Study On The Role Of The "Epac1-Piezo2" Axis In The Alleviation Of Visceral Pain In IBS By Acupuncture

Objective:Based on IBS with diarrhea(IBS-D)patients or trinitrobenzene sulfonic acid(TNBS)induced IBS visceral pain mice,this study was conducted to investigate the mechanisms of Epacl and Piezo2 in the colon or dorsal root ganglion(DRG)in the relief of visceral pain sensitivity in IBS by acupuncture or electroacupuncture(EA).Methods:1.Clinical study:Samples from the clinical sample bank of IBS-D patients treated with acupuncture(15 IBS-D patients who underwent colonoscopy twice before and after acupuncture and ten healthy participants who underwent colonoscopy)were used.The expressions of colonic Epacl and Piezo2 were measured by immunohistochemistry in healthy participants and IBS-D patients before and after acupuncture treatment.2.Animal experiment Ⅰ:IBS visceral pain mouse model was constructed by using TNBS.The mice were divided into four groups:control group,model group,EA group and sham EA group.The abdominal withdrawal reflex(AWR)and the paw withdrawal threshold(PWT)were applied to evaluate the intervention effect of EA on visceral and somatic hypersensitivity.Immunofluorescence,Western Blot and qPCR were used to detect the expressions of Epac1,Piezo2,5-HT3 receptor(5-HT3R)and 5-HT in the distal colon,and expressions of Epac1 and Piezo2 in the L5-S2 DRGs.3.Animal experiments Ⅱ:(1)mice were divided into control group,model group,model+inhibition of Epac1(shEpac1)group,model+shEpac1+EA group,and EA group to observe whether inhibition of colonic Epac1 would influence the effect of EA in alleviating visceral hypersensitivity in IBS and the expression of Piezo2;(2)mice were divided into control group,model group,model+inhibition of Piezo2(shPiezo2)group,model+ shPiezo2+ EA group,and EA group to observe whether inhibition of colonic Piezo2 would influence the effect of EA in alleviating visceral hypersensitivity in IBS and the expression of Epac1;(3)mice were divided into control group,model group,model+shEpac1+shPiezo2 group,model+shEpac1+ shPiezo2+EA group,and EA group to observe whether combined inhibition of Epac1 and Piezo2 would influence the effect of EA in alleviating visceral hypersensitivity in IBS.Results:1.Clinical study:Acupuncture significantly relieved the severity of abdominal pain and reduced the number of days of abdominal pain.There was a tendency for colonic Epac1 expression of IBS-D patients to be higher than healthy participants,and a trend for colonic Epac1 expression of patients after acupuncture treatment to be lower than that before acupuncture treatment.The differences were not statistically significant.There was no significant correlation between Epac1 expression and the severity of abdominal pain in patients with IBS-D.Piezo2 positive expression was significantly higher in the colon tissue of IBS-D patients than in healthy participants.There was a significant positive correlation between Piezo2 expression and the severity of abdominal pain in patients.12 weeks of acupuncture intervention significantly reduced Piezo2 positive expression in the colon.The decrease of Piezo2 positive expression before and after acupuncture was significantly positively correlated with the reduction of abdominal pain score.2.Animal experiment Ⅰ:(1)TNBS-induced IBS model mice had significantly higher AWR scores and a lower PWT than the control.The EA group had lower AWR scores than the model group.The AWR scores in the EA group were significantly lower than those in the sham EA group at 15,30 and 60 mmHg.EA treatment increased PWT compared to the model group.(2)The expression levels of colonic protein and mRNA levels of Epac1 and Piezo2 in the model group were significantly higher compared with the control group.They lowered in the EA group than in the model group.Compared with the sham EA group,Epac1 and Piezo2 expressions were lower in the EA group.(3)Compared with the control group,colonic 5-HT expression was significantly increased in the model group and significantly decreased after EA intervention.The colonic 5-HT expression level in the EA group was lower than that in the sham EA group.Compared with the control group,the model group’s colonic 5-HT3R protein and mRNA expression levels were significantly higher,and EA could dramatically decrease the 5-HT3R protein and mRNA expression levels.(4)Epac1 mRNA and Piezo2 mRNA in L5-S2 DRGs were significantly increased in the model group compared with the control group,and the EA intervention reduced the expression of Epac 1 mRNA.Epac1 and Piezo2 expressions in the model group were higher than in the control group at the protein level.Epac1 and Piezo2 expressions in the EA and sham EA groups were lower than the model group.Epac1 and Piezo2 expressions in the EA group were lower than the sham EA group.3.Animal experiment Ⅱ:(1)AWR scores were significantly higher in the model group compared with the control.The AWR scores in the EA or shEpac1 group were lower than the model group at 30,45 and 60mmHg.The shEpac1+EA group had no statistically significant difference in AWR scores compared with the EA group.The PWT in the model group was lower than in the control group,and EA could increase the PWT.The shEpac1 group had no statistically significant difference from the model group,and the shEpac1+EA group had no statistically significant difference from the EA group.Colonic Piezo2 protein and mRNA expressions in the model group were higher than the control group,while the Piezo2 protein and mRNA expressions in both the EA and shEpac1 groups were lower than the model group.Compared with the EA group,the expression of colonic Piezo2 protein in the shEpac1+EA group was lower,while the expression of colonic Piezo2 mRNA in the shEpac1+EA group was higher.Compared with the control group,colonic 5-HT expression was significantly increased in the model group.5-HT expression in colonic tissues tended to decrease after shEpac1 or EA intervention,but the difference was not statistically significant.The colonic 5-HT expression level in the shEpac1+EA group had no statistically significant compared with the EA group.(2)AWR scores were significantly higher in the model group compared with the control.The AWR scores of the shPiezo2 group were lower than those of the model group.The AWR scores of the EA group were lower than those of the model group under 30,45 and 60 mmHg.The AWR score of the shPiezo2 group was lower than that of the EA group under 45 mmHg.The shPiezo2+EA group had no statistically significant differences in the AWR scores compared with the EA group.The PWT in the model group was lower than in the control group,and EA increased the PWT.The expressions of colonic Epac1 protein and mRNA in the model group were higher than those of the control.Those expressions in both the EA and shPiezo2 groups were lower than the model group.The shPiezo2+EA group showed a lower expression of Epac1 protein than that of the EA group.The difference in the Epac1 mRNA in the shPiezo2+EA and the EA groups was not statistically significant.Compared with the control,the colonic 5-HT expression in the model group was significantly increased,and 5-HT was significantly decreased after EA or shPiezo2 intervention.The shPiezo2+EA group had no statistically significant in the colonic 5-HT expression level compared with the EA group.(3)The AWR scores of the model group were higher than those of the control.The AWR scores in the EA group or shEpac1+shPiezo2 group were lower than those in the model group under 30,45 and 60 mmHg;the differences in AWR scores between the shEpac1+shPiezo2 group and the EA group were not statistically significant.At 60 mmHg pressure,the shEpac1+shPiezo2+EA group had lower AWR scores than the EA group.EA increased the PWT,and the PWT of the shEpac1+shPiezo2+EA group was lower than the EA group.The expression of 5-HT in colonic tissues was significantly reduced after EA intervention.Combined inhibition of Epac1 and Piezo2 also significantly reduced the colonic 5-HT and the 5-HT expression level of mice in the shEpac1+shPiezo2+EA lower than that in the EA group.Conclusions:1.The positive expression of Piezo2 in the colon tissue of IBS-D patients was higher than healthy participants,and Piezo2 was positively correlated with the severity of abdominal pain.Acupuncture could significantly reduce the positive expression of Piezo2 in the colon.2.EA at ST36 and ST37 reduced the expressions of Epac1 and Piezo2 in colonic tissues and decreased 5-HT and 5-HT3R expressions,which may be related to the effect of EA in relieving visceral and somatic hypersensitivity.3.Inhibition of colonic Epac1 or Piezo2 did not eliminate the effect of EA in alleviating visceral hypersensitivity.Still,it could eliminate the effect of EA in alleviating somatic hypersensitivity in IBS model mice.4.Combined inhibition of colonic Epac1 and Piezo2 synergized with the effect of EA in relieving visceral hypersensitivity.It could eliminate the effect of EA in alleviating somatic hypersensitivity in IBS model mice.5.EA may alleviate visceral and somatic hypersensitivity in IBS mice by reducing the expression of Epac1 and Piezo2 in DRG,and the mechanism should be further investigated.