| Objective:To explore the effect of intestinal hyperpolarization-activated cyclic nucleotide gated cation channel(HCN)subtype 2 on visceral hypersensitivity and dynamic abnormality in rats with Irritable Bowel Syndrome(IBS),thus further elucidate the pathogenesis of IBS visceral pain and provide new targets for the therapy of IBS.Methods:(1)IBS model rats were established by colorectal dilatation(CRD)with 20 mm × 2.5 mm human blood vessel reconstruction balloon during postnatal days 8-14.The 60 mmHg pressure of colorectal dilatation was given for 1 min once a day.The normal control rats were treated as the same as the IBS model rats except CRD.Visceral hypersensitivity was assessed by electromyographic(EMG)responses to graded colorectal distention(CRD)in adult rats(8 week).The constipation and diarrhea rats were removed from the experiment by abdominal palpation and observation of perianal cleanliness.(2)Western bolt was used to test the expression of HCN2 subtypes of colorectum and ileum in IBS and control rats.(3)HCN2 blocking agent(niflumic acid)0.1 ml was administered via intraperitoneal injection,with different concentration(5 ?mol/L,10 ?mol/L,50 ?mol/L).EMG response to graded CRD was recorded after administration to assess the visceral hypersensitivity in IBS model rats and the control rats.(4)HCN nonselective inhibitor(ZD7288)was administered via intrathecal injection,30 minutes later,niflumic acid(50 ?mol/L)was administered via intraperitoneal injection.EMG response to graded CRD was recorded after administration to assess the visceral hypersensitivity in IBS model rats and the control rats.(5)The time of the glass bead output and the number of the fecal pellet output in 2 hours were observed before and after intraperitoneal injection of niflumic acid,which investigate the effects of niflumic acid on intestinal movement of IBS rats.(6)Intestinal movements are recorded in vitro,to observe the effects of HCN2 inhibitor(niflunic acid)and HCN channel non-selective inhibitor(Chloride chloride)on the frequency and amplitude of intestinal movement.Results:(1)The sensitivity of visceral pain in IBS model rats was significantly higher than that in the control group(p<0.05).(2)The results of Western blot showed that the expression of HCN2 subtypes of colorectum and ileum in IBS model rats was significantly higher than that in the control group(p<0.05).(3)Inhibitor of HCN2(niflumic acid),which was given intraperitoneal injection,clearly inhibited the sensitivity of visceral pain in IBS rats(p<0.05),and showed a significant dose dependent effect.(4)Niflumic acid(i.p)has no inhibitive effect on pain sensitivity in IBS rats after the HCN channel was blocked by intrathecal injection of ZD7288.(5)The time required to excrete glass bead from IBS rats was shorter than that of normal control rats(p<0.05),and the number of fecal pellets discharged at 2 h was higher than that of normal rats(p<0.01).After intraperitoneal injection of niflumic acid,compared with before administration,there was no significant change in the time of the glass bead output and the number of fecal pellets discharged in 2 h in the control rats;however,time of the glass bead output in IBS model rats was significantly prolonged(p<0.05);The number of fecal pellets discharged in IBS model rats decreased,the difference was statistically significant(p<0.01).(6)The experiment of isolated intestinal perfusion showed that compared with normal group,the frequency and amplitude of the intestinal movement of the rats in the IBS model group increased with a significant difference(p < 0.05).After the administration of niflumic acid or cesium chloride in the perfusion fluid,the intestinal movement frequency of the rats in the model group decreased and the movement amplitude lower significantly(p<0.05).Conclusion:There could occur visceral hyperalgesia,intestinal dynamic abnormality in adult rats if neonatal rats undergoing colorectal distension.Niflumic acid could improve these symptoms.The mechanism may be related to blocking HCN2 channel activity. |
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