Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

Endoplasmic reticulum (ER) homeostasis is regulated by ER-resident E3 ubiquitin ligase Hrd1, which has been implicated in inflammatory bowel disease (IBD). Ginsenoside Rb1 (GRb1) is the major ginsenoside in ginseng with multiple pharmacological activities. In this study we investigated the role of Hrd1 in IBD and its regulation by GRb1. Two mouse colitis models were established to mimic human IBD: drinking water containing dextran sodium sulfate (DSS) as well as intra-colonic infusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Colitis mice were treated with GRb1 (20, 40 mg·kg-1·d-1, ig) or a positive control drug sulfasalazine (500 mg·kg-1·d-1, ig) for 7 days. The model mice showed typical colitis symptoms and pathological changes in colon tissue. In addition to significant inflammatory responses and cell apoptosis in colon tissue, colon epithelial expression of Hrd1 was significantly decreased, the expression of ER stress markers GRP78, PERK, CHOP, and caspase 12 was increased, and the expression of Fas was increased (Fas was removed by Hrd1-induced ubiquitination). These changes were partially, or completely, reversed by GRb1 administration, whereas injection of Hrd1 inhibitor LS102 (50 mg·kg-1· d-1, ip, for 6 days) exacerbated colitis symptoms in colitis mice. GRb1 administration not only normalized Hrd1 expression at both the mRNA and protein levels, but also alleviated the ER stress response, Fas-related apoptosis, and other colitis symptoms. In intestinal cell line IEC-6, the expression of Hrd1 was significantly decreased by LPS treatment, but was normalized by GRb1 (200 µM). GRb1 alleviated LPS-induced ER stress and cell apoptosis in IEC-6 cells, and GRb1 action was inhibited by knockdown of Hrd1 using small interfering RNA. In summary, these results reveal a pathological role of Hrd1 in colitis, and provide a novel insight into alternative treatment of colitis using GRb1 activating Hrd1 signaling pathway.

Enhancement of epithelial cell autophagy induced by sinensetin alleviates epithelial barrier dysfunction in colitis.

Intestinal epithelial barrier dysfunction is a key pathology of colitis. Autophagy of epithelial cells maintains homeostasis of the intestinal barrier by inhibiting apoptosis and stimulating degradation of the tight junction protein claudin-2. This study investigated the effects and mechanism of activity of sinensetin, a polymethylated flavonoid isolated from tangerine peel and citrus, on intestinal barrier dysfunction in colitis. Animal model of colitis were established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid and oral treatment with dextran sulfate sodium. Epithelial barrier function was evaluated by measuring the serum recovery of fluorescein isothiocyanate-4 kD dextran in vivo and transepithelial electrical resistance in Caco-2 cells, respectively. Epithelial cell autophagy assayed by autophagosome formation and expression of autophagy-related protein. Sinensetin reversed colitis-associated increase in intestinal permeability, significantly promoted epithelial cell autophagy, and further decreased epithelial cell apoptosis, and reduced mucosal claudin-2. Sinenstetin alleviated colitis symptoms rats and mice with colitis. Knockdown of 5' adenosine monophosphate-activated protein kinase (AMPK) reversed the promotion of epithelial autophagy by sinensetin. In conclusion, sinensetin significantly alleviated intestinal barrier dysfunction in colitis by promoting epithelial cell autophagy, and further inhibiting apoptosis and promoting claudin-2 degradation. The results highlighted novel potential benefits of sinensetin in colitis.

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 Ca(2+) 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 Ca(2+)-dependent MLC phosphorylation.

Characteristics of diprophylline-induced bidirectional modulation on rat jejunal contractility.

In this study, we propose that diprophylline exerts bidirectional modulation (BM) on the isolated rat jejunal segment depending on its contractile state. The results supported the hypothesis. Diprophylline (20 µM) exerted stimulatory effects on the contractility of jejunal segment in six low contractile states while inhibitory effects in six high contractile states, showing the characteristics of BM. Diprophylline-induced stimulatory effect was significantly blocked by atropine, indicating the correlation with cholinergic activation. Diprophylline-induced inhibitory effect was partially blocked by phentolamine, propranolol, and L-N-Nitro-Arginine respectively, indicating their correlation with sympathetic activation and nitric oxide-mediated relaxing mechanisms. Diprophylline-induced BM was abolished by tetrodotoxin or in a Ca(2+) free condition or pretreated with tyrosine kinase inhibitor imatinib, suggesting that diprophylline-induced BM is Ca(2+) dependent, and that it requires the presence of enteric nervous system as well as pacemaker activity of interstitial cells of Cajal. Diprophylline significantly increased the reduced MLCK expression and myosin extent in constipation-prominent rats and significantly decreased the increased MLCK expression and myosin extent in diarrhea-prominent rats, suggesting that the change of MLCK expression may also be involved in diprophylline-induced BM on rat jejunal contractility. In summary, diprophylline-exerted BM depends on the contractile states of the jejunal segments, requires the presence of Ca(2+), enteric nervous system, pacemaker activity of interstitial cells of Cajal, and MLCK-correlated myosin phosphorylation. The results suggest the potential implication of diprophylline in relieving alternative hypo/hyper intestinal motility.

Characteristics of evodiamine-exerted stimulatory effects on rat jejunal contractility.

This study was designed to characterise the effects of evodiamine on intestinal contractility and reveal the correlated mechanisms. Evodiamine (2.5-80.0 µM) increased normal jejunal contractility and jejunal hypocontractility established under a variety of experimental conditions. Evodiamine-exerted stimulatory effects were blocked by the L-type Ca(2+) channel blocker nifedipine or abolished in the Ca(2+)-free assay condition. The stimulatory effects of evodiamine on jejunal contractility were partially blocked in the presence of neurotoxin tetrodotoxin or endogenous acetylcholine synthesis blocker hemicholinium-3 or muscarinic receptor antagonist atropine, respectively. Evodiamine-exerted stimulatory effects were blocked by c-kit receptor tyrosine kinase inhibitor imatinib. Evodiamine increased myosin phosphorylation in jejunal smooth muscle of constipation-prominent rats. These results showed that evodiamine-exerted stimulatory effects on jejunal segments are Ca(2+)-dependent, need the presence of interstitial cell of Cajal, requirement of cholinergic neuron and correlate with increased myosin phosphorylation, implicating the potential value of evodiamine in relieving hypo-motility disorders.

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.

The characteristics of genistin-induced inhibitory effects on intestinal motility.

Genistin belongs to isoflavones. Based on the facts that genistin exerts inhibitory effects on the contractility of vascular smooth muscle,the present study was designed to characterize the effects of genistin on intestinal contractility and evaluate its potential clinical implication. Ex vivo [isolated jejunal segment (IJS) of rat], in vitro, and in vivo assays were used in the study. The results indicated that genistin (5-80 µmol/L) inhibited the contraction of IJS in a dose-dependent manner and inhibited the increased-contractility of IJS induced by acetylcholine (ACh), histamine, high Ca(2+), and erythromycin, respectively. The inhibitory effects of genistin were correlated with the stimulation of alpha adrenergic and beta adrenergic receptors since these inhibitory effects were significantly blocked in the presence of phentolamine and propranolol respectively. No further inhibitory effects of genistin were observed in the presence of verapamil or in Ca(2+)-free condition, indicating genistin-induced inhibitory effects are Ca(2+)-dependent. Genistin decreased myosin light chain kinase (MLCK) protein contents and MLCK mRNA expression in IJS, and inhibited both phosphorylation and Mg(2+)-ATPase activity of purified myosin, implicating that the decrease of MLCK contents and inhibition of MLCK activity are involved in the genistin-induced inhibitory effects. The study suggests the potential clinical implications of genistin in relieving intestinal hypercontractility.

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.

Characteristics of deslanoside-induced modulation on jejunal contractility.

AIM: To characterize the dual effects of deslanoside on the contractility of jejunal smooth muscle. METHODS: Eight pairs of different low and high contractile states of isolated jejunal smooth muscle fragment (JSMF) were established. Contractile amplitude of JSMF in different low and high contractile states was selected to determine the effects of deslanoside, and Western blotting analysis was performed to measure the effects of deslanoside on myosin phosphorylation of jejunal smooth muscle. RESULTS: Stimulatory effects on the contractility of JSMF were induced (45.3% ± 4.0% vs 87.0% ± 7.8%, P < 0.01) by deslanoside in 8 low contractile states, and inhibitory effects were induced (180.6% ± 17.8% vs 109.9% ± 10.8%, P < 0.01) on the contractility of JSMF in 8 high contractile states. The effect of deslanoside on the phosphorylation of myosin light chain of JSMF in low (78.1% ± 4.1% vs 96.0% ± 8.1%, P < 0.01) and high contractile state (139.2% ± 8.5% vs 105.5 ± 7.34, P < 0.01) was also bidirectional. Bidirectional regulation (BR) was abolished in the presence of tetrodotoxin. Deslanoside did not affect jejunal contractility pretreated with the Ca(2+) channel blocker verapamil or in a Ca(2+)-free assay condition. The stimulatory effect of deslanoside on JSMF in a low contractile state (low Ca(2+) induced) was abolished by atropine. The inhibitory effect of deslanoside on jejunal contractility in a high contractile state (high Ca(2+) induced) was blocked by phentolamine, propranolol and L-NG-nitro-arginine, respectively. CONCLUSION: Deslanoside-induced BR is Ca(2+) dependent and is related to cholinergic and adrenergic systems when JSMF is in low or high contractile states.