroptosis for neuroprotection against TBI, implying that PTSG2 seems to be a potential pharmacological target in ferulic acid against TBI before experimental validation.In comparison with previous similar studies, our bioinformatics report contributes to develop the new drug research methodology in ferulic acid against TBI.However, due to existing limitations, further preclinical experiments in vivo should be performed to validate these bioinformatics findings prior to clinical application of ferulic acid for treating TBI.

Conclusion
Network pharmacology and molecular docking analyses are successfully subjected to this study for exploring and revealing the targets and mechanism of ferulic acid for the potential treatment of TBI, a life-threatening brain injury.Our present study demonstrates that the pharmacological effect of ferulic acid against TBI may be mostly associated with ferroptosis targeting signaling pathways.More importantly, ferulic acid may directly regulate PTSG2 activity via targeting ferroptosis for anti-TBI action.Additionally, these bioinformatics findings also indicate that the network pharmacology and molecular docking methods are useful for revealing the pharmacological targets and mechanisms of promising bioactive ingredients in Ferula sinkiangensis K. M. Shen in China.Although current report well presents the bioinformatics findings to predict the potential action of the ferulic acid in TBI, however, this study needs to be validated with in vitro and in vivo studies.

Figure 1 .
Figure 1.Correlative gene identification and network establishment in ferulic acid, TBI, and ferroptosis in Venn diagram analysis (a).Core target gene network identified in ferulic acid against TBI in relation to ferroptosis (b).

Figure 2 .
Figure 2. Bar diagram (a) and Circos graph (b) of the GO-enriched biological functions in ferulic acid against TBI in relation to ferroptosis.

Figure 3 .
Figure 3. Bar diagram (a) and Circos graph (b) of the KEGG-enriched signaling mechanisms in ferulic acid against TBI in relation to ferroptosis.

Figure 4 .
Figure 4. Integrated summarizing diagram in ferulic acid-ferroptosis-TBI findings based on current bioinformatics analysis.

Figure 5 .
Figure 5.In silico findings characterized the ferulic acid docking PTSG2 protein in TBI.