Yongjian Xiong

Citrus nobiletin ameliorates experimental colitis by reducing inflammation and restoring impaired intestinal barrier function.

SCOPE Inflammatory bowel disease is a chronic inflammatory disorder of the gastrointestinal tract. Citrus nobiletin can exert robust anti-inflammatory effects in vivo and in vitro. We evaluated the impact of nobiletin on the excessive inflammatory response and impaired barrier function in a rodent colitis model. METHODS AND RESULTS Colitis was established by infusion with 1 mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol (40% v/v) in rats at a 125 mg/kg dose. Caco-2 cell monolayer exposed to LPSs is used as a culture model for intestinal permeability measurements. Nobiletin decreased rat epithelial proinflammatory cytokines and mediators production. Nobiletin restored impaired barrier function in colitic rats and Caco-2 monolayer. Nobiletin decreased protein expressions of Akt, nuclear factor-kappa B (NF-κB), and myosin light chain kinase (MLCK) isolated from rat intestinal epithelial tissue and Caco-2 cell, respectively. PI3K inhibitor or siRNA targeting of either Akt or NF-κB mitigated the impact of nobiletin on MLCK expression and barrier function in Caco-2 monolayer, respectively. CONCLUSION Nobiletin exerted anti-inflammatory effects in TNBS-induced colitis through the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression. Nobiletin restored barrier function, which had been damaged after TNBS administration, through the inhibition of the Akt-NF-κB-MLCK pathway.

Hesperidin alleviates rat postoperative ileus through anti-inflammation and stimulation of Ca 2+ -dependent myosin phosphorylation

Aim:Postoperative ileus (POI) is a postoperative dysmotility disorder of gastrointestinal tract, which remains one of the most perplexing problems in medicine. In the present study we investigated the effects of hesperidin, a major flavonoid in sweet oranges and lemons, on POI in rats.Methods:SD rats were administered hesperidin (5, 20, and 80 mg·kg−1·d−1, ig) for 3 consecutive days. POI operation (gently manipulating the cecum for 1 min) was performed on d 2. The gastrointestinal motility and isolated intestinal contraction were examined 1 d after the operation. Then the myosin phosphorylation and inflammatory responses in cecum tissue were assessed. Smooth muscle cells were isolated from rat small intestine for in vitro experiments.Results:The gastric emptying and intestinal transit were significantly decreased in POI rats, which were reversed by administration of hesperidin. In ileum and cecum preparations of POI rats in vitro, hesperidin (2.5–160 μmol/L) dose-dependently increased the spontaneous contraction amplitudes without affecting the contractile frequency, which was blocked by the myosin light chain kinase (MLCK) inhibitor ML-7 or verapamil, but not by TTX. Furthermore, administration of hesperidin increased the phosphorylation of MLC20 in the cecum tissue of POI rats. Moreover, administration of hesperidin reversed the increased levels of inflammatory cytokines, iNOS and COX-2 in cecum tissue of POI rats. In freshly isolated intestinal smooth muscle cells, hesperidin (5–80 μmol/L) dose-dependently increased the intracellular Ca2+ concentration as well as the phosphorylation of MLC20, which was abrogated by ML-7 or siRNA that knocked down MLCK.Conclusion:Oral administration of hesperidin effectively alleviates rat POI through inhibition of inflammatory responses and stimulation of Ca2+-dependent MLC phosphorylation.

Salvianolic Acid B Restored Impaired Barrier Function via Downregulation of MLCK by microRNA-1 in Rat Colitis Model

Salvianolic acid B (Sal B) is isolated from the traditional Chinese medical herb Salvia miltiorrhiza and is reported to have a wide range of therapeutic benefits. The aim of this study was to investigate the effects of Sal B on epithelial barrier dysfunction in rat colitis and to uncover related mechanisms. Rat colitis model was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). The intestinal barrier function was evaluated by measuring the serum recovery of fluorescein isothiocyanate-4 kD dextran in vivo and transepithelial electrical resistance in vitro respectively. The protein expression related to intestinal barrier function was studied using western blotting. The effects of Sal B on inflammatory responses, oxidative damage and colitis recurrence were also studied in this study. The intestinal barrier dysfunction in colitis was reversed by Sal B, accompanied with the decrease of tight junction proteins, and the effect could be blocked by microRNA-1(miR-1) inhibition. The inflammatory responses, oxidative damage and colitis recurrence were also decreased by Sal B. The colitis symptoms and recurrences were ameliorated by Sal B, and restoration of impaired barrier function via downregulation of MLCK by miR-1 maybe involved in this effect. This study provides some novel insights into both of the pathological mechanisms and treatment alternatives of inflammatory bowel disease.

Effects of berberine on rat jejunal motility

The aim of the study was to evaluate berberine‐induced bidirectional regulation on the contractility of jejunum.

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.

Epithelial MLCK and Smooth Muscle MLCK May Play Different Roles in the Development of Inflammatory Bowel Disease

In the manuscript entitled ‘‘Claudin-2 Regulates Colorectal Inflammation via Myosin Light Chain Kinase-Dependent Signaling,’’ Nishida et al. [1] found that myosin light-chain kinase (MLCK) protein content in the entire mucosal layer of the colon tissue isolated from TNF-a-treated claudin-2 (-/-) mice was increased compared with that in the claudin-2 (?/?) mice. They concluded that claudin-2 could regulate colon inflammation via MLCK-dependent signaling pathway and claudin-2 as well as MLCK was the potential therapeutic targets for treating inflammatory bowel disease. Nishida et al. did not clarify whether epithelial MLCK (long MLCK, 220 kDa) or smooth muscle MLCK (short MLCK, 130 kDa) was observed in their study. It is well known that up-regulation of epithelial MLCK is involved in the defects of intestinal barrier function, which is critical to the initiation and progression of inflammatory bowel disease [2, 3]. The relationship between TNF-a-induced enhancement of epithelial MLCK expression and barrier dysfunction in inflammatory bowel disease is extensively studied; epithelial MLCK expression in different stages of inflammation may vary because the degree of inflammation and epithelial damage are different at different times [4]. Though claudin-2 up-regulation is prevented by epithelial MLCK inhibition [2], there is still an incomplete understanding of the causal relationship between the epithelial MLCK expression and claudin-2 expression in inflammatory bowel disease. Smooth muscle MLCK is transcribed from the same gene as epithelial MLCK, and the gene is the mylk 1 gene [5]. Smooth muscle MLCK plays an important role in the regulation of smooth muscle contraction, and variation of smooth muscle MLCK content leads to dysmotility [6]. The motility disorders secondarily cause abnormal growth of the intestinal flora, and the induced disturbance of this flora aggravates the pathogenesis of intestinal inflammation [7]. Based on the above information, it is critical to reveal that the epithelial and smooth muscle MLCK contents in different tissue layers and in different stages of intestinal inflammation can be observed. Furthermore, anti-MLCK antibody cross-reacts with both the short MLCK and long MLCK [5], and there should be two bands in the Western blotting image for determination of MLCK, which represent epithelial MLCK (220 kDa) and smooth muscle MLCK (130 kDa), respectively. Thanks very much to Digestive Diseases and Sciences for giving us the chance to discuss the hot topic regarding treating inflammatory bowel disease; thanks too for the contribution by Nishida et al.

Inhibitory effects of daidzein on intestinal motility in normal and high contractile states

Context: Daidzein is a naturally occurring compound and has various health benefits. However, its effects on intestinal smooth muscle contractility remain unknown. Aims: The present study was to characterize the effects of daidzein on the contractility of isolated jejunal smooth muscle and its underlying mechanisms. Methods: Ex vivo assay was selected as the major method to determine the effects of daidzein on the contractility of isolated jejunal smooth muscle fragment (JSMF). Results: Daidzein (5–160 µmol/L) inhibited the contractility of JSMF in normal contractile state and in a dose-dependent manner. Daidzein also inhibited the contractility of JSMF induced by ACh, histamine, erythromycin and high Ca2+, respectively, and decreased charcoal propulsion in the small intestine in vivo. The inhibitory effects of daidzein were partially blocked by phentolamine or propranolol and were abolished in the presence of varapamil or at Ca2+-free assay condition. However, the inhibitory effects of daidzein on jejunal contraction were not significantly influenced by nitric oxide (NO) synthase inhibitor L-NG-nitro-arginine (l-NNA). Daidzein was also found to directly inhibit the phosphorylation and Mg2+-ATPase activity of smooth muscle myosin. Discussion and Conclusion: The results implicated that α- and β-adrenergic receptors were involved in the inhibitory effects produced by daidzein rather than via NO pathway. As a phytoestrogen, daidzein has shown its potential value in relieving the hypercontractility of small intestine.

Activation of sirtuin 1 by catalpol-induced down-regulation of microRNA-132 attenuates endoplasmic reticulum stress in colitis

&NA; Defective expression of NAD‐dependent protein deacetylase sirtuin 1 (SIRT1) triggers endoplasmic reticulum (ER) stress and epithelial cell apoptosis in inflammatory bowel disease. MicroRNA‐132 (miR‐132) has been shown to regulate inflammatory processes through down‐regulating SIRT1. Catalpol is a potential antioxidant and anti‐apoptotic agent in inflammatory disease. This study aimed to investigate the signaling mechanisms underlying catalpol‐induced SIRT1 activation and inhibition of ER stress in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6‐trinitrobenzene sulfonic acid. miR‐132 expression was measured by quantitative real‐time polymerase chain reaction and in situ hybridization, and the regulation of SIRT1 by miR‐132 was examined by dual luciferase reporter assay. Protein expression related to ER stress and apoptosis was measured by western blotting. The ER stress marker proteins ATF6, CHOP, and caspase12, and acetylation of heat‐shock factor‐1 were increased in colitis and these increases were significantly reversed by catalpol, while the colitis‐induced reduction in GRP78 was also reversed by catalpol. The inhibition of ER stress by catalpol was significantly inhibited by small interfering RNA targeting SIRT1 or miR‐132. Moreover, other colitis symptoms including infiltration of inflammatory cells, cytokine profiles, oxidative responses, and epithelial cell apoptosis were also significantly decreased by catalpol. Mechanistically, the defective expression of SIRT1 in colitis was significantly counteracted by catalpol, while miR‐132, which is a negative targeting regulator of SIRT1, was confirmed as the potential target of catalpol. These results support a link between ER stress and the miR‐132/SIRT1/heat‐shock factor‐1 signaling pathway, and the modulation of this pathway by catalpol in colitis. Graphical abstract Figure. No caption available. ER, endoplasmic reticulum; HSF1, heat‐shock factor1; IBD, inflammatory bowel disease; SIRT1, sirtuin 1.

Effects of ginsenosides on rat jejunal contractility

Abstract 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 Ca2+ or by using tetrodotoxin, implicating the requirement of Ca2+ 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.

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.