Nociceptive Stimuli Activate the Hypothalamus-Habenula Circuit to Inhibit the Mesolimbic Reward System and Cocaine-Seeking Behaviors.

Nociceptive signals interact with various regions of the brain, including those involved in physical sensation, reward, cognition, and emotion. Emerging evidence points to a role of nociception in the modulation of the mesolimbic reward system. The mechanism by which nociception affects dopamine (DA) signaling and reward is unclear. The lateral hypothalamus (LH) and the lateral habenula (LHb) receive somatosensory inputs and are structurally connected with the mesolimbic DA system. Here, we show that the LH-LHb pathway is necessary for nociceptive modulation of this system using male Sprague Dawley rats. Our extracellular single-unit recordings and head-mounted microendoscopic calcium imaging revealed that nociceptive stimulation by tail pinch excited LHb and LH neurons, which was inhibited by chemical lesion of the LH. Tail pinch increased activity of GABA neurons in ventral tegmental area, decreased the extracellular DA level in the nucleus accumbens ventrolateral shell in intact rats, and reduced cocaine-increased DA concentration, which was blocked by disruption of the LH. Furthermore, tail pinch attenuated cocaine-induced locomotor activity, 22 and 50 kHz ultrasonic vocalizations, and reinstatement of cocaine-seeking behavior, which was inhibited by chemogenetic silencing of the LH-LHb pathway. Our findings suggest that nociceptive stimulation recruits the LH-LHb pathway to inhibit mesolimbic DA system and drug reinstatement.SIGNIFICANCE STATEMENT The LHb and the LH have been implicated in processing nociceptive signals and modulating DA release in the mesolimbic DA system. Here, we show that the LH-LHb pathway is critical for nociception-induced modulation of mesolimbic DA release and cocaine reinstatement. Nociceptive stimulation alleviates extracellular DA release in the mesolimbic DA system, cocaine-induced psychomotor activities, and reinstatement of cocaine-seeking behaviors through the LH-LHb pathway. These findings provide novel evidence for sensory modulation of the mesolimbic DA system and drug addiction.

Activation of a hypothalamus-habenula circuit by mechanical stimulation inhibits cocaine addiction-like behaviors.

BACKGROUND: Mechanoreceptor activation modulates GABA neuron firing and dopamine (DA) release in the mesolimbic DA system, an area implicated in reward and substance abuse. The lateral habenula (LHb), the lateral hypothalamus (LH), and the mesolimbic DA system are not only reciprocally connected, but also involved in drug reward. We explored the effects of mechanical stimulation (MS) on cocaine addiction-like behaviors and the role of the LH-LHb circuit in the MS effects. MS was performed over ulnar nerve and the effects were evaluated by using drug seeking behaviors, optogenetics, chemogenetics, electrophysiology and immunohistochemistry. RESULTS: Mechanical stimulation attenuated locomotor activity in a nerve-dependent manner and 50-kHz ultrasonic vocalizations (USVs) and DA release in nucleus accumbens (NAc) following cocaine injection. The MS effects were ablated by electrolytic lesion or optogenetic inhibition of LHb. Optogenetic activation of LHb suppressed cocaine-enhanced 50 kHz USVs and locomotion. MS reversed cocaine suppression of neuronal activity of LHb. MS also inhibited cocaine-primed reinstatement of drug-seeking behavior, which was blocked by chemogenetic inhibition of an LH-LHb circuit. CONCLUSION: These findings suggest that peripheral mechanical stimulation activates LH-LHb pathways to attenuate cocaine-induced psychomotor responses and seeking behaviors.

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.

Acupuncture reduces cocaine psychomotor responses by activating the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg), a GABAergic afferent to midbrain dopamine (DA) neurons, has emerged as an integral player in both rewarding and nociceptive responses. While previous studies have demonstrated that acupuncture modulates DA transmission in the mesolimbic reward system originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc) and can reduce drug self-administration, the central links between peripheral acupuncture signals and brain reward systems are not well-characterized. Thus, we hypothesised that acupuncture would elicit inhibitory signals from RMTg neurons to brain reward systems. Acupuncture reduced acute cocaine-induced locomotor activity and DA release in a point-specific manner, which was blocked by optogenetic silencing or chemical lesion of the RMTg. The acupuncture effect was mimicked by chemical activation of the RMTg. Acupuncture activated RMTg GABA neurons. In addition, the inhibitory effects of acupuncture on acute cocaine-induced locomotor activity were prevented by electrolytic lesions of the lateral habenula (LHb) or fasciculus retroflexus (FR), areas known to project to the RMTg. These findings suggest that acupuncture recruits the RMTg to reduce the psychomotor responses enhanced by acute cocaine.

Electroacupuncture at Neurogenic Spots in Referred Pain Areas Attenuates Hepatic Damages in Bile Duct-Ligated Rats.

Visceral pain frequently produces referred pain at somatic sites due to the convergence of somatic and visceral afferents. In skin overlying the referred pain, neurogenic spots characterized by hyperalgesia, tenderness and neurogenic inflammation are found. We investigated whether neurogenic inflammatory spots function as acupoints in the rat model of bile duct ligation-induced liver injury. The majority of neurogenic spots were found in the dorsal trunk overlying the referred pain and matched with locations of acupoints. The spots, as well as acupoints, showed high electrical conductance and enhanced expression of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP). Electroacupuncture at neurogenic spots reduced serum hepatocellular enzyme activities and histological patterns of acute liver injury in bile duct ligation (BDL) rats. The results suggest that the neurogenic spots have therapeutic effects as acupoints on hepatic injury in bile-duct ligated rats.

Hemistepsin a Induces Apoptosis of Hepatocellular Carcinoma Cells by Downregulating STAT3.

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.

Role of Lateral Hypothalamus in Acupuncture Inhibition of Cocaine Psychomotor Activity.

Acupuncture modulates the mesolimbic dopamine (DA) system; an area implicated in drug abuse. However, the mechanism by which peripheral sensory afferents, during acupuncture stimulation, modulate this system needs further investigation. The lateral hypothalamus (LH) has been implicated in reward processing and addictive behaviors. To investigate the role of the LH in mediating acupuncture effects, we evaluated the role of LH and spinohypothalamic neurons on cocaine-induced psychomotor activity and NAc DA release. Systemic injection of cocaine increased locomotor activity and 50 kHz ultrasonic vocalizations (USVs), which were attenuated by mechanical stimulation of needles inserted into HT7 but neither ST36 nor LI5. The acupuncture effects were blocked by chemical lesions of the LH or mimicked by activation of LH neurons. Single-unit extracellular recordings showed excitation of LH and spinohypothalamic neurons following acupuncture. Our results suggest that acupuncture recruits the LH to suppress the mesolimbic DA system and psychomotor responses following cocaine injection.

Luteolin prevents liver from tunicamycin-induced endoplasmic reticulum stress via nuclear factor erythroid 2-related factor 2-dependent sestrin 2 induction.

Endoplasmic reticulum (ER) stress designates a cellular response to the accumulation of misfolded proteins, which is related to disease progression in the liver. Luteolin (3',4',5,7-tetrahydroxyflavone) is a phytochemical found frequently in medicinal herbs. Although luteolin has been reported to possess the therapeutic potential to prevent diverse stage of liver diseases, its role in hepatic ER stress has not been established. Thus, the present study aimed to determine the role of luteolin in tunicamycin (Tm)-induced ER stress, and to identify the relevant mechanisms involved in its hepatoprotective effects. In hepatocyte-derived cells and primary hepatocytes, luteolin significantly decreased Tm- or thapsigargin-mediated C/EBP homologous protein (CHOP) expression. In addition, luteolin reduced the activation of three canonical signaling pathways related to the unfolded protein response, and decreased mRNA levels of glucose-regulated protein 78, ER DNA J domain-containing protein 4, and asparagine synthetase. Luteolin also significantly upregulated sestrin 2 (SESN2), and luteolin-mediated CHOP inhibition was blocked in SESN2 (+/-) cells. Moreover, luteolin resulted in phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as increased nuclear Nrf2 expression. Deletion of the antioxidant response element in the human SESN2 promoter inhibited increased luciferase activation by luteolin, suggesting that Nrf2 is a critical transcription factor for luteolin-dependent SESN2 expression. In a Tm-mediated liver injury model, luteolin decreased serum alanine aminotransferase and aspartate aminotransferase activities, prevented degenerative changes and apoptosis of hepatocytes, and inhibited CHOP and glucose-regulated protein 78 expression in hepatic tissues. Therefore, luteolin may be an effective phytochemical to manage ER stress-related liver injury.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine.

BACKGROUND: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. RESULTS: BHSST (20-50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. CONCLUSION: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function.

Rikkunshito Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through Ghrelin Receptors in Murine Small Intestine.

BACKGROUND: Rikkunshito has been used to treat gastrointestinal (GI) disorders. The purpose of this study was to investigate the effects of Rikkunshito, a traditional Japanese herbal medicine, on the pacemaker potentials of interstitial cells of Cajal (ICCs) from the small intestines of mice. METHODS: We isolated ICCs from the small intestines of mice, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs and membrane currents. RESULTS: Rikkunshito depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with GSK1614343 or (D-Lys3)-growth hormone-releasing peptide-6 inhibited Rikkunshito-induced depolarization of pacemaker potentials. Intracellular GDP-ß-S inhibited Rikkunshito-induced effects. In Ca2+-free solution or in the presence of thapsigargin, Rikkunshito did not depolarize pacemaker potentials. Moreover, in the presence of U-73122 or xestospongin C, Rikkunshito-induced effects were inhibited. However, in the presence of staurosporine, Go6976 or Rottlerin, Rikkunshito depolarized pacemaker potentials. Furthermore, Rikkunshito inhibited both transient receptor potentials melastatin 7 (TRPM7) and Ca2+-activated Cl- channels (ANO1) currents. CONCLUSION: Rikkunshito depolarized pacemaker potentials of ICCs via ghrelin receptor and G protein through internal or external Ca2+-, phospholipase C-, and inositol triphosphate-dependent and protein kinase C-, TRPM7-, and ANO1-independent pathways. The study shows that Rikkunshito may alleviate GI motility disorders through its depolarizing effects on ICCs.

Effects of Chaihu-Shugan-San on Small Intestinal Interstitial Cells of Cajal in Mice.

Chaihu-Shugan-San (CSS) has been widely used as an alternative treatment for gastrointestinal (GI) diseases in East Asia. Interstitial cells of Cajal (ICCs) are pacemakers in the GI tract. In the present study, we examined the action of CSS on pacemaker potentials in cultured ICCs from the mouse small intestine in vitro and on GI motility in vivo. We used the electrophysiological methods to measure the pacemaker potentials in ICCs. GI motility was investigated by measuring intestinal transit rates (ITR). CSS inhibited the pacemaker potentials in a dose-dependent manner. The capsazepine did not block the effect of CSS. However, the effects of CSS were blocked by glibenclamide. In addition, NG-nitro-L-arginine methyl ester (L-NAME) also blocked the CSS-induced effects. Pretreatment with SQ-22536 or with KT-5720 did not suppress the effects of CSS; however, pretreatment with ODQ or KT-5823 did. Furthermore, CSS significantly suppressed murine ITR enhancement by neostigmine in vivo. These results suggest that CSS exerts inhibitory effects on the pacemaker potentials of ICCs via nitric oxide (NO)/cGMP and ATP-sensitive K+ channel dependent and transient receptor potential vanilloid 1 (TRPV1) channel independent pathways. Accordingly, CSS could provide the basis for the development of new treatments for GI motility dysfunction.

Methanol extract of semen Ziziphi Spinosae attenuates ethanol withdrawal anxiety by improving neuropeptide signaling in the central amygdala.

BACKGROUND: Ethanol withdrawal (EtOHW) anxiety is a crucial risk factor for alcoholic relapse. The neuropeptide nociceptin/orphanin FQ (N/OFQ) acts upon its receptor (NOP) to antagonize corticotropin-releasing factor (CRF) and elicit anxiolytic actions. Semen Ziziphi Spinosae (SZS), a prototypical hypnotic-sedative herb in Oriental medicine, exhibits anxiolytic effects during nicotine withdrawal by improving amygdaloid CRF/CRF1 receptor (CRFR1) signaling. Therefore, we evaluated the effects of SZS on EtOHW anxiety and the involvement of amygdaloid CRF/CRFR1 and N/OFQ/NOP pathways. METHODS: Male Sprague Dawley rats received intraperitoneal injections of 2 g/kg EtOH (20% v/v) once daily for 28 d followed by a 3-d withdrawal. During EtOHW, the rats were given once-daily intragastric treatments of a methanol extract of SZS (MESZS, 60 or 180 mg/kg/d). Anxiety-like behaviors were measured with the open field (OF) and elevated plus maze (EPM) tests, and plasma corticosterone (CORT) levels were examined by an enzyme-linked immunosorbent assay. mRNA and protein expression levels of the neuropeptides and their receptors were determined by quantitative polymerase chain reaction and Western blot assays. RESULTS: MESZS increased the distance traveled in the center zone of the OF and dose-dependently elongated the duration of staying in the center zone in EtOHW rats. MESZS increased both the number of entries into and the time spent in the open arms of the EPM by EtOHW rats. And, MESZS inhibited the over secretion of plasma CORT during EtOHW. EtOHW enhanced CRF and CRFR1 gene and protein expression in the central nucleus of the amygdala (CeA), which were inhibited by 180 mg/kg/d MESZS. EtOHW increased amygdaloid NOP mRNA and protein expression but spared N/OFQ mRNA expression, and 180 mg/kg/d MESZS further promoted these increases. Additionally, a post-MESZS intra-CeA infusion of either CRF or the selective NOP antagonist UFP-101 abolished the expected anxiolytic effect of 180 mg/kg/d MESZS. CONCLUSIONS: These results suggest that MESZS ameliorates EtOHW anxiety by improving both CRF/CRFR1 and N/OFQ/NOP transmissions in the CeA.

Activating transcription factor 6-dependent sestrin 2 induction ameliorates ER stress-mediated liver injury.

Endoplasmic reticulum (ER) stress is characterized by an accumulation of misfolded proteins, and ER stress reduction is essential for maintaining tissue homeostasis. However, the molecular mechanisms that protect cells from ER stress are not completely understood. The present study investigated the role of sestrin 2 (SESN2) on ER stress and sought to elucidate the mechanism responsible for the hepatoprotective effect of SESN2 in vitro and in vivo. Treatment with tunicamycin (Tm) increased SESN2 protein and mRNA levels and reporter gene activity. Activating transcription factor 6 (ATF6) bound to unfolded protein response elements of SESN2 promoter, transactivated SESN2, and increased SESN2 protein expression. In addition, dominant negative mutant of ATF6α and siRNA against ATF6α blocked the ER stress-mediated SESN2 induction, whereas chemical inhibition of PERK or IRE1 did not affect SESN2 induction by Tm. Ectopic expression of SESN2 in HepG2 cells inhibited CHOP and GRP78 expressions by Tm. Moreover, SESN2 decreased the phosphorylations of JNK and p38 and PARP cleavage, and blocked the cytotoxic effect of excessive ER stress. In a Tm-induced liver injury model, adenoviral delivery of SESN2 in mice decreased serum ALT, AST and LDH activities and the mRNA levels of CHOP and GRP78 in hepatic tissues. Moreover, SESN2 reduced numbers of degenerating hepatocytes, and inhibited caspase 3 and PARP cleavages. These results suggest ATF6 is essential for ER stress-mediated SESN2 induction, and that SESN2 acts as a feedback regulator to protect liver from excess ER stress.

Salinomycin ameliorates oxidative hepatic damage through AMP-activated protein kinase, facilitating autophagy.

Salinomycin, a monocarboxylic ionophore in Streptomyces albus, has been studied as an anti-cancer agent. However, we wondered whether salinomycin has another effect such as an anti-oxidant and hepatic protectant, because some chemical drugs treating human diseases were sometimes related with their toxic effects. Therefore, this study was conducted to examine the effects of salinomycin against oxidative stress and mitochondrial impairment in vivo and in vitro as well as the cellular mechanisms of action. In hepatocyte, salinomycin inhibited arachidonic acid (AA) + iron-induced apoptosis, mitochondrial dysfunction and ROS production. As a molecular mechanism, salinomycin induced autophagy through AMP-activated protein kinase (AMPK) activation, as assessed by the accumulation of acidic vesicle organelles, p62 and LC3-II. Moreover, these protective effects were blocked by AMPK inhibition, which indicates the importance of AMPK in the process of salinomycin's effects. In mice, oral administration of salinomycin protected against carbon tetrachloride (CCl4)-induced oxidative stress and liver injury, and also activated AMPK as well as autophagy-related proteins in the liver. Collectively, salinomycin had the ability to protect hepatocytes against AA+iron-induced reactive oxygen species production and mitochondrial dysfunction, as well as CCl4-induced liver injury. Although this beneficial effect was demonstrated under severe oxidative stress, this study showed that salinomycin protected the liver against the oxidative stress and liver damage through AMPK and autophagy, and suggest that salinomycin has a possibility to treat a broad range of diseases.

Effects of Gamisoyo-San Decoction, a Traditional Chinese Medicine, on Gastrointestinal Motility.

BACKGROUND: Gamisoyo-San decoction (GSS), a traditional Chinese medicine, has been used to treat various gastrointestinal (GI) symptoms and diseases such as functional dyspepsia. The purpose of this study was to investigate the effect of GSS on GI motility functions in mice. METHODS: Percent intestinal transit rate (ITR%) and gastric emptying (GE) values were measured using Evans Blue and phenol red, respectively, in normal mice and in mice with experimentally induced GI motility dysfunction (GMD). RESULTS: In normal mice, GSS (0.01-1 g/kg) induced higher GE values than non-treated controls. Also, GSS could increase GE in loperamide-induced and cisplatin-induced GE delay models. In addition, GSS increased ITR% in a dose-dependent manner. Loperamide decreased ITR% and GSS recovered this loperamide-induced decrease in ITR%. To examine the effect of GSS on GMD, we used acetic acid (AA)-induced and streptozotocin (STZ)-induced mouse GMD models. The AA mouse model showed a significant decrease in ITR%. However, intragastric treatment with GSS significantly recovered this inhibition. Furthermore, STZ-induced diabetic mice showed a significant reduction in ITR%, which was also significantly inhibited by GSS. CONCLUSION: These results demonstrate that GSS can modulate bowel activity and that it could be used as a gastroprokinetic agent in the treatment of GI motility diseases.

Modulation of Pacemaker Potentials in Murine Small Intestinal Interstitial Cells of Cajal by Gamisoyo-San, a Traditional Chinese Herbal Medicine.

BACKGROUND: The Gamisoyo-san (GSS) has been used for -improving the gastrointestinal (GI) symptoms. The purpose of this study was to investigate the effects of GSS, a traditional Chinese herbal medicine, on the pacemaker potentials of mouse small intestinal interstitial cells of Cajal (ICCs). METHODS: ICCs from the small intestines were dissociated and cultured. Whole-cell patch-clamp configuration was used to record pacemaker potentials and membrane currents. RESULTS: GSS depolarized ICC pacemaker potentials in a dose-dependent manner. Pretreatment with 4-diphenylacetoxypiperidinium iodide completely inhibited GSS-induced pacemaker potential depolarizations. Intracellular GDP-ß-S inhibited GSS-induced effects, and in the presence of U-73122, GSS-induced effects were inhibited. Also, GSS in the presence of a Ca2+-free solution or thapsigargin did not depolarize pacemaker potentials. However, in the presence of calphostin C, GSS slightly depolarized pacemaker potentials. Furthermore, GSS inhibited both transient receptor potential melastatin7 and Ca2+-activated Cl- channel (anoctamin1) currents. CONCLUSION: GSS depolarized pacemaker potentials of ICCs via G protein and muscarinic M3 receptor signaling pathways and through internal or external Ca2+-, phospholipase C-, and protein kinase C-dependent and transient receptor potential melastatin 7-, and anoctamin 1-independent pathways. The study shows that GSS may regulate GI tract motility, suggesting that GSS could be a basis for developing novel prokinetic agents for treating GI motility dysfunctions.

Fucoxanthin, the constituent of Laminaria japonica, triggers AMPK-mediated cytoprotection and autophagy in hepatocytes under oxidative stress.

BACKGROUND: Laminaria japonica has frequently been used as a food supplement and drug in traditional oriental medicine. Among the major active constituents responsible for the bioactivities of L. japonica, fucoxanthin (FX) has been considered as a potential antioxidant. This study was conducted to examine the effects of L. japonica extract (LJE) or FX against oxidative stress on hepatocytes and to elucidate the overall their cellular mechanisms of the effects. METHODS: We constructed an in vitro model with the treatment of arachidonic acid (AA) + iron in HepG2 cells to stimulate the oxidative damage. The cells were pre-treated with LJE or FX for 1 h, and incubated with AA + iron. The effect on oxidative damage and cellular mechanisms of LJE or FX were assessed by cytological examination and several biochemical assays under conditions with or without kinase inhibitiors. RESULTS: LJE or FX pretreatment effectively blocked the pathological changes caused by AA + iron treatment, such as cell death, altered expression of apoptosis-related proteins such as procaspase-3 and poly (ADP-ribose) polymerase, and mitochondria dysfunction. Moreover, FX induced AMPK activation and AMPK inhibitor, compound C, partially reduced the protective effect of FX on mitochondria dysfunction. Consistent with AMPK activation, FX increased the protein levels of autophagic markers (LC3II and beclin-1) and the number of acridine orange stained cells, and decreased the phosphorylation of mTOR and simultaneously increased the phosphorylation of ULK1. And the inhibition of autophagy by 3-methylanine or bafilomycin A1 partially inhibited the protective effect of FX on mitochondria dysfunction. CONCLUSION: These findings suggest that FX have the function of being a hepatic protectant against oxidative damages through the AMPK pathway for the control of autophagy.

Combination of Pelargonium sidoides and Coptis chinensis root inhibits nuclear factor kappa B-mediated inflammatory response in vitro and in vivo.

BACKGROUND: Pelargonium sidoides (PS) and Coptis chinensis root (CR) have traditionally been used to treat various diseases, including respiratory and gastrointestinal infections, dysmenorrhea, and hepatic disorders. The present study was conducted to evaluate the anti-inflammatory effects of a combination of PS and CR in vitro and in vivo. METHODS: The in vitro effects of PS + CR on the induction of inflammation-related proteins were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The levels of nitric oxide (NO) and of inflammatory cytokines and prostaglandin E2 (PGE2) were measured using the Griess reagent and enzyme-linked immunosorbent assay (ELISA) methods, respectively. The expression of inflammation-related proteins was confirmed by Western blot. Additionally, the effects of PS + CR on paw edema volume, skin thickness, and numbers of infiltrated inflammatory cells, mast cells, COX-2-, iNOS-, and TNF-α-immunoreactive cells in dorsum and ventrum pedis skin were evaluated in a rat model of carrageenan (CA)-induced paw edema. RESULTS: PS + CR significantly reduced production of NO, PGE2 and three pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6) and also decreased levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Treatment with PS + CR significantly reduced the protein expression levels of LPS-stimulated nuclear factor kappa B (NF-κB) and phosphorylated inhibitor of NF-κB (p-I-κBα). Additionally, PS + CR significantly inhibited the increases in paw swelling, skin thickness, infiltrated inflammatory cells, mast cell degranulation, COX-2-, iNOS-, and TNF-α-immunoreactive cells in the rat model of CA-induced acute edematous paw. CONCLUSIONS: These results demonstrate that PS + CR exhibits anti-inflammatory properties through decreasing the production of pro-inflammatory mediators (NO, PGE2, TNF-α, IL-1ß, and IL-6), suppressing NF-κB signaling in LPS-induced RAW 264.7 cells. Additionally, the results of the CA-induced rat paw edema assay revealed an anti-edema effect of PS + CR. Furthermore, it is suggested that PS + CR also inhibits acute edematous inflammation by suppressing mast cell degranulation and inflammatory mediators (COX-2, iNOS, and TNF-α). Thus, PS + CR may be a potential candidate for the treatment of various inflammatory diseases, and it may also contribute to a better understanding of the molecular mechanisms underlying inflammatory response regulation.

AMPK activation by liquiritigenin inhibited oxidative hepatic injury and mitochondrial dysfunction induced by nutrition deprivation as mediated with induction of farnesoid X receptor.

PURPOSE: Nutrition is indispensable for cell survival and proliferation. Thus, loss of nutrition caused by serum starvation in cells could induce formation of reactive oxygen species (ROS), resulting in cell death. Liquiritigenin (LQ) is an active flavonoid in licorice and plays a role in the liver as a hepatic protectant. METHODS: This study investigated the effect of LQ, metformin [an activator of activated AMP-activated protein kinase (AMPK)] and GW4064 [a ligand of farnesoid X receptor (FXR)] on mitochondrial dysfunction and oxidative stress induced by serum deprivation as well as its molecular mechanism, as assessed by immunoblot and flow cytometer assays. RESULTS: Serum deprivation in HepG2, H4IIE and AML12 cells successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, and altered expression of apoptosis-related proteins such as procaspase-3, poly(ADP-ribose) polymerase, and Bcl-2. However, LQ pretreatment significantly blocked these pathological changes and mitochondrial dysfunction caused by serum deprivation. Moreover, LQ activated AMPK in HepG2 cells and mice liver, as shown by phosphorylation of AMPK and ACC, and this activation was mediated by its upstream kinase (i.e., LKB1). Experiments using a chemical inhibitor of AMPK with LKB1-deficient Hela cells revealed the role of the LKB1-AMPK pathway in cellular protection conferred by LQ. LQ also induced protein and mRNA expression of both FXR as well as small heterodimer partner, which is important since treatment with FXR ligand GW4064 protected hepatocytes against cell death and mitochondrial damage induced by serum deprivation. CONCLUSION: AMPK activators such as LQ can protect hepatocytes against oxidative hepatic injury and mitochondrial dysfunction induced by serum deprivation, and the beneficial effect might be mediated through the LKB1 pathway as well as FXR induction.

Moutan Cortex Protects Hepatocytes against Oxidative Injury through AMP-Activated Protein Kinase Pathway.

Moutan Cortex, the root bark of Paeonia suffruticosa ANDREWS in Ranunculaceae, has widely demonstrated analgesic, anti-spasmodic, and anti-inflammatory effects in various cancer and immune cell lines. Oxidative stress is associated with development of several diseases, including liver disease. We prepared the water extract of Moutan Cortex (MCE) to investigate the cytoprotective activities and its mechanism. MCE protected hepatocytes from arachidonic acid (AA)+iron induced oxidative stress, as indicated by reactive oxygen species (ROS) production and cell viability analysis. MCE also suppressed mitochondrial dysfunction in AA+iron-treated human hepatocyte-derived hepatocellular carcinoma cell line, HepG2 cells. In addition, MCE treatment induces AMP-activated protein kinase (AMPK) and liver kinase B1 phosphorylation, which play a role in inhibition of oxidative stress induced cell death. Moreover, one of the MCE compounds, chlorogenic acid, exerted protective effects against oxidative stress and apoptosis. Taken together, MCE protected hepatocytes against AA+iron-induced oxidative stress through AMPK activation, and may be a candidate for the treatment of liver disease.