Monoamine serotonin is a major neurotransmitter that acts on a wide range of central nervous system and peripheral nervous system functions and is known to have a role in various processes. Recently, it has been found that 5-HT is involved in cognitive and memory functions through interaction with cholinergic pathways. The natural flavonoid kaempferol (KAE) extracted from Cudrania tricuspidata is a secondary metabolite of the plant. Recently studies have confirmed that KAE possesses a neuroprotective effect because of its strong antioxidant activity. It has been confirmed that KAE is involved in the serotonergic pathway through an in vivo test. However, these results need to be confirmed at the molecular level, because the exact mechanism that is involved in such effects of KAE has not yet been elucidated. Therefore, the objective of this study is to confirm the interaction of KAE with 5-HT3A through electrophysiological studies at the molecular level using KAE extracted from Cudrania tricuspidata. This study confirmed the interaction between 5-HT3A and KAE at the molecular level. KAE inhibited 5-HT3A receptors in a concentration-dependent and voltage-independent manner. Site-directed mutagenesis and molecular-docking studies confirmed that the binding sites D177 and F199 are the major binding sites of human 5-HT3A receptors of KAE.
Transient receptor potential vanilloid member 1 (TRPV1) is activated in response to capsaicin, protons, temperature, and free reactive oxygen species (ROS) released from inflammatory molecules after exposure to harmful stimuli. The expression level of TRPV1 is elevated in the dorsal root ganglion, and its activation through capsaicin and ROS mediates neuropathic pain in mice. Its expression is high in peripheral and central nervous systems. Although pain is a response evolved for survival, many studies have been conducted to develop analgesics, but no clear results have been reported. Here, we found that naringin selectively inhibited capsaicin-stimulated inward currents in Xenopus oocytes using a two-electrode voltage clamp. The results of this study showed that naringin has an IC50 value of 33.3 μM on TRPV1. The amino acid residues D471 and N628 of TRPV1 were involved in its binding to naringin. Our study bridged the gap between the pain suppression effect of TRPV1 and the preventive effect of naringin on neuropathic pain and oxidation. Naringin had the same characteristics as a model selective antagonist, which is claimed to be ideal for the development of analgesics targeting TRPV1. Thus, this study suggests the applicability of naringin as a novel analgesic candidate through antioxidative and analgesic effects of naringin.
DDX3 is a host viral factor that can inhibit the hepatitis B virus‐induced innate immune responses. In this study, the 20 bioactive compounds have screened the effects on DDX3 and we found that 5‐HT upregulated DDX3 promoter activity via the 5‐HT7 receptor on liver hepatocellular cells (HepG2 cells) by using a luciferase assay, reverse transcription‐polymerase chain reaction analysis, and Western blot analysis. Furthermore, we are trying to elucidate the pathways involved in the stimulating effect of 5‐HT on DDX3 expression to induce innate immune responses against hepatitis B virus infection. A knockdown of the 5‐HT7 receptor by transfection si‐5‐HT7 receptors or si‐control into HepG2 cells treated by 5‐HT (or 5‐HT plus agonist) confirmed the role of the 5‐HT7 receptor in DDX3 expression. The IFN‐β‐Luc expression and level of hepatitis B virus surface Antigen (HBsAg) showed that DDX3 mediated by the 5‐HT7 agonist (AS‐19) increased IFN‐β expression and inhibited HBV replication. Luciferase assays showed the involvement of 5‐HT7 receptors in DDX3 expression via cAMP/AC/PKA pathways by using protein kinase A (PKA) and adenylyl cyclase inhibitor (MDL 12330A). AS‐19 mediated DDX3 promoter activated PKA extracellular signal‐regulated kinase ERK signaling the p53 phosphorylation (‐1080/‐1070) resulted in upregulation of DDX3 promoter transactivation via the 5‐HT7 receptors agonist. Overall, 5‐HT7 was found to be a new potential target to inhibit hepatitis B infection by activating AC/PKA/ERK pathways by phosphorylating p53 via the 5‐HT7 agonist response by mediating DDX3 expression.
Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3β4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3β4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3β4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and β4 subunits on lupeol binding with nAChRs. Double mutants of α3β4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3β4 nAChRs. A characteristic of α3β4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3β4 nAChRs. The interaction between lupeol and α3β4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3β4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3β4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.
Irritable bowel syndrome (IBS) is a chronic disease that causes abdominal pain and an imbalance of defecation patterns due to gastrointestinal dysfunction. The cause of IBS remains unclear, but intestinal-brain axis problems and neurotransmitters have been suggested as factors. In this study, chanoclavine, which has a ring structure similar to 5-hydroxytryptamine (5-HT), showed an interaction with the 5-HT3A receptor to regulate IBS. Although its derivatives are known to be involved in neurotransmitter receptors, the molecular physiological mechanism of the interaction between chanoclavine and the 5-HT3A receptor is unknown. Electrophysiological experiments were conducted using a two-electrode voltage-clamp analysis to observe the inhibitory effects of chanoclavine on Xenopus oocytes in which the h5-HT3A receptor was expressed. The co-application of chanoclavine and 5-HT resulted in concentration-dependent, reversible, voltage-independent, and competitive inhibition. The 5-HT3A response induced by 5-HT was blocked by chanoclavine with half-maximal inhibitory response concentration (IC50) values of 107.2 µM. Docking studies suggested that chanoclavine was positioned close F130 and N138 in the 5-HT3A receptor-binding site. The double mutation of F130A and N138A significantly attenuated the interaction of chanoclavine compared to a single mutation or the wild type. These data suggest that F130 and N138 are important sites for ligand binding and activity. Chanoclavine and ergonovine have different effects. Asparagine, the 130th amino acid sequence of the 5-HT3A receptor, and phenylalanine, the 138th, are important in the role of binding chanoclavine, but ergonovine has no interaction with any amino acid sequence of the 5-HT3A receptor. The results of the electrophysiological studies and of in silico simulation showed that chanoclavine has the potential to inhibit the hypergastric stimulation of the gut by inhibiting the stimulation of signal transduction through 5-HT3A receptor stimulation. These findings suggest chanoclavine as a potential antiemetic agent for excessive gut stimulation and offer insight into the mechanisms of 5-HT3A receptor inhibition.
Sleep is an essential component of quality of life. The majority of people experience sleep problems that impact their quality of life. Melatonin is currently a representative sleep aid. However, it is classified as a prescription drug in most countries, and consumers cannot purchase it to improve their sleep. This sleep induction experiment in mice aimed to identify a natural combination product (NCP) that can create synergistic sleep-promoting effects. Based on the mechanism of action of sleep, we investigated whether phenomenological indicators of sleep quality change according to the intake of NCP. The sleep onset and sleep time of the mice that consumed the NCP found by this study were improved compared to the existing sleep aids. The mean melatonin level in the blood increased by 197% compared to the control. To our knowledge, this is the first study to demonstrate that Rosa multiflora Thunb. (Yeongsil) can promote sleep similarly to Zizyphus jujuba Miller (Sanjoin). The results indicate a preclinical study of NCPs containing Rosa multiflora Thunb and Zizyphus jujuba Miller developed by us showed significant differences in sleep incubation and duration depending on melatonin concentrations. Our results also suggest that increased melatonin concentrations in the blood are likely to improve sleep quality, especially regarding incubation periods.
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