| Purpose:To detect colonic EC cells by morphology, serum and colon 5-HT,5-HIAA by ELISA, colon TPH1, SERT,5-HT3R,5-HT4R mRNA and protein expression by molecular biology, and to explore the 5-HT molecular mechanism of OAT on IBS and to provide experimental evidence for OAT on IBS.Material and method:1. Setting up animal model and given OAT30 SPF Wistar male rats were randomly divided into control, IBS model and OAT groups. IBS model was established by method of restraint stress and combining bound. IBS model and OAT groups were put into selfmade cages 1 h and given a stimulus alternately daily①water deprivation,24h②40℃,5min③electric shock,30V,5s aff and on 5s, totally120s④swimming 14℃,5min⑤fasting,24h⑥tail clipped 180s; a total of 18 days 3 cycles. Control group had no treatment.OAT group was given OAT from 2nd day after model set endding. With No.31, 13mm needles, OAT was given 3mm deep acupuncture in parallel for 20min, during retention the needles were moved softly 3 times×1min,2 times everyday for 7 days.2. Indexes were detected Immunohistochemistry was used to detect and analyze colonic EC cells. ELISA was used to detect serum and colon 5-HT and 5-HIAA. Western-Blot and RT-PCR were used to detect and analysis colon TPH1, SERT,5-HT 3 R,5-HT 4 R mRNA and protein.Results:1. The general state of ratsNo animals died during the experiment. Compared with control group, irritable aggressive IBS model rats appeared dull coat, loss of appetite, watery feces and chronic diarrhea. Based on the model standard to observe colonic pathology, transit function and sensitivity, we confirmed that IBS model was successful. OAT significantly improved IBS model rats general state.2. Effects of OAT on colonic 5-HT and 5-HIAA levels in IBS modelCompared with control group, the levels of 5-HT and 5-HIAA in IBS model increased remarkably (P<0.05).Percent conversion of 5-HT in IBS model increased remarkably too. Compared with those in IBS model, the levels of 5-HT and 5-HIAA and percent conversion of 5-HT in OAT decreased remarkably (P<0.05).3. Effects of OAT on colonic EC cells in IBS modelCompared with control group, the stained intensity and the number of EC cells in IBS model increased remarkably (P<0.05). The stained intensity and the number of EC cells in OAT decreased remarkably (P<0.05)compared with those in IBS model.4. Effects of OAT on colonic TPH1 and SERT expression in IBS modelCompared with control group, IBS model TPH1 expression increased (P<0.05) and SERT protein and mRNA expression reduced(P<0.05); OAT TPH1 expression reduced (P<0.05) and SERT protein and mRNA expression upregulated(P<0.05) compared with IBS model group. 5. Effects of OAT on colonic 5-HT 3 R and 5-HT 4 R expression in IBS modelCompared with control group, IBS model 5-HT 3 R expression increased (P<0.05) and 5-HT 4 R protein and mRNA expression reduced (P<0.05); OAT 5-HT 3 R expression reduced (P<0.05) and 5-HT4 R protein and mRNA expression upregulated(P<0.05) compared with IBS model group.OAT decreased 5-HT synthesis by downregulated EC cells and TPH1 expression, while the expression of SERT was upregulated to improve the level of 5-HT catabolism, so that the 5-HT synthesis and catabolism can be matched and coordinated,5-HT levels returned to normal, then IBS rat colon to restore normal function. OAT downregulated the expression of 5-HT3R, upregulated 5-HT4R expression, so that the two returned to normal levels, which may be the mechanism of OAT for IBS.Conclusion:1. The levels of colon and serum 5-HT,5-HIAA, and Percent conversion of 5-HT increase in IBS model rats. OAT can downregulate those.2. IBS model rats have increase in EC number and stained intensity, increase TPH1 expression and reduce SERT expression. OAT can reduce EC number and stained intensity, TPH1 expression and increase expression of SERT.3. IBS model rats increase 5-HT 3 R expression and reduce expression of 5-HT 4 R. OAT can reduce 5-HT 3 R expression and increase expression of 5-HT 4 R.
|
PDF Full Text Request