| There are two parts in this paper, including preliminary study of establishing a new disease-syndrome-symptom integrated IBS animal model and exploration of brain-gut axis mechanism of Chang'anyihao in treatment of IBS animal model.1 Preliminary study of establishing a new disease-syndrome-symptom integrated IBS animal modelObjective:To establish a new disease-syndrome-symptom integrated animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency.Methods:(1)Model establishment:we combined mother-infant separation, chronic restraint and senna gavage to establish a new animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency. (2)Model evaluation:we used pain threshold indicating visceral hypersensitivity to evaluate "disease" model; open field test and serum D-xylose level to evaluate "syndrome" model; defecation grain number and loose stool rate to evaluate "symptom" model.Results:(1)In the end, rat body weight change was significantly different (F=10.132, p=0.000<0.05). Compared with normal group, body weight gain of the three-factor rats was significantly decreased (p<0.05). (2)In the end, the pain threshold in different groups was significantly different (F=41.299,p=0.000<0.05), pain threshold of the three-factor rats was significantly declined compared with the normal group (p<0.05). (3)In the end, there was significant difference in open field test between groups (p<0.05). Compared with the normal group, total cross number, standing number and decoration number of three-factor rats were significantly dropped (p<0.05). (4)In the end, the serum D-xylose level of three-factor rats significantly went down (F=143.614, p=0.000<0.05). (5)In the end, defecation grain number and loose stool rate of the three-factor rats significantly went up (p<0.05). Conclusion:we combined mother-infant separation, chronic restraint and senna gavage and successfully established a new disease-syndrome-symptom integrated animal model of IBS-D due to syndrome of liver stagnation and spleen deficiency.2 Exploration of brain-gut interaction mechanism of Chang'anyihao in treatment of IBS animal modelObjective:To explore the brain-gut axis mechanism of Chang'anyihao in treatment of IBS animal model. Experiment 1.Chang'anyihao on body weight and visceral sensitivityMethods:(1)Model establishment and evaluation:we combined chronic restraint stress and forced swimming to replicate IBS visceral hypersensitivity rat model and used weight change, visceral sensitivity and histopathological biopsy as evaluation indicators. (2)Chang'anyihao in treatment:we used body weight change to assess weight gain and pain threshold to assess changes in visceral sensitivity.Conclusions:(1)In the end, weight changes between groups were significantly different (F=3.551,p=0.000<0.05). Compared with the normal group, body weight gain in the model group was significantly reduced (p=0.019). There were significant differences in pain threshold between groups (F=8.311,p=0.000<0.05). Compared with the normal group, pain threshold of the model group was significantly decreased (p<0.05). Histopathological HE staining in each group showed no significant changes. (2)On the 4th,8th and 12th day and at the end of treatment, the weight changes in different groups were not statistically different (p=0.730,0.804,0.137,0.444). In the end, there were significant differences in pain threshold between different groups (X 2=30.933,p=0.000<0.05). Compared with the normal group, pain threshold of the model group significantlty decreased (x 2=24.8472;p=0.0000<0.05). Compared with the model group, pain thresholds of fluoxetine group, Chang'anyihao high, medium and low-dose groups were significantly increased (x2=8.7969,13.9562,12.7508, 13.8110;p=0.0123,0.0009,0.0017,0.0010<0.05).Experiment 2.Chang'anyihao on colon 5-HT levelMethods:We used immunohistochemical staining method to evaluate 5-HT level in rat colon.Results:In the end,5-HT levels in rat colon were significantly different between groups (x2=53.144,p=0.000<0.05). Compared with the normal group, the 5-HT level in model group significantly went up (x2=38.2112;p=0.0000<0.05). Compared with the model group,5-HT levels of Schutte group, Chang'anyihao high and medium-dose groups significantly decreased (X 2=13.9439,14.6154,15.8966;p=0.0009,0.0004,0.0007<0.05).Experiment 3.Chang'anyihao on serum 5-HT level of IBS rat modelMethods:We used ELISA to detect serum 5-HT level.Results:In the end, serum 5-HT levels were significantly different between groups (x2=53.125,p=0.000<0.05). Compared with the normal group,5-HT level in the model group significantly went up (x2=34.4853;p=0.0000<0.05). Compared with the model group,5-HT levels of Schutte group, Chang'anyihao high and medium-dose groups significantly decreased (x 2=19.6384,16.0064,16.6231;p=0.0001,0.0003,0.0002<0.05).Experiment 4. Chang'anyihao on hippocampal 5-HT1a and BDNF gene expressionMethods:We used fluorescent quantitative real-time PCR to detect 5-HTla and BDNF mRNA gene expression level in rat hippocampal.Results:(1)In the end,5-HT1a mRNA expression levels were significantly different between groups (x2=59.606, p=0.000<0.05). Compared with the normal group,5-HTla mRNA expression level in the model group significantly went up (x2=40.5840;p=0.0000<00.05). Compared with the model group,5-HTla mRNA expression levels of Chang'anyihao high, medium and low-dose groups significantly decreased (x2=11.6823,25.7737,14.5150; p=0.0029,0.0000,0.0007<0.05). (2)In the end, BDNF mRNA expression levels were significantly different between groups (x2=45.914,p=0.000<00.05). Compared with the normal group, BDNF mRNA expression level in the model group significantly went up (x2=36.7966;p=0.0000<0.05). Compared with the model group, BDNF mRNA expression levels of fluoxetine group, Chang'anyihao high, medium and low-dose groups significantly decreased (x2=17.2327,10.9142,10.2546,13.6571;p=0.0002,0.0043,0.0059,0.0011<0.05).Experiment 5.Correlation between visceral sensitivity and colon 5-HT levelObjective:To investigate the relationship between visceral sensitivity and colon 5-HT level.Methods:We used Spearman correlation to assess the correlations between two parameters, differences were considered significant at p<0.05.Results:There was a linear negative correlation between pain threshold and colon 5-HT level (rs=-0.435,p=0.000).Experiment 6.Correlation between visceral sensitivity and serum 5-HT levelObjective:To investigate the relationship between visceral sensitivity and serum 5-the HT level.Results:Pain threshold and serum 5-HT level showed a linear negative correlation (rs=-0.484, p=0.000).Experiment 7.Correlation between visceral sensitivity and 5-HT1a mRNA level in rat hippocampalObjective:To investigate correlation between visceral sensitivity and BDNF mRNA expression level in rat hippocampal.Results:Pain threshold and 5-HT1a mRNA level in rat hippocampal showed a linear negative correlation (rs=-0.550, p=0.000).Experiment 8. Correlation between visceral sensitivity and BDNF mRNA level of rat hippocampalObjective:To investigate the correlations between visceral sensitivity in rats and hippocampal BDNF mRNA gene expression level.Results:Pain threshold and 5-HT1a mRNA level in rat hippocampal showed a linear negative correlation in all rats, model rats and treated rats (rs=-0.629,-0.773,-0.324,p=0.000,0.015, 0.030).Conclusion:Chang'anyihao can reduce serum and colon 5-HT levels and lower BDNF and 5-HT1a mRNA expression in rat hippocampus. |
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