| Objective: The present study aims to screen out the miRNAs which may regulate mast cell tryptase/ PAR2 signal pathway in the mechanism of IBS-D,and verify it in an animal model.Methods: Several mi RNAs which may involve in the signal pathway of mast cell tryptase/PAR-2 via the online database mi RDB(http://www.mirdb.org/miRDB/)were screened out based on our previous findings[2].An IBS-D rat model was established with the induction of acetic acid and restraint stress.The visceral sensitivity,faeces characteristics,and gastrointestinal through time of each rat were detected respectively to evaluate the IBS-D model.Histological changes were evaluated by HE staining.The selected mi RNAs were verified in the colonic tissue of IBS-D rats with one mi RNA selected as the target mi RNA.The target miRNA was silenced by the lentivirus miRNAs inhibitor vector.Subsequently,the visceral sensitivity,faeces characteristics,and gastrointestinal through time of each rat were detected again after injecting lentivirus.HE staining was performed again to detect the histological changes of the four groups.Finally,the colonic level of the target miRNA was assessed by qRT-PCR,and the expression of tryptase and PAR2 were analyzed by q RT-PCR and western blot.Results:1.MiR490-5p and mi R29b-1-5p were found to conform to the selection criteria.2.Rats with IBS-D exhibited shorter whole gut transit time than control rats(P<0.05).The nociceptive threshold was about 0.945 ml in the control group,and it was lowered to around 0.699 ml in the model group(P<0.05).The number of fecal pellet output for 6hr was negligible in the control rats.However,the defecation of the rats with IBS-D was significantly increased(P<0.05).All the data demonstrated above indicated that the model of IBS-D was successfully established.3.Compared with control group,the staining process of H&E showed that there was no remarkable inflammatory feature in IBS-D group.4.mi R490-5p was up-regulated in rats with IBS-D.By contrast,mi R29b-1-5p was down-regulated.MiR490-5p was selected as our target mi RNA.5.On day 3 following intravenous injections of lenti-mi R490-5p inhibition into IBS-D rats,the whole gut transit time was significantly prolonged the visceral response threshold to colonic distension was significantly increased,and the number of all fecal pellet and formless stool output was significantly decreased,indicating a reversion of syndrome in rats with IBS-D following inhibition of miR490-5p expression.6.Photomicrographs of colons from control rats,IBS-D rats,Lenti-miR-control rats and Lenti-miR490-5p inhibition rats exhibited no remarkable inflammatory feature in all groups.7.The expression of tryptase and PRA2 were significantly increased in the colon tissue of IBS-D rats.miR490-5p in vivo decreased by intravenous injection of lenti-miR490-5p inhibitor vector.The symptoms of IBS-D rats were reversed,and the expression of tryptase and PAR2 in colonic tissue from IBS-D rats decreased.Conclusions:1.Our results showed that increased colonic miR490-5p expression contributes to visceral hypersensitivity,abnormal2.gastrointestinal dynamics and intestinal permeability in rats with IBS-D,indicating miR490-5p involve in the mechanism of IBS-D.3.mi R490-5p can regulate the expression of tryptase and PAR2 in IBS-D rats' colonic tissue,indicating that miR490-5p associate with the signal pathway of mast cell tryptase/PAR2.4.miR490-5p may modulate visceral sensitivity,gastrointestinal motility and intestinal permeability via regulating the signal pathway of mast cell tryptase/ PAR2. |
PDF Full Text Request