A Study on the Effects of Muscarinic and Serotonergic Regulation by Bojanggunbi-tang on the Pacemaker Potential of the Interstitial Cells of Cajal in the Murine Small Intestine.

In traditional Korean medicine, the 16-herb concoction Bojanggunbi-tang (BGT) is used to treat various gastrointestinal (GI) diseases. In this study, we investigated the regulatory mechanism underlying the influence of BGT on the interstitial cells of Cajal (ICCs), pacemaker cells in the GI tract. Within 12 h of culturing ICCs in the small intestines of mice, the pacemaker potential of ICCs was recorded through an electrophysiological method. An increase in the BGT concentration induced depolarization and decreased firing frequency. This reaction was suppressed by cholinergic receptor muscarinic 3 (CHRM3) antagonists, as well as 5-hydroxytryptamine receptor (5HTR) 3 and 4 antagonists. Nonselective cation channel inhibitors, such as thapsigargin and flufenamic acid, along with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors, also suppressed the BGT reaction. Guanylate cyclase and protein kinase G (PKG) antagonists inhibited BGT, but adenylate cyclase and protein kinase A antagonists had no effect. In conclusion, we demonstrated that BGT acts through CHRM3, 5HTR3, and 5HTR4 to regulate intracellular Ca2+ concentrations and the PKC, MAPK, guanylate cycle, and PKG signaling pathways.

A study of the therapeutic mechanism of Jakyakgamcho-Tang about functional dyspepsia through network pharmacology research.

Herbal medicines have traditionally been used as an effective digestive medicine. However, compared to the effectiveness of Herbal medicines, the treatment mechanism has not been fully identified. To solve this problem, a system-level treatment mechanism of Jakyakgamcho-Tang (JGT), which is used for the treatment of functional dyspepsia (FD), was identified through a network pharmacology study. The two components, paeoniae radix alba and licorice constituting JGT were analyzed based on broad information on chemical and pharmacological properties, confirming 84 active chemical compounds and 84 FD-related targets. The JGT target confirmed the relationship with the regulation of various biological movements as follows: cellular behaviors of muscle and cytokine, calcium ion concentration and homeostasis, calcium- and cytokine-mediated signalings, drug, inflammatory response, neuronal cells, oxidative stress and response to chemical. And the target is enriched in variety FD-related signaling as follows: MAPK, Toll-like receptor, NOD-like receptor, PI3K-Akt, Apoptosis and TNF signaling pathway. These data give a new approach to identifying the molecular mechanisms underlying the digestive effect of JGT.

[6]-Gingerol induces Caspase-Dependent Apoptosis in Bladder Cancer cells via MAPK and ROS Signaling.

The anti-cancer effects of [6]-gingerol ([6]-GIN), the main active polyphenol of ginger (Zingiber officinale), were investigated in the human bladder cancer cell line 5637. [6]-GIN inhibited cell proliferation, increased sub­G1 phase ratios, and depolarized mitochondrial membrane potential. [6]-GIN-induced cell death was associated with the downregulation of B­cell lymphoma 2 (BCL­2) and survivin and the upregulation of Bcl­2­associated X protein (Bax). [6]-GIN activated caspase­3 and caspase-9 and regulated the activation of mitogen-activated protein kinases (MAPKs). Further, [6]-GIN also increased the intracellular reactive oxygen species (ROS) levels and TG100-115 or tranilast increased [6]-GIN­induced cell death. These results suggest that [6]-GIN induced apoptosis in the bladder cancer cell line 5637 and therefore has the potential to be used in the development of new drugs for bladder cancer treatment.

Grape seed powder increases gastrointestinal motility.

Grape seed is an important natural bioactive product with various health benefits. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract. The present study investigated the effects of grape seed powder (GSP) on ICC properties and GI motility. GSP depolarized the pacemaker potentials of ICCs in a dose­dependent manner. Y25130 or SB269970 slightly inhibited GSP­induced effects. However, Y25130 and SB269970 together completely blocked GSP-induced effects. In the presence of inhibitors of protein kinase C, protein kinase A, or mitogen-activated protein kinase, GSP­induced ICC depolarization was inhibited. GSP increased the intestinal transit rate in normal mice and in mice with acetic acid-induced GI motility disorder. In addition, the levels of motilin and substance P were elevated after GSP dosing. These results demonstrate that GSP can regulate GI motility, and therefore, it is a potential therapeutic agent for treating GI motility disorders.

The novel resveratrol derivative 3,5-diethoxy-3',4'-dihydroxy-trans-stilbene induces mitochondrial ROS-mediated ER stress and cell death in human hepatoma cells in vitro.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells in vitro. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 µmol/L, respectively). Co-treatment with the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that pro-apoptotic ER stress and/or ROS may govern the Res-006-induced HepG2 cell death. We further revealed that treatment of HepG2 cells with Res-006 (65 µmol/L) immediately elicited the dysregulation of mitochondrial dynamics and the accumulation of mitochondrial ROS. It also collapsed the mitochondrial membrane potential and further induced ER stress and cell death. These events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300-1000 µmol/L). The results suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol derivative involves crosstalk between the mitochondria and ER stress mechanisms.

Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.

Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.

Atractylodes macrocephala Koidz Alleviates Symptoms in Zymosan-Induced Irritable Bowel Syndrome Mouse Model through TRPV1, NaV1.5, and NaV1.7 Channel Modulation.

(1) Background: Irritable bowel syndrome (IBS) is a common disease in the gastrointestinal (GI) tract. Atractylodes macrocephala Koidz (AMK) is known as one of the traditional medicines that shows a good efficacy in the GI tract. (2) Methods: We investigated the effect of AMK in a network pharmacology and zymosan-induced IBS animal model. In addition, we performed electrophysiological experiments to confirm the regulatory mechanisms related to IBS. (3) Results: Various characteristics of AMK were investigated using TCMSP data and various analysis systems. AMK restored the macroscopic changes and weight to normal. Colonic mucosa and inflammatory factors were reduced. These effects were similar to those of amitriptyline and sulfasalazine. In addition, transient receptor potential (TRP) V1, voltage-gated Na+ (NaV) 1.5, and NaV1.7 channels were inhibited. (4) Conclusion: These results suggest that AMK may be a promising therapeutic candidate for IBS management through the regulation of ion channels.

Exploring the Therapeutic Effects of Atractylodes macrocephala Koidz against Human Gastric Cancer.

Atractylodes macrocephala Koidz (AMK) is a traditional herbal medicine used for thousands of years in East Asia to improve a variety of illnesses and conditions, including cancers. This study explored the effect of AMK extract on apoptosis and tumor-grafted mice using AGS human gastric adenocarcinoma cells. We investigated the compounds, target genes, and associated diseases of AMK using the Traditional Chinese Medical Systems Pharmacy (TCMSP) database platform. Cell viability assay, cell cycle and mitochondrial depolarization analysis, caspase activity assay, reactive oxygen species (ROS) assay, and wound healing and spheroid formation assay were used to investigate the anti-cancer effects of AMK extract on AGS cells. Also, in vivo studies were conducted using subcutaneous xenografts. AMK extract reduced the viability of AGS cells and increased the sub-G1 cell fraction and the mitochondrial membrane potential. Also, AMK extract increased the production of ROS. AMK extract induced the increased caspase activities and modulated the mitogen-activated protein kinases (MAPK). In addition, AMK extract effectively inhibited AGS cell migration and led to a notable reduction in the growth of AGS spheroids. Moreover, AMK extract hindered the growth of AGS xenograft tumors in NSG mice. Our results suggest that AMK has anti-cancer effects by promoting cell cycle arrest and inhibiting the proliferation of AGS cancer cells and a xenograft model through apoptosis. This study could provide a novel approach to treat gastric cancer.

Study on the Therapeutic Effects and Mechanisms of Gintonin in Irritable Bowel Syndrome and Its Relationship with TRPV1, TRPV4, and NaV1.5.

Irritable bowel syndrome (IBS) is a gastrointestinal (GI) disease accompanied by changes in bowel habits without any specific cause. Gintonin is a newly isolated glycoprotein from ginseng that is a lysophosphatidic acid (LPA) receptor ligand. To investigate the efficacy and mechanisms of action of gintonin in IBS, we developed a zymosan-induced IBS murine model. In addition, electrophysiological experiments were conducted to confirm the relevance of various ion channels. In mice, gintonin restored colon length and weight to normal and decreased stool scores, whilst food intake remained constant. Colon mucosal thickness and inflammation-related tumor necrosis factor-α levels were decreased by gintonin, along with a reduction in pain-related behaviors. In addition, the fecal microbiota from gintonin-treated mice had relatively more Lactobacillaceae and Lachnospiraceae and less Bacteroidaceae than microbiota from the control mice. Moreover, gintonin inhibited transient receptor potential vanilloid (TRPV) 1 and TRPV4 associated with visceral hypersensitivity and voltage-gated Na+ 1.5 channels associated with GI function. These results suggest that gintonin may be one of the effective components in the treatment of IBS.

Analysis of Network Pharmacological Efficacy and Therapeutic Effectiveness in Animal Models for Functional Dyspepsia of Foeniculi fructus.

For centuries, Foeniculi fructus (F. fructus) has been used as a traditional herbal medicine in China and Europe and is widely used as a natural therapy for digestive disorders, including indigestion, flatulence, and bloating. The mechanism of F. fructus that alleviates functional dyspepsia was analyzed through network pharmacology, and its therapeutic effect on an animal model of functional dyspepsia were investigated. The traditional Chinese medicine systems pharmacology (TCMSP) database was used to investigate the compounds, targets, and associated diseases of F. fructus. Information on the target genes was classified using the UniProtdatabase. Using the Cytoscape 3.9.1 software, a network was constructed, and the Cytoscape string application was employed to examine genes associated with functional dyspepsia. The efficacy of F. fructus on functional dyspepsia was confirmed by treatment with its extract in a mouse model of loperamide-induced functional dyspepsia. Seven compounds targeted twelve functional dyspepsia-associated genes. When compared to the control group, F. fructus exhibited significant suppression of symptoms in a mouse model of functional dyspepsia. The results of our animal studies indicated a close association between the mechanism of action of F. fructus and gastrointestinal motility. Based on animal experimental results, the results showed that F. fructus provided a potential means to treat functional dyspepsia, suggesting that its medical mechanism for functional dyspepsia could be described by the relationship between seven key compounds of F. fructus, including oleic acid, ß-sitosterol, and 12 functional dyspepsia-related genes.

Network Pharmacological Analysis and Experimental Validation of the Effect of Smilacis Glabrae Rhixoma on Gastrointestinal Motility Disorder.

Gastrointestinal motility disorder (GMD) is a disease that causes digestive problems due to inhibition of the movement of the gastrointestinal tract and is one of the diseases that reduce the quality of life of modern people. Smilacis Glabrae Rhixoma (SGR) is a traditional herbal medicine for many diseases and is sometimes prescribed to improve digestion. As a network pharmacological approach, we searched the TCMSP database for SGR, reviewed its constituents and target genes, and analyzed its relevance to gastrointestinal motility disorder. The effects of the SGR extract on the pacemaker activity in interstitial cells of Cajal (ICC) and gastric emptying were investigated. In addition, using the GMD mouse model through acetic acid (AA), we investigated the locomotor effect of SGR on the intestinal transit rate (ITR). As a result of network pharmacology analysis, 56 compounds out of 74 candidate compounds of SGR have targets, the number of targets is 390 targets, and there are 904 combinations. Seventeen compounds of SGR were related to GMD, and as a result of comparing the related genes with the GMD-related genes, 17 genes (active only) corresponded to both. When looking at the relationship network between GMD and SGR, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were most closely related to GMD. In addition, the SGR extract regulated the pacemaker activity in ICC and recovered the delayed gastric emptying. As a result of feeding the SGR extract to AA-induced GMD mice, it was confirmed that the ITR decreased by AA was restored by the SGR extract. Through network pharmacology, it was confirmed that quercetin, resveratrol, SCN5A, TNF, and FOS were related to GMD in SGR, and these were closely related to intestinal motility. Based on these results, it is suggested that SGR in GMD restores digestion through the recovery of intestinal motility.

The traditional herbal medicines mixture, Banhasasim-tang, relieves the symptoms of irritable bowel syndrome via modulation of TRPA1, NaV1.5 and NaV1.7 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: The cause of irritable bowel syndrome (IBS), a functional gastrointestinal (GI) disorder, remains unclear. Banhasasim-tang (BHSST), a traditional herbal medicines mixture, mainly used to treat GI-related diseases, may have a potential in IBS treatment. IBS is characterized by abdominal pain as the main clinical symptom, which seriously affects the quality of life. AIM OF THE STUDY: We conducted a study to evaluate the effectiveness of BHSST and its mechanisms of action in treating IBS. MATERIALS AND METHODS: We evaluated the efficacy of BHSST in a zymosan-induced diarrhea-predominant animal model of IBS. Electrophysiological methods were used to confirm modulation of transient receptor potential (TRP) and voltage-gated Na+ (NaV) ion channels, which are associated mechanisms of action. RESULTS: Oral administration of BHSST decreased colon length, increased stool scores, and increased colon weight. Weight loss was also minimized without affecting food intake. In mice administered with BHSST, the mucosal thickness was suppressed, making it similar to that of normal mice, and the degree of tumor necrosis factor-α was severely reduced. These effects were similar to those of the anti-inflammatory drug-sulfasalazine-and antidepressant-amitriptyline. Moreover, pain-related behaviors were substantially reduced. Additionally, BHSST inhibited TRPA1, NaV1.5, and NaV1.7 ion channels associated with IBS-mediated visceral hypersensitivity. CONCLUSIONS: In summary, the findings suggest that BHSST has potential beneficial effects on IBS and diarrhea through the modulation of ion channels.

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.

Reduced EGFR Level in eIF2α PhosphorylationDeficient Hepatocytes Is Responsible for Susceptibility to Oxidative Stress.

Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylationdeficient hepatocytes' vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylationdeficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.

eIF2α phosphorylation is required to prevent hepatocyte death and liver fibrosis in mice challenged with a high fructose diet.

BACKGROUND: Dietary fructose can rapidly cause fatty liver in animals through de novo lipogenesis (DNL) and contribute to the development and severity of nonalcoholic fatty liver disease (NAFLD). In response to diverse cellular insults including endoplasmic reticulum (ER) and oxidative stress, phosphorylation of the eukaryotic translation initiation factor 2 alpha subunit (eIF2α) attenuates general translation initiation, allowing cells to conserve resources and initiate adaptive gene expression to restore homeostasis. The present study aimed to investigate the role of eIF2α phosphorylation in protecting against NAFLD induced by high fructose ingestion in a hepatocyte-specific eIF2α-phosphorylation-deficient mouse model. METHODS: Hepatocyte-specific non-phosphorylatable (S51A) eIF2α knock-in (A/A;fTg/0;CreHep/0, A/AHep ) mice were generated by crossing A/A;fTg/fTg mice with the floxed WT eIF2α transgene (fTg) with Alfp-Cre recombinase transgenic S/A;CreHep/0 (S/A-CreHep ) mice. Hepatocyte-specific eIF2α-phosphorylation-deficient 3-month-old mice or 12-month-old mice were fed a 60% high fructose diet (HFrD) for 16 or 5 wks, and the effects of eIF2α-phosphorylation deficiency on NADP/NADPH and GSSG/GSH levels, ROS-defense gene expression, oxidative damage, cell death, and fibrosis were observed. RESULTS: Prolonged fructose feeding to mice caused dysregulation of the unfolded protein response (UPR) sensor activation and UPR gene expression, and then led to decreased expression of several ROS defense genes including glutathione biogenesis genes. Nonetheless, these changes were not sufficient to induce the death of eIF2α phosphorylation-sufficient hepatocytes. However, there was a substantial increase in hepatocyte death and liver fibrosis in fructose-fed middle-aged mice deficient in hepatocyte-specific eIF2α phosphorylation because of diminished antioxidant capacity due to reduced expression of antioxidant enzymes (GPX1 and HO-1) and lower NADPH and glutathione levels, as well as a possible increase in ROS-induced damage from infiltrating NOX2-expressing leukocytes; all this led to a vicious cycle of hepatocyte death and leukocyte infiltration. CONCLUSION: Our findings suggest that eIF2α phosphorylation maintains NADPH and GSH levels and controls the expression of ROS-defense genes, thereby protecting hepatocytes from oxidative stresses induced by fructose metabolism.

TRIPLE (Insulin, Glucagon and EGFP) Immunofluorescence Staining Protocol in Pancreas.

This protocol aims to introduce methods for immunostaining two endogenous proteins insulin and glucagon and one exogenous transgene driven EGFP in mouse pancreatic islet. The immunostaining results of insulin and glucagon indirectly tell functionality of pancreatic beta cells and alpha cells respectively. Furthermore, the protocol provides immunostaining steps for the third protein which can be applicable to any other endogenous proteins with a specific antibody generated in mouse.