| Objective To investigate the effect of hyperpolarization activated cyclic nucleotide gated channels on contractile function in irritable bowel syndrome rats and to provide experimental evidence for the pathogenesis of irritable bowel syndrome.Methods1.IBS model rats: SD rats were given 60 mmHg pressure colorectal distention stimulation for 1 min,at a fixed time every day on the 8-14 d after birth.The control group had the same process as the model group except for no colorectal stimulation.After 8 weeks,the visceral pain sensitization of SD rats was evaluated by the score of pain and the amplitude of extra abdominal oblique muscle discharge.The frequency and amplitude of colon movement in control group and IBS group were compared by perfusion of colonic tissue in vitro;3.The expression of HCN1 and HCN2 in rat colon were detected by Western bolt;4.The localization of HCN1,HCN2 and c-kit(the interstitial cell of Cajal,marker)in rat colon was detected by immunofluorescence double labeling.5.After the whole layer of colonic tissue was prepared,the localization of HCN1,HCN2 and c-kit on the myenteric plexus of rat colon smooth muscle was detected by immunofluorescence double labeling.6.The effects of NFA (HCN2-specific inhibitor)and ZD7288(non-selective inhibitor of HCN channel)on the frequency and amplitude of colonic movement were observed.Results1.In adult IBS rats,the pain threshold was significantly lower than that of the control group.Under the pressure of 40 mmHg to 60 mmHg,the amplitude of EMG in IBS rats increased and the pain response enhanced(p < 0.01).2.In the perfusing experiment of isolated colonic tissue,the frequency and amplitude of colonic movement in IBS group were higher than those in control group,and there was significant difference(p < 0.01).3.Western blot showed that the expression of HCN1 and HCN2 in colon of IBS rats was higher than that of control rats(p < 0.05).4.Immunofluorescence double labeling showed that there was a co-localization relationship between HCN1,HCN2 and ICCs marker c-kit in the myenteric plexus of rat colon.The average fluorescence intensity of IBS group was significantly higher than that of control group(p < 0.01).5.After the whole layer of colonic tissue was prepared,the localization of HCN1,HCN2 and ICCs marker c-kit on myenteric plexus of colonic smooth muscle in rats was detected by immunofluorescence double labeling.6.In isolated colonic perfusion:(1)Both 30 μmol/L NFA and 100 μmol/L NFA reduced the colonic motility frequency and decreased the amplitude of IBS rats,which was significantly different from the control group(p < 0.05).10 μmol/L NFA for IBS rats,there was no significant difference in the frequency and amplitude of colonic movement(p > 0.05).However,the frequency of colonic movement in thecontrol group was increased by 10 μmol/L NFA.The difference was statistically significant(p < 0.05).While 10 μmol/L NFA for the the amplitude of control group,there was no significant effect(p > 0.05).(2)50 μmol/L ZD7288 reduced the colonic motility frequency and decreased the amplitude of IBS rats,which was significantly different between before and after administration(p < 0.05),then 30 μmol/L NFA has no significant effect on the frequency and amplitude(p > 0.05).While 50 μmol/L ZD7288 had no significant effect on the frequency and amplitude of colonic movement in the control group.There was no significant difference between before and after administration(p > 0.05),then 30 μmol/L NFA has no significant effect on the frequency and amplitude(p > 0.05).The blocking effect of ZD7288 on NFA indicated that the colonic motility of IBS rats act through the HCN channels.Conclusions The up-regulation of HCN1 and HCN2 in colonic increased the excitability of interstitial cells of Cajal,which may lead to abnormal intestinal motility in rats with irritable bowel syndrome. |
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