An Investigation of the Molecular Mechanisms Underlying the Analgesic Effect of Jakyak-Gamcho Decoction: A Network Pharmacology Study

Herbal drugs have drawn substantial interest as effective analgesic agents; however, their therapeutic mechanisms remain to be fully understood. To address this question, we performed a network pharmacology study to explore the system-level mechanisms that underlie the analgesic activity of Jakyak-Gamcho decoction (JGd; Shaoyao-Gancao-Tang in Chinese and Shakuyaku-Kanzo-To in Japanese), an herbal prescription consisting of Paeonia lactiflora Pallas and Glycyrrhiza uralensis Fischer. Based on comprehensive information regarding the pharmacological and chemical properties of the herbal constituents of JGd, we identified 57 active chemical compounds and their 70 pain-associated targets. The JGd targets were determined to be involved in the regulation of diverse biological activities as follows: calcium- and cytokine-mediated signalings, calcium ion concentration and homeostasis, cellular behaviors of muscle and neuronal cells, inflammatory response, and response to chemical, cytokine, drug, and oxidative stress. The targets were further enriched in various pain-associated signalings, including the PI3K-Akt, estrogen, ErbB, neurotrophin, neuroactive ligand-receptor interaction, HIF-1, serotonergic synapse, JAK-STAT, and cAMP pathways. Thus, these data provide a systematic basis to understand the molecular mechanisms underlying the analgesic activity of herbal drugs.


Materials and Methods
where m is the number of chemical compounds, n is the number of targets of chemical compound j, d i is the number of links of target i of chemical compound j, and c i (or c j ) is the number of previous studies having "pain" and component i (or j) in their title or abstract searched from the PubMed database (https://pubmed.ncbi.nlm.nih.gov/). e chemical compounds with the highest CIs were regarded as contributing more to the pharmacological activity of a certain herbal drug [81].

Functional Enrichment Analysis.
Gene ontology (GO) enrichment analysis was performed with g:Profiler [106]. Pathway enrichment analysis was performed with Kyoto Encyclopedia of Genes and Genomes database [107]. Functional association analysis was conducted using Gen-eMANIA [108].

Molecular Docking
Analysis. e structures of chemical compounds of JGd and their targets were obtained from the PubChem [109] and RCSB Protein Databank [110] databases, respectively. en, the molecular docking scores between the chemical compounds and the targets were assessed using AutoDock Vina [111]. Of note, a certain chemical compound is regarded as having high binding affinity to a target if the corresponding docking score is less than or equal to −5.0 [112,113].

Results
e network pharmacology study for the exploration of analgesic mechanisms of JGd was conducted as follows ( Figure 1). Detailed information regarding the chemical constituents of JGd was obtained from the comprehensive biomolecular databases, and the bioactive compounds were investigated using their ADME characteristics ( Figure 1). e human targets of the active chemical compounds were identified from various databases and models that assess chemical-protein interactions ( Figure 1). en, we integrated the extensive herbal drug-related data into networks and performed network pharmacology analysis ( Figure 1).

Network Pharmacology-Based Analysis of Jakyak-Gamcho
Decoction. To perform network pharmacology-based analysis of the pharmacological features of JGd, we constructed an herbal medicine-active chemical compound-target (H-C-T) network composed of 129 nodes (two herbal medicines, 57 active chemical compounds, and 70 pain-associated targets) and 217 links (Figure 2 and Supplementary Table S3) using comprehensive information regarding the herbal drug. We found that quercetin (number of targets � 36) and kaempferol (number of targets � 11) have relatively many targets ( Figure 2 and Supplementary Table S3), implying that they might be important active compounds for the therapeutic activity of JGd. In addition, 27 human genes/proteins were found to be targeted by two or more active chemical compounds of JGd (Figure 2), suggesting a polypharmacological mechanism.
To investigate the biological interaction relationship between the JGd targets, we generated a PPI network (58 nodes and 174 links) comprising the targets ( Figure 3). Next, we searched for hubs, specific nodes with a high degree in the network that are shown to have crucial biological functions and promising therapeutic potential [129,130]. In the analysis, hubs were determined as nodes for which the degree was greater than or equal to twice the average node degree of the network [131,132]. e results showed that PIK3R1 (degree � 25), HSP90AA1 (degree � 15), EGFR (degree � 14), AKT1 (degree � 13), LPAR1 (degree � 13), LPAR2 (degree � 13), and LPAR3 (degree � 13) were hubs (Figure 3), implying that they might be the key targets responsible for the analgesic activity of JGd. ese hubs were shown to be involved in the regulation of pain-related processes and could function as potent targets to induce analgesic effects. e PIK3R1 gene was suggested to have the potential to function as a painrelated regulator according to the genetic interaction analysis [133], and its expression level might be associated with osteoarthritis pathogenesis [134]. Upregulation of the HSP90AA1 gene was observed in patients with fibromyalgia [135][136][137], and pharmacological inhibition of heat shock protein 90 (HSP90; encoded by HSP90AA1) was shown to alleviate monoarthritis-induced pain [138]. e activation of epidermal growth factor receptor (EGFR; encoded by EGFR) and AKT (encoded by AKT1) is associated with the development and enhancement of diverse types of pain, and their therapeutic modulation might be associated with analgesic properties [139][140][141][142][143][144][145][146][147][148][149][150][151][152][153][154][155][156]. Lysophosphatidic acid receptor 1 (encoded by LPAR1) activity is involved in pain behavior arising from bone cancer, inflammation, diabetes, and neuropathy, and its pharmacological or genetic ablation might reduce the pain response [157][158][159][160][161][162][163][164][165]. Lysophosphatidic acid receptor 3 (encoded by LPAR3) plays crucial roles in the development and maintenance of neuropathic pain, and its blockade exerts analgesic effects [163,166,167].
We further assessed the CIs of the active chemical compounds of JGd to assess their pharmacological contribution to the analgesic effect of the herbal drug as described earlier [81,168]. As a result, quercetin was shown to have the highest CI (91.83%) (Supplementary Figure S1), which suggests that this chemical compound might be the primary contributor to the analgesic activity of JGd.
Together, these data indicate the system-level pharmacological properties of the analgesic activity of JGd.

Functional Enrichment Investigation of Jakyak-Gamcho Decoction Networks.
To investigate the molecular mechanisms underlying the analgesic effect of JGd, we carried out GO enrichment analysis of the targets. As a result, the JGd targets were enriched in GO terms involved in the modulation of a variety of biological activities, such as calcium-and cytokine-mediated signalings, calcium ion concentration and homeostasis, cellular behaviors of muscle and neuronal cells, inflammatory response, and response to chemical, cytokine, drug, and oxidative stress (Supplementary Figure S2), which are in accordance with the previously reported molecular mechanisms of the herbal drug [40, 41, 44, 46, 49, 55, 58-60, [169][170][171][172]. In addition, GeneMANIA analysis indicated that the JGd targets might functionally interact via diverse mechanisms (Supplementary Figure S3), implying the similarity in their pharmacological roles.
Collectively, these results demonstrate the molecularand pathway-level mechanisms underlying the analgesic activity of JGd.

Molecular Docking Evaluation.
To investigate the binding potential of the chemical compounds of JGd components for the targets, we evaluated their molecular docking activity. As a result, 95.09% of the binding interactions between the active chemical components of JGd and the hub targets was found to have docking scores equal to or lower than −5.0 ( Figure 5 and Supplementary Table S4), indicating their therapeutic binding potential. Of note, the protein structures for LPAR2 and LPAR3 were unavailable in the RCSB Protein Databank [110]; therefore, they were excluded from the analysis.

Discussion
Herbal medicines are increasingly being acknowledged as effective analgesic and pain-relieving agents owing to their promising therapeutic activity with fewer side effects [29][30][31][32][33][34][35][36]. JGd is a well-known herbal drug that alleviates pain induced by multiple diseases such as peripheral neuropathy, myalgia, arthralgia, and diabetes [30, 37-40, 44, 46-48], and it is one of the most frequently prescribed oral analgesics in East Asia [56]. Previous studies have attempted network pharmacology analyses to investigate the mechanisms underlying JGd for the treatment of osteoarthritis and Parkinson's disease [293,294]; however, its network-perspective analgesic properties have not been fully elucidated.
To conclude, we investigated the systems' perspective pharmacological properties of JGd, a widely prescribed analgesic herbal drug [56]. Based on the network pharmacological approach, we investigated 57 active chemical compounds and their 70 pain-related targets responsible for the analgesic activity of JGd. e targets of JGd were associated with the modulation of biological functions such as calcium-and cytokine-mediated signalings, calcium ion concentration and homeostasis, cellular behaviors of muscle and neuronal cells, inflammatory response, and response to chemical, cytokine, drug, and oxidative stress, which suggests the molecular mechanisms of JGd treatment. In addition, the enrichment analysis indicated that the targets are involved in various pathways that are associated with the pathophysiology of pain, including the PI3K-Akt, estrogen, ErbB, neurotrophin, neuroactive ligand-receptor interaction, HIF-1, serotonergic synapse, JAK-STAT, and cAMP pathways. e overall data offer a novel systematic view of the polypharmacological characteristics of herbal drugs and a mechanistic basis for their clinical implications for pain treatment.

Data Availability
e data used to support the findings of this study are included within the article and supplementary materials file.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Supplementary Materials
Supplementary Figure S1: contribution index analysis for the analgesic activity of active chemical compounds of Jakyak-Gamcho decoction. Supplementary Figure S2: functional enrichment analyses for the pain-associated targets of Jakyak-Gamcho decoction. Supplementary Figure S3: functional interaction analysis of the pain-associated targets of Jakyak-Gamcho decoction. Supplementary Table S1: list of the chemical compounds contained in Jakyak-Gamcho decoction. Supplementary Table S2: list of the active chemical compounds contained in Jakyak-Gamcho decoction. Supplementary Table S3: list of the targets of active chemical compounds of Jakyak-Gamcho decoction. Supplementary Table S4: