Prediction of the Medicinal Mechanisms of Pinellia ternata Breitenbach, a Traditional Medicine for Gastrointestinal Motility Disorders, through Network Pharmacology

Pinellia ternata Breitenbach (PTB) is a widely used herbal medicine in China, Japan, and South Korea. It has antiemetic, anti-inflammatory, antitussive, and sedative properties. The raw material is toxic, but can be made safer using alum solution or by boiling it for a long time. In addition, PTB seems to be effective for gastrointestinal motility disorders (GMDs), but this is yet to be conclusively proven. Herein, PTB compounds, targets, and related diseases were investigated using the traditional Chinese medical systems pharmacology database and an analysis platform. Information on target genes was confirmed using the UniProt database. Using Cytoscape 3.8.2, a network was established and GMD-related genes were searched using the Cytoscape stringApp. The effects of the PTB extract on the pacemaker potential of interstitial cells of Cajal and GMD mouse models were investigated. In total, 12 compounds were found to target 13 GMD-related genes. In animal experiments, PTB was found to better regulate pacemaker potential in vitro and inhibit GMD signs compared to control groups in vivo. Animal studies showed that the mechanism underlying the effects of PTB is closely related to gastrointestinal motility. The results obtained demonstrated that PTB offers a potential means to treat GMDs, and we suggested that the medicinal mechanism of GMDs can be explained by the relationship between 12 major components of PTB, including oleic acid, and 13 GMD-related genes.


Introduction
Gastrointestinal motility disorders (GMDs) can occur anywhere in the gastrointestinal tract. Therefore, such conditions can exhibit a variety of chronic symptoms that significantly affect a patient's quality of life, including nausea and vomiting [1][2][3]. Currently, there is no cure for GMD, and management may involve lifestyle changes and drugs. The gastrointestinal process occurs in each segment of this organ, and the contents move between each segment through active and passive peristaltic movements, which are slow waves of muscle contraction and relaxation [4,5]. Smooth muscle cells, intestinal neurons, including telosites, and interstitial cells of Cajal (ICCs) play an important role in gastrointestinal motility. Various studies have shown that ICCs act as special gastrointestinal pacemaker cells [6,7]. Thus, abnormalities in ICCs are related to several GMDs such as gastroparesis [8].
Pinellia ternata Breitenbach (PTB) is one of the main ingredients in traditional herbal medicine and has been used for antiemetic, antitussive, anti-inflammatory, and sedative pacemaker cells [6,7]. Thus, abnormalities in ICCs are related to several GMDs such as gastroparesis [8].
Pinellia ternata Breitenbach (PTB) is one of the main ingredients in traditional herbal medicine and has been used for antiemetic, antitussive, anti-inflammatory, and sedative purposes since ancient times [9]. Previously characterized phytochemicals in this herb include alkaloids [10], volatile oils [11], and polysaccharides [12]. To investigate the effects of PTB on GMDs and the medicinal mechanisms, we used a network-based system pharmacological approach. We used the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP, https://tcmsp-e.com/tcmsp.php. accessed date: 10 January 2022 to 15 March 2022), which provides comprehensive information on the relationship between herbs and their components, molecular targets, and diseases. TCMSP provides information on the absorption, distribution, metabolism, and excretion (ADME), which is useful for developing new drugs or analyzing the efficacy of herbal medicines [13]. A schematic of the research protocol is shown in Figure 1. In this study, a network-based pharmacological analysis of PTB was performed. In addition, PTB extracts were examined for their pacemaking activity and evaluated for their therapeutic effects in mouse models of GMDs. Figure 1. Schematic of the study protocol for network pharmacology exploration. ADME: absorption, distribution, metabolism, and excretion.

Information for 366 Targets Derived through Correlation Investigation between Compounds and Targets
We identified 116 potentially active compounds in PTB using the TCMSP database (Supplementary Materials 1). Among these, 85 compounds contained the target information (Supplementary Materials 2), and it was found that these 85 compounds and 366 targets interacted with each other through a combination of 1226 compounds ( Figure 2). As shown in Figure 2, GLY was linked to the most target genes (161 genes), followed by DAL (65 genes), succinic acid (57 genes), L-serine (54 genes), ASI (47 genes), oleic acid (43 Figure 1. Schematic of the study protocol for network pharmacology exploration. ADME: absorption, distribution, metabolism, and excretion.

Figure 2.
Compound-target network of Pinellia ternate Breitenbach. The size of the node depends on the number of connected edges. The compounds are expressed as red square nodes, and the targets are expressed as blue round nodes.

Molecule Name
Gene Name Disease Name

All 52 GI Disease-Related Compounds, Except 6-Shogaolin, in PTB Are Related to GMDs
To determine the relationship between PTB and GMDs, we used the Cytoscaping app to check for GMD-related genetic information. First, 100 GMD-related genes were identified by applying a reliability (score) cutoff of 0.40 and a maximum of 100 proteins (Supplementary Materials Table S3). From the results obtained, a network of GMD-related genes and PTB target genes was created (Figure 4). It was confirmed that there were 13 genes corresponding to both sets of genes. The GMD-related genes targeted by PTB were actin, cytoplasmic 1 (ACTB), cholecystokinin (CCK), C-reactive protein (CRP), proto-oncogene c-Fos (FOS), glucagon (GCG), insulin (INS), myeloperoxidase (MPO), Mu-type opioid receptor (OPRM1), prostaglandin G/H synthase 2 (PTGS2), peptide YY (PYY), sodium channel protein type 5 subunit alpha (SCN5A), sodium-dependent serotonin transporter (SLC6A4), and transient receptor potential cation channel subfamily V member 1 (TRPV1).

All 52 GI Disease-Related Compounds, except 6-Shogaolin, in PTB Are Related to GMDs
To determine the relationship between PTB and GMDs, we used the Cytoscaping app to check for GMD-related genetic information. First, 100 GMD-related genes were identified by applying a reliability (score) cutoff of 0.40 and a maximum of 100 proteins (Supplementary Materials 3). From the results obtained, a network of GMD-related genes and PTB target genes was created (Figure 4). It was confirmed that there were 13 genes corresponding to both sets of genes. The GMD-related genes targeted by PTB were actin, cyto-  . Network of gastrointestinal motility disorder-related genes and Pinellia ternate Breitenbach-targeting genes. The 13 genes included in both "genes related to gastrointestinal motility disorder" and "Pinellia ternate Breitenbach-target genes" are collected in the center. Figure 5 illustrates the network of relationships between PTB compounds and GMDrelated target genes. The results showed that oleic acid and PTGS2 are most closely related to GMD. In summary, EIC, 6-shogaol, cavidine, baicalein, beta-sitosterol, methyl palmitelaidate, (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl)piperazine-2,5-quinone, linolenic acid, stigmasterol, coniferin, oleic acid, pedatisectine a, and cyclo-(val-tyr) were determined to be active compounds that target GMD-related genes, indicating that these compounds might be potential medicinal candidates. However, as shown in Figure 2, PTB contained many compounds with multi-targeting characteristics; thus, the synergistic effects of PTB compounds on GMD were investigated in vitro and in vivo. . Network of gastrointestinal motility disorder-related genes and Pinellia ternate Breitenbachtargeting genes. The 13 genes included in both "genes related to gastrointestinal motility disorder" and "Pinellia ternate Breitenbach-target genes" are collected in the center. Figure 5 illustrates the network of relationships between PTB compounds and GMDrelated target genes. The results showed that oleic acid and PTGS2 are most closely related to GMD. In summary, EIC, 6-shogaol, cavidine, baicalein, beta-sitosterol, methyl palmitelaidate, (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl)piperazine-2,5-quinone, linolenic acid, stigmasterol, coniferin, oleic acid, pedatisectine a, and cyclo-(val-tyr) were determined to be active compounds that target GMD-related genes, indicating that these compounds might be potential medicinal candidates. However, as shown in Figure 2, PTB contained many compounds with multi-targeting characteristics; thus, the synergistic effects of PTB compounds on GMD were investigated in vitro and in vivo.

Effects of PTB Extract on the Pacemaker Potential of ICCs
ICCs regulate gastrointestinal motility by acting as pacemakers in the GI tract [6][7][8].

Importance of Ca 2+ in PTB Extract-Induced Pacemaker Potential Depolarization of ICCs
To investigate the importance of Ca 2+ in PTB extract-induced responses, we used external Ca 2+ -free conditions or thapsigargin. Pre-treatment with an external Ca 2+ -free solution or thapsigargin suppressed pacemaker potentials and inhibited PTB extract-induced responses ( Figure 7A,B). The average depolarization was 1.1 ± 0.5 mV (p < 0.0001) with Ca 2+ -free solution and 1.2 ± 0.3 mV (p < 0.0001) with thapsigargin ( Figure 7C). However, the average amplitude did not change significantly ( Figure 7D). These results indicated that the PTB extract-induced responses are controlled by Ca 2+ .

Importance of Ca 2+ in PTB Extract-Induced Pacemaker Potential Depolarization of ICCs
To investigate the importance of Ca 2+ in PTB extract-induced responses, we used external Ca 2+ -free conditions or thapsigargin. Pre-treatment with an external Ca 2+ -free solution or thapsigargin suppressed pacemaker potentials and inhibited PTB extract-induced responses ( Figure 7A,B). The average depolarization was 1.1 ± 0.5 mV (p < 0.0001) with Ca 2+ -free solution and 1.2 ± 0.3 mV (p < 0.0001) with thapsigargin ( Figure 7C). However, the average amplitude did not change significantly ( Figure 7D). These results indicated that the PTB extract-induced responses are controlled by Ca 2+ .

Discussion
PTB is a monocotyledonous perennial herbaceous plant that is widely used in traditional herbal medicine in China, Japan, and South Korea. It has been used for antiemetic, anti-inflammatory, antitussive, and sedative purposes [18]. In addition, experiments using a mouse model have shown that PTB induces efferent activity in the gastric branches of the vagus nerve, which is known to be effective in GMDs, but the pharmacological mechanisms underlying this have yet to be studied [19]. Therefore, this study was conducted using a combination of network-based pharmacological analyses and experimental verification to identify the physiologically active ingredients and medicinal mechanisms of PTB. Additionally, 116 compounds were identified in PTB, including 19 active compounds (Sup. 1). Of these 116 compounds, 85 had target information and 365 target genes were collected (Sup. 2). Twelve genes were associated with GMD ( Figure 5 Table 2 and Figure 5, PTGS2 was the target of all GMD-related PTB compounds, suggesting that PTB compounds can adjust PTGS2 levels synergistically. PTGS2 plays the most important role in the treatment of mucosal defense and gastrointestinal inflammation and ulcers. PTGS2 also contributes to normalization of gastrointestinal function after inflammation [20]. In addition, SCN5A and OPRM1 were found to be targets of multiple PTB compounds. The voltage-gated mechanosensitive Na + channel NaV1.5, encoded by SCN5A, is present in ICCs and human gastrointestinal smooth muscle cells. SCN5A contributes to the electrical slow wave and mechanical sensitivity of smooth muscles [21]. OPRM1 is linked to the inhibition of acetylcholine release from intestinal and motor neurons and the inhibition of purine and nitrogen release from motor neurons, thereby inhibiting propulsion kinetic patterns [22]. OPRM1 activation induces the inhibition of submucosal secretion by motor neurons, which reduces active Cl secretion and passive liquid movement to the colon lumen [23]. This effect on mobility and secretion results in constipation induced by OPRM1. These results indicate that the medicinal mechanism of GMDs is related to the effects of PTB on PTGS2, SCN5A, and ORPM1. The GMD-related active

Discussion
PTB is a monocotyledonous perennial herbaceous plant that is widely used in traditional herbal medicine in China, Japan, and South Korea. It has been used for antiemetic, anti-inflammatory, antitussive, and sedative purposes [18]. In addition, experiments using a mouse model have shown that PTB induces efferent activity in the gastric branches of the vagus nerve, which is known to be effective in GMDs, but the pharmacological mechanisms underlying this have yet to be studied [19]. Therefore, this study was conducted using a combination of network-based pharmacological analyses and experimental verification to identify the physiologically active ingredients and medicinal mechanisms of PTB. Additionally, 116 compounds were identified in PTB, including 19 active compounds (Sup. 1). Of these 116 compounds, 85 had target information and 365 target genes were collected (Sup. 2). Twelve genes were associated with GMD ( Figure 5), including ACTB, CCK, CRP), FOS, GCG, INS, MPO, OPRM1, PTGS2, PYY, SCN5A, SLC6A4, and TRPV1 ( Figure 4). These results are the same as those of past studies. Specifically, as shown in Table 2 and Figure 5, PTGS2 was the target of all GMD-related PTB compounds, suggesting that PTB compounds can adjust PTGS2 levels synergistically. PTGS2 plays the most important role in the treatment of mucosal defense and gastrointestinal inflammation and ulcers. PTGS2 also contributes to normalization of gastrointestinal function after inflammation [20]. In addition, SCN5A and OPRM1 were found to be targets of multiple PTB compounds. The voltage-gated mechanosensitive Na + channel NaV1.5, encoded by SCN5A, is present in ICCs and human gastrointestinal smooth muscle cells. SCN5A contributes to the electrical slow wave and mechanical sensitivity of smooth muscles [21]. OPRM1 is linked to the inhibition of acetylcholine release from intestinal and motor neurons and the inhibition of purine and nitrogen release from motor neurons, thereby inhibiting propulsion kinetic patterns [22]. OPRM1 activation induces the inhibition of submucosal secretion by motor neurons, which reduces active Cl secretion and passive liquid movement to the colon lumen [23]. This effect on mobility and secretion results in constipation induced by OPRM1. These results indicate that the medicinal mechanism of GMDs is related to the effects of PTB on PTGS2, SCN5A, and ORPM1. The GMD-related active compounds EIC, (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl)piperazine-2,5-quinone, baicalein, beta-sitosterol, cavidine, coniferin, cyclo-(val-tyr), linolenic acid, methyl palmitelaidate, oleic acid, pedatisectine a, and stigmasterol were identified ( Figure 5). In addition, 12 compounds were found to target PTGS2, and oleic acid was found to target PTGS2, MPO, CCK, CRP, GCG, INS, and PYY. Beta-sitosterol targets PTGS2, SCN5A, OPRM1, and SLC6A4, and cavidine targets PTGS2, SCN5A, OPRM1, and SLC6A4. Several studies have reported a relationship between the major components of PTB and GMDs. Emulsions containing oleic acid ac-tivate nutrient-induced inhibitory feedback mechanisms in the small intestine, slowing down gastrointestinal passage and reducing diarrhea [24]. beta-sitosterol has significant antibacterial activity and improved DSS-induced colitis in mice [25]. Cavidine improved ulcerative colitis by regulating the oxidation and antioxidant balance and inhibiting NF-κB signaling pathways and pro-inflammatory cytokines, such as TNF-α and IL-6, in colonic tissue [26]. As shown in Figure 2, compounds interacted with an average of 14 target genes upon identifying the multi-compound multi-target properties of herbal medicines, and PTB was predicted to be a therapeutic medicine for GMDs based on the synergies among several compounds contained in PTB.
We investigated the restorative effects of PTB in a mouse model of GMDs. First, we checked the effects of PTB extract on the pacemaker potentials of ICCs. PTB extract was found to regulate the pacemaker potential of ICCs, and this response was controlled by Ca 2+ (Figures 6 and 7). In addition, PTB reactions occurred through M3, 5-HT3, and 5-HT7 receptors ( Figure 8). Further, PTB extract increased the normal ITR and restored the ITR in GMD mice (Figure 9). In summary, our results showed that PTB has potential for the treatment of GMDs, and that its medicinal effect may be due to the regulation of ICCs. They also suggested that these mechanisms are related to the interactions among 12 key components of PTB, such as oleic acid, and 13 GMD-related genes, including PTGS2.

Identification of PTB Compounds
Analysis platforms were used to identify the potential active compounds of PTB. We entered 'Pinellia ternata Breitenbach' as a query to search for the herb name.

Analysis of Targets
The target information of the compound was determined by searching TCMSP [13]. The target proteins were linked to the official gene names using the UniProtKB database (https://www.uniprot.org/uniprot. accessed date: 10 January 2022 to 15 March 2022) [27].

Preparation of the PTB Extract
Dried PTB was purchased from Herb Farm Co. (Wonju, Korea). A voucher specimen (PJW-058) was deposited at the Department of Herbology, College of Korean Medicine, Kyung Hee University, Korea. Dried PTB (400 g) was extracted using distilled water (4000 mL) for 2 h at 100 • C. The water extract of PTB was passed through a membrane filter (0.45 µm; EMD Millipore). After evaporation, the remaining aqueous extract was freeze-dried to yield 8.5% of the dried weight (w/w).

Preparation of ICCs
A total of 74 ICR mice (37 males and 37 females; 3-8 days-old) were used for the experiments. Small intestinal cells were cultured with smooth muscle growth medium (Clonetics, San Diego, CA, USA).

Intestinal Transit Rate (ITR)
A total of 45 ICR mice (males, 5-6 weeks-old) were used for ITR experiments. Evans blue (5%, w/v) was administered after the administration of PTB extract into the stomach. After 30 min of Evans blue administration, the ITR was measured.

GMD Model Mice
Acetic acid (AA, 0.6%, w/v, in saline)-induced peritoneal stimulation was used to generate a GMD mouse model. AA was injected intraperitoneally, and other processes were the same as previous studies [30,31].

Statistical Analyses
Data are expressed as the mean ± standard error of the mean. Significant differences were evaluated using one-way analysis of variance or a student's t-test. Statistical significance was considered as p < 0.05.

Conclusions
PTB analysis using a network-based pharmacological approach showed that 12 compounds and 13 genes were associated with GMDs. Our animal studies showed that PTB regulates the pacemaker potential of ICCs and inhibits GMD-like signs in a mouse model of GMDs. These results indicate that PTB has therapeutic potential for GMD treatment. In addition, we proposed a mechanism responsible for the interactions among 12 PTB compounds and 13 GMD-related genes.