An Experimental Study On The Effects And Mechanisms Of Melationin On Dysfunction Of Intestinal Motility Induced By Stress

Background:Irritable bowel syndrome (IBS) is a common functional disease of the digestive tract. At present, the pathophysiology of IBS is incompletely known; factors thought to play a role in this disease include altered gastrointestinal motility, visceral hypersensitivity and dysregulation of the brain-gut axis and psychosocial factors. Changes of some bioactive substances have been proven in blood and tissues of IBS patients, such as cholecystokinin (CCK),5-HT. Melatonin is a hormone mainly produced by the pineal gland and the gastrointestinal tract. The abnormal concentration of melatonin and its metabolites were found in IBS patients. Melatonin was involved in some process of the physiological functions, and interacted with other hormones. That is worth focusing on the relationship of abnormal concentration of melatonin and the pathophysiology of IBS. Many studies found that the colonic contractile frequency and magnitude of IBS patients were significantly different from healthy people. Melatonin regulate gastrointestinal motility,this ability may be relate to the pathophysiology of IBS.So, this study focus on the changes of concentration of melatonin, the effect of melatonin on intestinal motility and the mechanisms involved in.Part IObjectives:To investigate the effect of melatonin on the frequencies of fecal output, the index of small intestinal transit and the levels of hormone in serum induced by acute and chronic water avoidance stress, and discuss the mechanisms involved in.Methods:60 rats were divided to control+vehicle (Control+V), chronic water avoidance stress+vehicle (cWAS+V), chronic water avoidance stress+melatonin (cWAS+MT), acute water avoidance stress+vehicle (aWAS+V) and acute water avoidance stress+melatonin (aWAS+MT) group. The changes in the frequencies of fecal output, the index of small intestinal transit and the levels of hormone in serum induced by acute and chronic water avoidance stress and effects of daily melatonin administration on them were observed.Results:The total distances and velocity were decreased by 10 days of chronic stress, but acute water avoidance stress increased them. Melatonin improved these changes of the behaviors in chronic water avoidance stress. The acute and chronic water avoidance stress induced significant accentuation of intestinal motility, which shown as increased fecal output. Melatonin decreased this augment induced by chronic and acute water avoidance stress. Melatonin also decreased the index of small intestinal transit in acute water avoidance stress. These effects were associated with the concentrations of melatonin and 5-HT in serum. The concentrations of melatonin was elevated (from 16.03±3.96pg/mL to 28.89±1.58pg/mL) and it inhibited the secretion of 5-HT in acute water avoidance stress. Melatonin further decreased the 5-HT which reduced by chronic water avoidance stress (from 692.72±67.03ng/mL to 588.73±36.61 ng/mL).Conclusions:These results suggested that melatonin may regulate the intestinal motility dysfunction through its direct effect on the smooth muscle and via the indirect effect on 5-HT secretary in acute and chronic water avoidance stress. In addition, the central nervous mechanism about expression of receptors may be involved in chronic stress.Part IIObjectives:To investigate the effects and the mechanisms of melatonin and others drugs on the spontaneously contractile activity in rat proximal colonic smooth muscle strips.Methods The proximal colon which near ileocecal junction about 3 cm was removed and placed in Ca2+-free PSS which was constantly oxygenated. After the mucosa was carefully dissected away, the smooth muscle strips were obtained. The fresh longitudinal muscle strip mounted in a 5ml Tyrode’s solution and connected to an isometric force transducer. Tyrode’s solutions were continuously warmed by a circulating water jacketed at 37 C and bubbled with carbogen (95% O2+5% CO2). The muscle strips were placed under 1 g load tension and allowed to equilibrate for 60 min, with solution change every 20 min. The relative effects on the contractile amplitude and frequency of strips were observed before and after the addition of drugs.Results:Melatonin inhibited the spontaneous contraction of colon in rat, the concentration of 10-4 mol/L,10"3 mol/L melatonin inhibited the amplitude of spontaneous contraction were (43.58±19.53)% and (79.18±17.77)% (P=0.046 and P=0.020, all n=6) compared to control group, respectively. The concentration of 10-5 mol/L,10-4 mol/L,10-3 mol/L melatonin inhibited the frequencies of spontaneous contraction were (52.54±22.13)% and (120.28±51.82)%, (252.49±46.50)% (P=0.043 and P=0.020, P=0.000, all n=6) compared to control group, respectively. When these strips were pretreated with 10-4 mol/L Ach, the amplitude of spontaneous contraction was (428.48±195.25)% (P=0.001),melatonin inhibited the reinforced spontaneous contraction induced by Ach, the amplitude of spontaneous contraction was (46.88+20.53)% (P=0.007,n=6) compared to Ach group. When these strips were pretreated with 3×10-2 mol/L KCL, the amplitude of spontaneous contraction was (239.87±33.98)% (P=0.018), melatonin inhibited the reinforced spontaneous contraction induced by KCL, the amplitude of spontaneous contraction was (14.32±7.34)% (P=0.001,n=6) compared to KCL group. When these strips were pretreated with 10-6mol/L 5-HT, the amplitude of spontaneous contraction was (102.00±44.13)% (P=0.033), melatonin inhibited the reinforced spontaneous contraction induced by 5-HT, the amplitude of spontaneous contraction was (35.59±6.44)% (P=0.003,n=6) compared to 5-HT group.Conclusions:Melatonin inhibited the colonic spontaneous contraction, and attenuated the reinforced spontaneous contraction induced by Ach, KCL and 5-HT. These effects may be through the enteric neural system and the smooth muscle cell. PartⅢObjectives:To examine the effect and its mechanism of melatonin on voltage-dependent potassium currents and calcium-activated potassium currents.Methods:The proximal colon which near ileocecal junction about 3cm was removed, the strips were cut into small pieces (2mm×2mm) and placed in Ca2+-free PSS solution at 37℃. After 20-30 min incubation, the segments were rinsed in Ca2+-free PSS solution five times to remove the enzyme. Single cells were dispersed by gentle agitation with a wide-pore pipette. Several drops of cell suspension were placed in a coverslip-overlayed chamber which was mounted on an Olympus invertedⅨ71 microscope. Cells were perfused with Tyrode’s (3 ml/min) after adhering to the coverslip. The ion channel currents were recorded by conventional whole-cell patch clamp method. Whole-cell currents were recorded with an EPC-10 amplifier. Drugs were added subsequently to the cell suspension to observe relative effects on voltage-dependent potassium currents and calcium-activated potassium currents when their values were stable.Results:Melatonin (10-4 mol/L) inhibited the amplitude of the currents of voltage-dependent potassium channel (P=0.011, n=6),from (829.31±114.08) pA to (430.06±52.48) pA, (47.47±8.20)% compared to control group. When these cells were pretreated with Luzindole, the amplitude of voltage-dependent potassium currents was also inhibited by melatonin (P=0.026, n=6),the inhibition percentage was (32.77±2.22)% and the amplitude was (557.55±18.39) pA. The amplitude of calcium-activated potassium currents was (1561.61±365.69) pA. Melatonin (10-4 mol/L) inhibited the calcium-activated potassium currents (P=0.011, n=6),from (1561.61±365.69) pA to (765.50±372.42) pA, the amplitude was (47.47±8.20) % compared to control group.Conclusions:Melatonin inhibited the voltage-dependent potassium currents and calcium-activated potassium currents. These effects may result from decreased the concentration of calcium in intracellular. The decreased currents of two channels may attenuate the repolarization and the frequencies of action potential.