Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella).

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 µg/kg diet.

Wei Chang An pill regulates gastrointestinal motility in a bidirectional manner.

CONTEXT: Wei Chang An (WCA) is a commercial prescription developed for the coordination of gastrointestinal movement. OBJECTIVE: To investigate the role of WCA in the regulation of diarrhoea and constipation in rats. MATERIAL AND METHODS: The diarrhoea and constipation models were prepared by gavage of Folium senna and diphenoxylate hydrochloride. Rats were randomized equally (n = 6) into the normal group given saline daily, the positive group given Pinaverium Bromide (13.5 mg/kg) or Sennoside A (0.1 mg/kg) and three WCA-treated groups (22, 44, and 88 mg/kg) by gavage daily for 7 consecutive days. The effects of WCA were assessed by a series of faecal symptoms and histopathology. Gastrointestinal parameters were determined by ELISA. The effect of WCA on gastrointestinal tissues was evaluated by strip assay. Expression of ROCK-1 and MLCK was measured by RT-PCR and Western blotting. RESULTS: Data from Bristol stool form scale, diarrhoea index, visceral sensitivity, defaecation time, and intestinal propulsive rate showed that WCA protected rats against diarrhoea and constipation (p < 0.01). The up-regulation of Substance P and 5-hydroxytryptamine in diarrhoea rats and down-regulation of Substance P and vasoactive intestinal polypeptide in constipation rats were inhibited by WCA (p < 0.05). WCA stimulated the gastrointestinal strip contractions but inhibited ACh-induced contractions (p < 0.01). The decreased ROCK-1 and MLCK expression in diarrhoea rats and increased in constipation rats were suppressed by WCA (p < 0.01). CONCLUSIONS: WCA has both antidiarrhea and anti-constipation effects, suggesting its bidirectional role in gastrointestinal modulation, and providing evidence of WCA for irritable bowel syndrome treatment.

Evaluation of Alpha-Ketoglutarate Supplementation on the Improvement of Intestinal Antioxidant Capacity and Immune Response in Songpu Mirror Carp (Cyprinus carpio) After Infection With Aeromonas hydrophila.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear. AIM OF THE STUDY: This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease. MATERIALS AND METHODS: The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D. RESULTS: The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2). CONCLUSIONS: AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway.

Pharmacological activation of ERß by arctigenin maintains the integrity of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal epithelial barrier dysfunction is deeply involved in the pathogenesis of inflammatory bowel diseases (IBD). Arctigenin, the main active constituent in Fructus Arctii (a traditional Chinese medicine), has previously been found to attenuate colitis induced by dextran sulfate sodium (DSS) in mice. The present study investigated whether and how arctigenin protects against the disruption of the intestinal epithelial barrier in IBD. Arctigenin maintained the intestinal epithelial barrier function of mice with DSS- and TNBS-induced colitis. In Caco-2 and HT-29 cells, arctigenin lowered the monolayer permeability, increased TEER, reversed the abnormal expression of tight junction proteins, and restored the altered localization of F-actin induced by TNF-α and IL-1ß. The specific antagonist PHTPP or shRNA of ERß largely weakened the protective effect of arctigenin on the epithelial barrier function of Caco-2 and HT-29 cells. Molecular docking demonstrated that arctigenin had high affinity for ERß mainly through hydrogen bonds as well as hydrophobic effects, and the protective effect of arctigenin on the intestinal barrier function was largely diminished in ERß-mutated (ARG346 and/or GLU305) Caco-2 cells. Moreover, arctigenin-blocked TNF-α induced increase of the monolayer permeability in Caco-2 and HT-29 cells and the activation of myosin light chain kinase (MLCK)/myosin light chain (MLC) pathway in an ERß-dependent manner. ERß deletion in colons of mice with DSS-induced colitis resulted in a significant attenuation of the protective effect of arctigenin on the barrier integrity and colon inflammation. Arctigenin maintained the integrity of the intestinal epithelial barrier under IBD by upregulating the expression of tight junction proteins through the ERß-MLCK/MLC pathway.

Banha-sasim-tang improves gastrointestinal function in loperamide-induced functional dyspepsia mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Banha-sasim-tang (BST; Hange-shashin-to in Kampo medicine; Banxia xiexin tang in traditional Chinese medicine) is a traditional Chinese harbal medicine that has been commonly used for gastrointestinal disorders. AIM OF THE STUDY: To investigate the pharmacological effects of BST, a standardized herbal drug, on main symptoms of functional dyspepsia including delayed gastric emptying, and underlying mechanisms of action in mouse model. METHODS AND MATERIALS: Balb/C mice were pretreated with BST (25, 50, 100 mg/kg, po) or mosapride (3 mg/kg, po) for 3 days, and then treated with loperamide (10 mg/kg, ip) after 19 h fasting. A solution of 0.05% phenol red (500 µL) or 5% charcoal diet (200 µL) was orally administered, followed by scarifying and assessment of gastric emptying or gastro-intestinal motility. C-kit (immunofluorescence), nNOS (western blot) and gastric contraction-related gene expression were examined in stomach tissue. RESULTS: The loperamide injection substantially delayed gastric emptying, while the BST pretreatment significantly attenuated this peristaltic dysfunction, as evidenced by the quantity of stomach-retained phenol red (p < 0.05 or 0.01) and stomach weight (p < 0.05 or 0.01). The BST pretreatment significantly tempered the loperamide-induced inactivation of c-kit and nNOS (p < 0.05 or 0.01) as well as the contraction-related gene expression, such as the 5HT4 receptor (5HT4R), anoctamin-1 (ANO1), ryanodine receptor 3 (RYR3) and smooth muscle myosin light chain kinase (smMLCK). The BST pretreatment also significantly attenuated the alterations in gastro-intestinal motility (p < 0.01). CONCLUSION: Our results are the first evidence of the prokinetic agent effects of Banha-sasim-tang in a loperamide-induced FD animal model. The underlying mechanisms of action may involve the modulation of peristalsis via activation of the interstitial cells of Cajal and the smooth muscle cells in the stomach.

Folic acid deficiency impairs the gill health status associated with the NF-κB, MLCK and Nrf2 signaling pathways in the gills of young grass carp (Ctenopharyngodon idella).

The aim of this study was to investigate the effect of dietary folic acid on fish growth, the immune and barrier functions of fish gills, and the potential mechanisms of these effects. Young grass carp (Ctenopharyngodon idella) were fed diets containing graded levels of folic acid at 0.10 (basal diet), 0.47, 1.03, 1.48, 1.88 and 3.12 mg kg(-1) diet for 8 weeks. The results showed that acid phosphatase and lysozyme activities and the complement component 3 content in fish gills decreased with folic acid deficiency (P < 0.05). Folic acid deficiency up-regulated liver-expressed antimicrobial peptide 1, interleukin 1ß, interleukin 8, tumor necrosis factor α, nuclear factor κB p65, IκB kinase α (IKK-α), IKK-ß and IKK-γ gene expression. Folic acid deficiency down-regulated interleukin 10, transforming growth factor ß, IκB and target of rapamycin gene expression in fish gills (P < 0.05). These results showed that limited folic acid decreased fish gill immune status. Furthermore, folic acid deficiency down-regulated claudin-b, claudin-c, claudin-3, occludin and zonula occludens 1 gene expression, whereas folic acid deficiency up-regulated claudin-12, claudin-15, myosin light chain kinase and p38 mitogen activated protein kinase gene expression in fish gills (P < 0.05). These results suggested that folic acid deficiency disrupted tight junction-mediated fish gill barrier function. Additionally, folic acid deficiency increased the content of reactive oxygen species, protein carbonyl and malondialdehyde (MDA); Mn superoxide dismutase activity and gene expression; and Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b gene expression (P < 0.05). Conversely, folic acid deficiency decreased Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione s-transferases and glutathione reductase activities and gene expression as well as NF-E2-related factor 2 gene expression in fish gills (P < 0.05). All of these results indicated that folic acid deficiency impaired fish gill health status via regulating gene expression of cytokines, tight junction proteins, antioxidant enzymes, NF-κB p65, MLCK and Nrf2. Based on percent weight gain, LZ activity and MDA content in the gills, the dietary folic acid requirements for young grass carp were 1.60, 2.07 and 2.08 mg kg(-1), respectively.

Effect of acupuncture at Fengchi (GB 20) on the activity of myosin light chain kinase in the middle meningeal artery of migraine modeled rats.

OBJECTIVE: To study the effect of acupuncture at Fengchi (GB 20) on the activation of myosin light chain kinase (MLCK) in the middle meningeal artery of migraine modeled rats. METHODS: Forty-four clean grade healthy female Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, blank control group, Fengchi (GB 20) acupuncture group, and Fengchi (GB 20) prevention group. Neurogenic inflammation of these rats was induced by electrical stimulation. The γ-32P infiltration method was then used to detect MLCK activation in the middle meningeal artery, and immunocytochemistry was applied to detect the structural protein expression of MLCK. RESULTS: The miaraine model was successfully established in the rats. Compared with the control group, MLCK activation was significantly decreased in the blank control group (P < 0.01). CONCLUSION: The activation of MLCK in the middle meningeal artery was increased by acupuncture at Fengchi (GB 20), indicating its effectiveness in preventing and curing on acute migraine attacks.

Dietary pantothenic acid deficiency and excess depress the growth, intestinal mucosal immune and physical functions by regulating NF-κB, TOR, Nrf2 and MLCK signaling pathways in grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary pantothenic acid (PA) on the growth, intestinal mucosal immune and physical barrier, and relative mRNA levels of signaling molecules in the intestine of grass carp (Ctenopharyngodon idella). A total of 540 grass carp (253.44 ± 0.69 g) were fed six diets with graded levels of PA (PA1, PA15, PA30, PA45, PA60 and PA75 diets) for 8 weeks. The results indicated that compared with PA deficiency (PA1 diet) and excess (PA75 diet) groups, optimal PA supplementation increased (P < 0.05): (1) percent weight gain (PWG), feed intake and feed efficiency; (2) lysozyme activity, complement 3 content, liver-expressed antimicrobial peptide 2 and hepcidin, interleukin 10, transforming growth factor ß1 and inhibitor of κBα mRNA levels in some intestinal segments; (3) activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase, and NF-E2-related factor 2 (Nrf2) mRNA level in the whole intestine; (4) Claudin b, Claudin 3, Claudin c, Occludin and ZO-1 mRNA levels in some intestinal segments of grass carp. Conversely, optimal PA supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1ß, interferon γ2, interleukin 8, nuclear factor κB P65 (NF-κB P65), IκB kinase α, IκB kinase ß, IκB kinase γ and target of rapamycin (TOR) mRNA expression levels in some intestinal segments; (2) reactive oxygen species, malondialdehyde and protein carbonyl contents, and Kelch-like ECH-associating protein 1a, Kelch-like ECH-associating protein 1b in the intestine; (3) Claudin 12, Claudin 15a and myosin light-chain kinase (MLCK) mRNA levels in some intestinal segments of grass carp. In conclusion, optimum PA promoted growth, intestinal mucosal immune and physical function, as well as regulated mRNA levels of signaling molecules NF-κB P65, TOR, Nrf2 and MLCK in grass carp intestine. Based on the quadratic regression analysis of PWG and intestinal lysozyme activity, the optimal PA levels in grass carp (253.44-745.25 g) were estimated to be 37.73 mg/kg and 41.38 mg/kg diet, respectively.

Capsaicin alleviates abnormal intestinal motility through regulation of enteric motor neurons and MLCK activity: Relevance to intestinal motility disorders.

SCOPE: Capsaicin is an active component of chili peppers, having diverse effects. However, the effects of capsaicin on intestinal motility are still controversial. The present study aimed to investigate the effects of capsaicin on intestinal motility disorder and uncover related mechanisms. MATERIALS AND RESULTS: A rat model with intestinal motility disorder was established in vitro through adding different stimuli into tissue bath; in vivo using constipation and diarrhea model, respectively. Capsaicin exerted dual effects on intestinal motility, i.e. the relaxation and contraction of jejunum induced by corresponding stimulus were, respectively, regulated to be normal contraction by capsaicin. The mechanisms underlined capsaicin-induced dual effects were investigated using Western blotting, qRT-PCR, and whole-cell patch clamp, respectively. Results showed that cholinergic excitatory nerves, adrenergic nerves, and neurons containing nitric oxide synthase, which are the main muscle motor neurons in enteric nervous system (ENS), are involved in capsaicin-induced dual effects. The competition for regulation of Ca(2+) influx by capsaicin induced the interaction with components of the ENS. Capsaicin significantly increased myosin light chain kinase (MLCK) expression and myosin phosphorylation extent in jejunal segments of constipation-prominent rats and significantly decreased MLCK expression and myosin phosphorylation extent in jejunal segments of diarrhea-prominent rats. CONCLUSION: In summary, capsaicin alleviates abnormal intestinal motility through regulating enteric motor neurons and MLCK activity, which is beneficial for the treatment of gastrointestinal motility disorders.

Pharmacokinetic difference of berberine between normal and chronic visceral hypersensitivity irritable bowel syndrome rats and its mechanism.

Berberine is one of active alkaloids from Rhizoma coptidis in traditional Chinese medicine. The pharmacokinetics of berberine in rat plasma were compared between normal and chronic visceral hypersensitivity irritable bowel syndrome rats (CVH-IBS) established by mechanical colon irritation using angioplasty balloons for 2 weeks after oral administration of berberine hydrochloride (25 mg/kg) with the equivalent dose of 22 mg/kg for berberine according to body weight. Immunohistochemical analysis of c-fos and myosin light chain kinase (MLCK) and immunofluorescence analysis of MLCK in rat colon were conducted. Quantification of berberine in rat plasma was achieved by using a sensitive and rapid UPLC-MS/MS method. Plasma samples were collected at 15 different points in time and the pharmacokinetic parameters were analyzed by WinNonlin software. The great different pharmacokinetic behavior of berberine was observed between normal and CVH-IBS model rats. Compared with normal group, T1/2 and AUC(0-t) of berberine in the model group were significantly increased, respectively (573.21 ± 127.53 vs 948.22 ± 388.57 min; 8,657.19 ± 1,562.54 vs 11,415.12 ± 1,670.72 min.ng/ml). Cl/F of berberine in the model group significantly decreased, respectively (13.89 ± 1.69 vs 9.19 ± 2.91 L/h/kg). Additionally, the expressions of c-fos and MLCK in model group were higher than those in normal group. The pharmacokinetic behavior of berberine was significantly altered in CVH-IBS pathological conditions, which indicated the dosage modification of berberine hydrochloride in CVH-IBS were necessary. Especially, improved exposure to berberine in rat plasma in CVH-IBS model rats was attributed to increased the expression of MLCK.

The effect of DA-9701 on 5-hydroxytryptamine-induced contraction of feline esophageal smooth muscle cells.

Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the gastrointestinal (GI) tract, and the central nervous system (CNS) of animals and humans. The signaling pathways of 5-hydroxytryptamine (5-HT)-induced contractions in cat esophageal smooth muscle cell (ESMC)s have been identified, but the downstream components of the 5-HT signaling pathway remain unclear. DA-9701 is the standardized extract of the Pharbitis nil Choisy seed (Pharbitidis Semen, Convolvulaceae) and the root of Corydalis yahusuo W.T. Wang (Corydalis Tuber, Papaveraceae). DA-9701 is known to have strong gastroprokinetic effects and a good safety profile. In this study, we investigated the 5-HT signaling pathway at the G-protein level, and we explored the mechanisms by which DA-9701 induces smooth muscle contraction. Freshly isolated smooth muscle cells were harvested from the feline esophagus, and cells were permeabilized to measure their length. 5-HT produced esophageal smooth muscle contractions in a dose-dependent manner. Furthermore, 5-HT produced a relatively long-acting contraction. 5-HT binds to the 5-HT2, 5-HT3 and 5-HT4 receptors to induce smooth muscle contraction in feline ESMCs. These receptors, which are located in esophageal smooth muscle, are coupled to Gαq, Gαo and Gαs. These G proteins activate PLC, which leads to Ca2+/calmodulin-dependent MLCK activation, resulting in MLC20 phosphorylation and cell contraction. Conversely, DA-9701 inhibits 5-HT-induced contraction by inhibiting MLC20 phosphorylation.

Characteristics of nobiletin-induced effects on jejunal contractility.

Nobiletin, a citrus polymethoxylated flavone, exhibits multiple biological properties including anti-inflammatory, anti-carcinogenic, and anti-insulin resistance effects. The present study found that nobiletin exerted significant stimulatory effects on the contractility of isolated rat jejunal segments in all 6 different low contractile states, and meanwhile significant inhibitory effects in all 6 different high contractile states, showing characteristics of bidirectional regulation (BR). Nobiletin-exerted BR on jejunal contractility was abolished in the presence of c-kit receptor tyrosine kinase inhibitor imatinib or Ca(2+) channel blocker verapamil. In the presence of neuroxin tetrodotoxin, nobiletin only exerted stimulatory effects on jejunal contractility in both low and high contractile states. Hemicholinium-3 and atropine partially blocked nobiletin-exerted stimulatory effects on jejunal contractility in low-Ca(2+)-induced low contractile state. Phentolamine or propranolol or l-NG-nitro-arginine significantly blocked nobiletin-exerted inhibitory effects on jejunal contractility in high-Ca(2+)-induced high contractile state respectively. The effects of nobiletin on myosin light chain kinase (MLCK) mRNA expression, MLCK protein content, and myosin light chain phosphorylation extent were also bidirectional. In summary, nobiletin-exerted BR depends on the contractile states of rat jejunal segments. Nobiletin-exerted BR requires the enteric nervous system, interstitial cell of Cajal, Ca(2+), and myosin phosphorylation-related mechanisms.

Effects of ginsenosides on rat jejunal contractility.

CONTEXT: Ginsenosides are primary active ingredients of ginseng, which are believed to have various health benefits. It is found that the biotransformation of ginsenosides mainly takes place in the gastrointestinal tract and the information about ginsenosides-exerted effects on intestinal contractility is not sufficient. AIMS: The present study proposed that ginsenosides could exert stimulatory or inhibitory effects on intestinal motility depending on the assay condition-related intestinal contractile states and was to characterize the effects of ginsenosides on intestinal motility. METHODS: Jejunal contractility determination, Western blotting analysis, and real-time polymerase chain reaction were performed to test the effects of total ginsenosides isolated from Panax ginseng C. A. Mey (Araliaceae) root. RESULTS: The results showed that ginsenosides at the fixed concentration of 20 mg/L exerted bidirectional regulation (BR) on the contractility of isolated jejunal segment (IJS), depending on the contractile states. The contractility of IJS was increased by ginsenosides in low contractile states, which were correlated to the cholinergic activation, and the contractility of IJS was decreased by ginsenosides in high contractile states, which were correlated to the adrenergic activation and nitric oxide related mechanisms. Ginsenosides-induced BR was abolished in the absence of Ca(2+) or by using tetrodotoxin, implicating the requirement of Ca(2+) and the enteric nervous system. Effects of ginsenosides on myosin light chain phosphorylation and the mRNA expression of myosin light chain kinase were also bidirectional. DISCUSSION AND CONCLUSION: Results suggest that ginsenosides may have the potential clinical implication for reliving the symptoms of alternative hypo- and hyper-intestinal motility.

Dietary selenium influences calcium release and activation of MLCK in uterine smooth muscle of rats.

We sought to elucidate the effects of different concentrations of dietary selenium on calcium ion release, MLCK levels, and muscle contraction in the uterine smooth muscle of rats. The selenium (Se) content of blood and of uterine smooth muscle tissues was detected by fluorescence spectrophotometry. Ca(2+) content was measured by atomic absorption spectroscopy. Calmodulin (CaM) and MLCK RNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Dietary Se intake increased the Se levels in the blood and in uterine smooth muscle tissues and increased the Ca(2+) concentration in uterine smooth muscle tissues. The addition of Se also promoted CaM expression and enhanced MLCK activation in uterine smooth muscle tissues. In conclusion, Ca(2+), CaM, and MLCK were regulated by Se in uterine smooth muscle; Se plays a major role in regulating smooth muscle contraction in the uterus.

Effects of berberine on rat jejunal motility.

OBJECTIVES: The aim of the study was to evaluate berberine-induced bidirectional regulation on the contractility of jejunum. METHODS: Different low and high contractile states of isolated jejunal segment from rat were established to investigate the effects of berberine. KEY FINDINGS: Stimulatory effects on jejunal segment were exerted by berberine in six low contractile states and inhibitory effects were produced on jejunal segment in six high contractile states. The effects of berberine on myosin light chain kinase (MLCK) mRNA expression, MLCK protein content, and myosin phosphorylation in jejunum were also bidirectional. Bidirectional regulation was not observed in the presence of tetrodotoxin. No regulatory effects of berberine on jejunal contractility were observed in the presence of verapamil. The stimulatory effects of berberine on jejunal contractility were blocked by atropine. The inhibitory effects of berberine on jejunal contractility were abolished by phentolamine, propranolol and L-NG-nitro-arginine, respectively. CONCLUSIONS: Berberine-induced bidirectional regulation needed the presence of the enteric nervous system, and depended on the influx of extracellular Ca(2+) , related to the cholinergic system while jejunum was in low contractile states, and related to the adrenergic system and nitric oxide relaxing mechanism while jejunum was in high contractile states. The results suggested the potential clinical implication of berberine for alternating-type irritable bowel syndrome.

Myosin light-chain kinase is necessary for membrane homeostasis in cochlear inner hair cells.

The structural homeostasis of the cochlear hair cell membrane is critical for all aspects of sensory transduction, but the regulation of its maintenance is not well understood. In this report, we analyzed the cochlear hair cells of mice with specific deletion of myosin light chain kinase (MLCK) in inner hair cells. MLCK-deficient mice showed impaired hearing, with a 5- to 14-dB rise in the auditory brainstem response (ABR) thresholds to clicks and tones of different frequencies and a significant decrease in the amplitude of the ABR waves. The mutant inner hair cells produced several ball-like structures around the hair bundles in vivo, indicating impaired membrane stability. Inner hair cells isolated from the knockout mice consistently displayed less resistance to hypoosmotic solution and less membrane F-actin. Myosin light-chain phosphorylation was also reduced in the mutated inner hair cells. Our results suggest that MLCK is necessary for maintaining the membrane stability of inner hair cells.

Rational design of new binding specificity by simultaneous mutagenesis of calmodulin and a target peptide.

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies the role of CaM as a shared participant in calcium-dependent signal transduction pathways but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/target pairs that retain high affinity for one another but lack affinity for wild-type CaM and its natural interaction partners would therefore be useful as sensor components and possibly also as elements of "synthetic" cellular-signaling networks. Here, we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal-muscle light-chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained an affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple-linked binding equilibria.

Identification, tissue expression and chromosomal localization of human Obscurin-MLCK, a member of the titin and Dbl families of myosin light chain kinases.

Members of the Dbl family of guanine nucleotide exchange factors (GEFs) have important roles in the organization of actin-based cytoskeletal structures of a wide variety of cell types. Through the activation of members of the Rho family of GTP signaling molecules, these exchange factors elicit cytoskeletal alterations that allow cellular remodeling. As important regulators of RhoGTPase activity, members of this family are candidates for mediating the RhoGTPase activation and cytoskeletal changes that occur during cardiac development and during the myocardial response to hypertrophic stimuli. In this study, we characterize a novel human gene that is expressed in skeletal and cardiac muscle and has putative functional domains similar to those found in members of both the Dbl family of GEFs and the titin family of myosin light chain kinases (MLCK). The cDNA sequence of this gene, which has been designated Obscurin-myosin light chain kinase (Obscurin-MLCK), would be predicted to encode for at least 68 immunoglobulin domains, two fibronectin domains, one calcium/calmodulin binding domain, a RhoGTP exchange factor domain, and two serine-threonine kinase domains. The combination of the putative Rho GEF and two kinase domains has not been noted in any other members of the titin or Dbl families. Alternative splicing allows the generation of a number of unique Obscurin-MLCK isoforms that contain various combinations of the functional domains. One group of isoforms is comparable to Unc-89, a Caenorhabditis elegans sarcomere-associated protein, in that they contain a putative RhoGEF domain and multiple immunoglobulin repeats. Other isoforms more closely resemble MLCK, containing one or both of the putative carboxy-terminal serine-threonine kinase domains. The modular nature of the Obscurin-MLCK isoforms indicates that it may have an array of functions important to cardiac and skeletal muscle physiology.

Gastrin induces over-expression of genes involved in human U373 glioblastoma cell migration.

Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.