Inhibitive Effect Of Niflumic Acid On Chronic Visceral Hypersensitivity In Rats

ObjectiveTo study the relationship of hyperpolarization-activated cyclic nucleotide gated cation channels and chronic visceral pain in rats, observing the effect of the early application of niflumic acid (the specific subtypes 2 blocker of this channels) to chronic visceral nociceptive sensitivity in rats, with the purpose of providing new targets for the drug treatment of chronic visceral pain.Methods(一)Chooseing SD rats newborn, first postnatal 8-14 days, every afternoon accept colon stimulation under 60mmHg pressure for a minute (colon stimulation CI) in order to establish chronic visceral pain model; the control group has the same processes execpt accepting CI. After Adult rats 8W, compare the expression and distribution of HCN1, HCN2 channels in lumbosacral spinal cord and thoracolumbar spinal cord, hippocampus, paraventricular nucleus, raphe nucli of brain between control and model group by immunohistochemical method.(二)The model group and control group is divided into two groups at random respectively, that is group A1B2 and group A1B1, group A2B1 and group A2B2. (8 rats in every group). Group A1B1 and A2B1 accept intraperitoneal injection of niflumic acid, while group A1B2 and A2B2 accept intraperitoneal injection of dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) solvent. Rats after age 8w using method of abdominal withdrawal reflex (abdominal withdrawal reflex, AWR) and pain threshold and external oblique discharge measurement for assessing intestinal sensitivity of chronic visceral pain, observe the effect of niflumic acid to chronic visceral pain in rats. Results1. Ways of evaluation of chronic visceral pain1.1 The scores of AWRThe scores of AWR in model rats were significantly increased under 20～60mmHg CRD compared with control rats, but the difference of the scores had no statistical significance under 80mmHg CRD.1.2 Pain thresholdThe pain threshold in model rats was significantly decreased compared with control rats.1.3 The spikes of EOMAThe spikes of EOMA in model rats were significantly increased under 20～80mmHgCRD compared with control rats..1.4 The colonic pathological changeDistal colon descendens topographic histology of control and model rats is observed and compared. No apparent change was observed by naked eyes and microscope in control and model rats2. Morphological study study of the relationship between HCN and chronic visceral hypersensitivity in rats2.1 HCN1 and HCN2 subunits stongly express in rat models of thoracolumbar spinal cordHCN1 observed and compared with model HCN2, in normal rats thoracolumbar spinal expression, the results show that the model rats HCN1, HCN2 expression in the model rats of the rats were enhanced. Rat models of thoracolumbar spinal HCN1, HCN2 visceral pain may involve chronic central sensitization mechanism.2.2 HCN1 and HCN2 subunits, in rat models for lumbosacral spinal cordHCN1 observed and compared with model HCN2, in normal rats lumbosacral section of the spinal cord, the results shows that the model rats were HCN1,HCN2 expression of contrast enhancement, rat rat models for HCN1 spinal lumbosacral HCN2, visceral pain may involve chronic central sensitization mechanism.2.3 HCN1 and HCN2 subunits in brain hippocampus in rat modelsHCN1 observed and compared with model HCN2, in normal rats brain hippocampal expression, the results shows that the model rats, HCN1 and HCN2 expression of the rats were enhanced, indicating that the rat models HCN1 and HCN2 hippocampal cerebral, visceral pain may involve chronic central sensitization mechanism.2.4 HCN1 and HCN2 subunits in the brain raphe nucli in rat modelsHCN1 observed and compared with model HCN2, in normal rats raphe brain expression of nucli, the results show that the model rats, HCN1 and HCN2 expression of the rats were enhanced, indicating that the rat models raphe nuclear of brain HCN1 and HCN2, chronic visceral pain may involve the central sensitization mechanism.2.5 HCN1 and HCN2 subunitsand in large paraventricular nucleus in rat modelsHCN1 observed and compared with model HCN2, in normal rats by large ventricle nuclear expression, results show that the model rats, HCN1 and HCN2 expression of the rats were enhanced, indicating that the rat models of large paraventricle nucleus HCN1 and HCN2, the chronic visceral pain may involve the central sensitization mechanism.3. The inhibitive effect of niflumic acid on algesic sensitivity in rats of chronic viseceral painRats newborn had CI, accept intraperitoneal injection of niflumic acid, the specific blocker of HCN channel subtybe 2 early (21-23 day after birth), find that the inhibitive effect of niflumic acid to algesic sensitivity of visceral pain is dose dependent and can improve its response to pain threshold.3.1 Niflumic acid improve the pain threshold of chronic visceral pain in ratsNewborn rat accepting CI intraperitoneal injection of niflumic acid at dose of 1mg, has no significant in changing pain threshold, while injecting dose at 5 and 10 mg, significantly increases pain threshold.3.2 Different doses of niflumic acid has different inhibitive efffect of external oblique discharge to chronic visceral pain in rats.Injection of niflumic acid at dose of 1mg at 20mmHg CRD has no significantly in changing external oblique discharge amplitude comparing with intra-abdominal injection DMSO group, while at 60 ~ 80 mmHg CRD is significantly reduced; Intra-abdominal injection of niflumic acid at dose of 5mg and 10mg rats at 20 ~ 80 mmHg CRD is significantly reduced external oblique discharge amplitude. Comparing with intra-abdominal injection niflumic acid at dose of 1mg group, injection niflumic acid at dose of 5mg and 10mg group at 20 ~ 80mmHg CRD is significantly reduced external oblique discharge amplitude. Comparing with intra-abdominal injection niflumic acid at dose of 5mg group, injection niflumic acid at dose of 5mg and 10mg group only at 40 mmHg CRD is significantly reduced external oblique discharge amplitude, while at at other stress CRD has no significant effect. Comparing with model group , intra-abdominal injection DMSO group at 20 ~ 80mgHg CRD has no significant effect in reducing external oblique discharge amplitude.3.3 The effect of niflumic acid at dose of 5mg to external oblique discharge amplitude between control and model groupA1B1 group giving niflumci acid at dose of 5mg, find that its external oblique discharge amplitude at any pressure of CRD comparing with control group A1B2 and solvent group A2B1 and A2B2 is affected by two factors of whether to accecpt CI and whether to accept niflumic acid. In 20~80mmHg CRD pressure, the main effect of building model group to external oblique discharge amplitude is 11.385, 18.915, 23.595, 22.425, that is the model group has raise its external oblique discharge amplitude about 11.385uV, 18.915 uV, 23.595 uV, 22.425 uV at 20~80mmHg pressure of CRD. The result above has its statistic significance. The main effect of the group giving niflumic acid at dose of 5mg to external oblique discharge amplitude is -11.055, -19.985, -25.25, -22.61, that is the group giving niflumic acid at dose of 5mg has reduced its external obique discharge amplitude about 11.055, 19.985, 25.25, 22.61uV respectively at 20~80mmHg pressure of CRD. The result above has its statistic significance. The effect of model ( rats newborn accepting CI ) and intra-abdominal injection of niflumic acid to external oblique discharge amplitude at 20~80mmHg pressure of CRD has interacted obviously (P﹤0.05). Conclusion1. The expression of HCN1 and HCN2 in thoracolumbar spinal cord, lumbosacral spinal cord, hippocampus, paraventricular nuclei, raphe nuclei is stongly enhanced in IBS model group comparing with control group, providing morphological basis for HCN1 and HCN2 participating in chronic visceral pain of central sensitization mechanism.2. Niflumic acid (specific blocker of HCN2 subtybe) can dose dependently inhibit the response to chronic visceral pain and improve its response to pain threshold suggesting that the early application of niflumic acid can effectively prevent chronic visceral pain in rats.