The Effects And Mechanisms Of Tong-Xie-Yao-Fang On Visceral Sensitivity And Dysfunction Of Intestinal Motility Induced By Chronic Stress

Objectives:1. To observe the biological characteristics of chronic water avoidance stress rats and explore the biological basis of this model.2. To observe the role of CORT in visceral sensitivity and dysfunction of intestinal motility induced by chronic stress and explore its mechanisms.3. To observe the influences of Tong-Xie-Yao-Fang on chronic stress rats and preliminary explore the mechanisms.Methods:1.1)Set up the chronic water avoidance stress model; 2)Observed the weight gain of the rats; 3)Measured the abdominal withdrawal reflex (abdominal withdrawal reflex, AWR) score caused by colorectal distension (colorectal distension, CRD) and visceral pain threshold of score 3 to evaluate visceral sensitivity of the rats; 4)Observed the stool form and count the fecal pellets during the period of water avoidance stress (WAS) and baseline per hour in order to evaluate the intestinal transit function of the rats; 5)The sugar water preference and open-field test were used to evaluate experimental rats’depression-and anxiety-like behavior; 6)The rats’colonic samples had been used for HE staining to observe the pathological changes; 7)Assayed plasma corticosterone (CORT) levels using Elisa and measured the gland/body weight ratio, in order to evaluate the hypothalamic-pituitary-adrenal axis (HPA axis) activity; 8)Applied Elisa to measure the plasma neurotransmitters (5-HT, SP, VIP, NO) levels to evaluate its endocrine basis; 9) Toluidine blue staining method was used for dyeing and counting the colon mast cells.2.1)Rebuilded the chronic water avoidance stress model and set up the normal control group, model group and CORT blockers group (RU-486 group); 2)Evaluated the visceral sensitivity, intestinal transit capacity, anxiety and depression-like behaviors of the three groups; 3)Assayed plasma corticosterone (CORT) levels using Elisa and measure the adrenal gland/body weight ratio; 4) RT-PCR was used to detect the mRNA levels of endogenous cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid receptor 1 (TRPV1) in the L6-S2 DRG and western-blot method for the CB1 and TRPV1 protein levels.3. 1)Rebuilded the chronic water avoidance stress model and set up the normal control group, model group and Tong-Xie-Yao-Fang high group, Tong-Xie-Yao-Fang middle group and Tong-Xie-Yao-Fang low group. 2)Compared between groups:weight gain, visceral sensitivity, intestinal transit capacity and anxiety and depression-like behavious.3) Evaluate activity of HPA axis of each group rats.4) Measured neurotransmitter levels of each group rats (plasma levels of 5-HT, SP, and NO).5) RT-PCR was used for detecting the mRNA levels of endogenous cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid receptor 1 (TRPV1) in the L6-S2 DRG of each group and western-blot method for the CB1 and TRPV1 protein levels.6) Toluidine blue staining method was used for dyeing and counting the colon mast cells of the each group rats.Results:1. Model rats compared with control group rats:1) Weight gain were significantly reduced.2) Presenced the status of visceral hyperalgesia (AWR score was significantly increased and visceral pain threshold was significantly lower).3) Intestinal transit notably enhanced (the fecal output number was obviously increased during the period of water avoidance stress (WAS) and baseline per hour).4) Presenced depression-and anxiety-like behaviors (sugar water preference ratio decreased significantly and the explore behaviors in the open field tank significantly reduced).5) Colonic samples were absence of any obvious histopathologic appearances.6) HPA axis activity increased (plasma CORT levels were significantly increased and the adrenal/body weight ratio was significantly increased).7) Plasma 5-HT and SP levels were significantly increased and NO levels were significantly decreased, while the VIP levels did not change.8) Colon mast cell number obviously increased.2.1) RU-486 could obviously improve the visceral hypersensitivity state of the model rats.2) RU-486 could significantly reduce the intestinal transit capacity of the models rats.3) To a certain extent, RU-486 could improve the depression and anxiety state of the model rats.4) The activity of HPA axis of the model rat was obviously increased and RU-486 could improve the biological behaviour of the rats by reducing the activity of the HPA axis.5) The CB1mRNA and protein levels in L6-S2DRG of the model rats were significantly reduced, while the TRPV1mRNA and protein levels were significantly increased.6) RU-486 could significantly increase the CB1mRNA and protein levels and reduce TRPV1mRNA and protein levels in L6-S2 DRG of the model rats3.1)Tong-Xie-Yao-Fang could improve the visceral hypersensitivity and high intestinal transit state of the chronic stress model rats,and had certain effect on the depression and anxiety-like behavious. With the improvement of IBS-like symptoms, its weight gain also increased.2) Tong-Xie-Yao-Fang could regulate the secretion of the neurotransmitters (reduced plasma 5-HT and SP levels, increased NO levels), reduce the number of mast cells in the colon, regulate the function of HPA axis (reduced plasma CORT levels and adrenal gland/body weight ratio) and then increased CB1mRNA and protein levels and reduced TRPV1mRNA and protein levels in L6-S2 DRG The above may be its internal mechanisms.Conclusions:1. The chronic water avoidance stress rats presenced the visceral hyperalgesia and dysfunction of intestinal motility that were the main clinical features of irritable bowel syndrome (IBS) patients. So, it could be used for related animal experimental research and HPA axis activity increased, endocrine abnormalities, the increased number of mast cells in the colon were the inherent biological basis of the model.2. The abnormal expression of the CB1 (down) and TRPV1 (raised) in the L6-S2 DRG were closely related to the visceral hyperalgesia and high intestinal transit state of the chronic water avoidance stress rats and the changes may be regulated by the CORT and its related receptors. The hyperactivity of the HPA axis (plasma CORT levels significantly Elevated) may be involved in the anxiety and depression like behaviours of the chronic water avoidance stress rats. The above findings would provide theoretical basis for further research.3. Tong-Xie-Yao-Fang had significant improvement on the intestinal visceral hypersensitivity and dysfunction of intestinal motility of the chronic stressrats. Regulating the endocrine, colon mast cells amount, HPA axis activityand CB 1 and TRPV1 mRNA and protein levels in the L6-S2DRGs may beits inherent mechanism. Those will provide certain theoretical basis for theclinical treatment of IBS, which has important clinical and theoreticalvalues.