| ObjectiveThe IBS rat model was established by intracolonic infusion of 0.5% acetic acid combined with restraint stress and nip trail. From the perspective of brain-gut axis interaction, the purpose of the present study is to (i) further investigate the relationship between the pathogenesis of IBS and serotonergic signaling system; (ii) explore the effect of ZSS on IBS and to investigate the role of the serotonergic signaling system as an underlying mechanism of ZSS on IBS rats.Methods1、Model establishment and behavioral evaluationAll the Sprague-Dawley rats were acclimatized for 1 week prior to behavioral tests and then were randomly divided into four groups (n=8 in each group):control group; model group; ZSS group; Dicetel group. except for the rats in control group, all the other rats received an intracolonic infusion of 1 mL 0.5% acetic acid in saline,2 cm from the anus for 14 d.7 d later, the rats were constrained for 1 hour and nipped tail for 30 min for 14 consecutive days. The rats in control group (n=8) only received an intracolonic infusiocn of equal volume of saline for 14 d. The modeling lasted 3 weeks.During the experiment, the behavior performances of all rats were measured weekly in a blind manner to evaluate the effects of induced stress and the model, including the body weight, defecation parameters, sucrose preference test, open-file test and the abdominal withdrawal reflex (AWR). In addition, the effects of ZSS on IBS rats were estimated at 35th day, including performing behavioral tests2、Biochemical index determination and histological examinationEnzyme-linked immunosorbent assay kits were used to measure the content of 5-HT in serum, colon and brain. Hematoxylin-eosin staining is used for histological assessment of of colon and hypothalamus.3、The immunoreactive intensity of 5-HT and CgA in colon and hypothalamusImmunohistochemistry staining is used to evaluate expression of the 5-HT and CgA in colon and brain tissue. The processes included fixation, trimming, embedding, sectioning, dewaxing to water, antigen repairing, blocking, incubating corresponding antibody and so on. The finished sections were visualized with an Optec BK-DM320 biological microscope and analyzed using Image processing plus software.The intensities of the immunoreactivity were expressed as integral optical density (IOD).4、TPH-1,5-HT3R,5-HT4R and SERT mRNA levels in colon and hypothalamusThe real-time quantitative PCR was used to detect relevant genes of the serotonin signal system, involving TPH-1,5-HT3R,5-HT4R and SERT mRNA levels. GAPDH was used as endogenous control and results were analyzed by 2-△△Ct method.5、TPH-1,5-HT3R,5-HT4R and SERT proteins expression in colon and hypothalamuswestern blot, a blot that consists of a sheet of cellulose nitrate containing spots of protein for identification by a suitable molecular probe and is used especially for the detection of antibodies. Western blot technology was carried out to determine TPH-1, 5-HT3R,5-HT4R and SERT proteins expression. Densitometric analysis of each band was quantified using NIH Image software and normalized to GAPDH level.6、Statistical analysisSPSS20.0 statistical software was used for data analysis. Data were shown as mean± SD. Student t-est was used to analyze differences between two groups, and comparisons among groups were analyzed using one-way ANOVA followed by Student-Newman-Keuls post hoc test. P<0.05 was considered statistically significant.Results1、The IBS rat model was established by intracolonic infusion of 0.5% acetic acid combined with restraint stress and nip trail. In this model, IBS rats, their stool form was more loose and formless, simultaneously their defecation frequency significantly increased compared with the controls. Moreover, the body weights of all rats were evaluated every week and the weight of IBS rats grew slowly compared to controls. In addition, IBS rats displayed decreases in performance for sucrose intake and locomotor activity. Moreover, compared to controls, the AWR scores in response to graded intensity of CRD were increased. After drug treatment for 14 consecutive days, ZSS decreased stool frequency, increased stool consistency and the body weight in IBS rats. Moreover, ZSS not only decreased the visceral hypersensitivity as assessed by colorectal distension (CRD), but also exhibited antidepressant-like effects as shown in the sucrose preference and open field tests.2、the 5-HT content in serum and colon were increased in IBS rats. Howerer, the 5-HT content in hypothalamus was decreased in Model group. Decreases in serum and colon levels of 5-HT were observed in IBS rats after treatment with ZSS. While 5-HT content of hypothalamus in IBS rats was elevated relative to control rats. The results of H-E staining of colon from IBS rats were no obvious inflammatory cells. While H-E staining of hypothalamus, the nerve cells of IBS rats were different from those of control rats.3, Immunohistochemistry stained of 5-HT and CgA in colon and hypothalamus① Expression of both 5-HT and CgA were significant higher in model group relative to the controls. After treatment of ZSS, the intensities of 5-HT and CgA in ZSS-treated rats were remarkably reduced relative to IBS rats. ② In hypothalamus, compared to control group, the immunoreactive intensity of 5-HT and CgA in Model group were downgraded. ZSS could adjust the expression 5-HT and CgA in hypothalamus.4、TPH-1,5-HT3R,5-HT4R, SERT mRNA levels in colon and hypothalamus① There were statistically significant differences in the mRNA levels of SERT, 5-HT4R and TPH-1 in model group compared with the controls, with mRNA levels of SERT being decreased 0.31-fold while 5-HT4R and TPH-1 being increased 2.25-fold and 2.27-fold. After treatment of ZSS, mRNA levels of 5-HT4R and TPH-1 were decreased compared with model group and SERT expression was increased. The mRNA level of 5-HT3R showed no significant differences among four groups. ② In hypothalamus, TPH-1 and 5-HT3R showed no significant differences among four groups.5-HT4R mRNA level was decreased in model group compared to control while SERT mRNA level was increased. After ZSS treatment, the mRNA levels of 5-HT4R and SERT were adjusted.5、TPH-1,5-HT3R,5-HT4R, SERT proteins expression in colon and hypothalamus① The results show that the protein expression of 5-HT3R,5-HT4R, and TPH-1 were significantly increased in model group above the controls while SERT protein expression was dramatically reduced. However, ZSS could down-regulate 5-HT4R, and TPH proteins levels, and increase SERT protein expression, but ZSS might have little effect on 5-HT3R.② In hypothalamus, proteins expression of TPH-land 5-HT3R showed no significant differences among four groups. Proteins expression of 5-HT4R was decreased in model group relative to control group, while proteins expression of SERT1 was increased. After drug treatment, ZSS could up-regulate 5-HT4R protein expression, and down-regulate SERT proteins levels. Dicetel had no significant effect on relative genes and proteins expression. Moreover, alterations in mRNA levels were majorly consistent with their protein expressions.ConclusionZSS intervened the mechanism of action of D-IBS rats possibly by adjusting the brain-gut axis interaction, reducing the content of 5-HT in serum and colon, improving the content of 5-HT in the hypothalamus, adjusting the TPH-1,5-HT3R,5-HT4R and SERT genes and proteins expression, so as to improve intestinal function, reduce the elevated visceral hypersensitivity and improve emotion. And the results suggest that there may be a certain relationship between the pathogenesis of IBS and serotonergic signaling pathways of the enteric nervous system, including TPH-1,5-HT3R,5-HT4R, SERT. IBS with psychological disorders such as depression may be related to the central nervous system in 5-HT4R, SERT. |
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