| Background:Irritable bowel syndrome is a common clinical digestive system dysfunction disease.The main features are abdominal pain and changes in bowel habits,which also bring great inconvenience to life and work,and even cause depression,anxiety,etc.The occurrence of emotional disorders.The pathogenesis of IBS is complicated.Modern research suggests that abnormal interaction of the brain and intestine axis leads to high sensitivity of the viscera and abnormal intestinal motility is the main pathogenesis of IBS.In the study of brain-intestinal axis interaction mechanism,5-HT is an important communication transmitter,and 95%of 5-HT in organisms is derived from the synthesis and release of intestinal chromaffin cells.It has been found that capsaicin activates colon TRPV1,making Increased 5-HT release is associated with phosphorylation of CaMKⅡ and activation of Tryptophan Hydroxylase 1（TPH1）;modern clinical studies and animal experiments have demonstrated that acupuncture can effectively reduce the expression of TRPV1 in the colon and improve it.IBS visceral pain and bowel habits and other symptoms,combined with the previous research results of this research group put forward hypothesis:colon tissue TRPV1regulates CaMKⅡ signal-mediated conductive needle to alleviate IBS visceral hypersensitivity.ObjectiveThis study was aim to preliminary reveals that TRPV1 regulates CaM/CaMKⅡ signaling pathway on EC cells and participates in the molecular biological mechanism of electroacupuncture at Zusanli to relieve visceral pain in IBS miceMethodIn this study,wild-type C57BL6 mice and TRPV-/-knockout mice were used as research objects,and the viewpoints were verified by three-part experiments.Experiment 1:The IBS visceral pain mice model was established by singular intrarectal administration of 246-trinitrobenzenesulfonic acid（TNBS）;And mice were studied after 0,3,7 and 28 days with AWR scores,macroscopic assessment of colitis and histopathology observation,to evaluate whether the model induced method is successful.Experiment 2:Wild type C57BL6 mice were randomly divided into three group.（1）Normal group（NG）:singular intrarectal administration of 0.9%Na Cl,28 days later,the same fixation method as the electroacupuncture group was fixed for 15 min,once a day for 7 consecutive days;（2）Model group（MG）:singular intrarectal administration of TNBS,28 days later,the same fixation method as the electroacupuncture group was fixed for 15 min,once a day for 7 consecutive days;（3）electroacupuncture group（EA）:singular intrarectal administration of TNBS,and after 28 days,the bilateral Zusanli acupoints were taken,followed by Han Electro-acupuncture instrument,parameter waveform:continuous wave,2Hz,1m A,15min/time,once a day for 7 consecutive days.The AWR score was used to score the visceral pain of the mice after the intervention.The positive expression of CaMKⅡ and the enterochromaffin-specific marker TPH1was observed by immunofluorescence（double labeling）.Using ELISA to observe the5-HT protein expression in colon;Using Western Blot to observe the TRPV1,p CaMKⅡ,TPH1 protein expression in colon;Using Real-time PCR to observe the TRPV1,CaMKⅡ,TPH1 mRNA expression in colon;Experiment 3:TRPV1-/-mice were randomly divided into four group.（1）Normal group（NG）:singular intrarectal administration of 0.9%Na Cl,28 days later,the same fixation method as the electroacupuncture group was fixed for 15 min,once a day for7 consecutive days;（2）Model group（MG）:singular intrarectal administration of TNBS,28 days later,the same fixation method as the electroacupuncture group was fixed for 15 min,once a day for 7 consecutive days;（3）Electroacupuncture group（EA）:singular intrarectal administration of TNBS,and after 28 days,the bilateral Zusanli points were taken,followed by Han Electro-acupuncture instrument,parameter waveform:continuous wave,2Hz,1m A,15min/time,once a day for 7 consecutive days.（4）Normal and electroacupuncture group（NE）:singular intrarectal administration of0.9%Na Cl,28 days later,the bilateral Zusanli acupoints were taken,followed by Han Electro-acupuncture instrument,parameter waveform:continuous wave,2Hz,1m A,15min/time,once a day for 7 consecutive days.Using ELISA to observe the 5-HT protein expression in colon;Using Western Blot to observe the CaMKⅡ,TPH1 protein expression in colon;Using Real-time PCR to observe the CaMKⅡ,TPH1 mRNA expression in colon;ResultExperiment 1:1)By comparing the AWR scores of mice with different intensity of CRD before and after modeling,the stress levels of 30,45,60 mm Hg were significantly higher in the 3,7 and 28 days than in the 0 day group,3,7,and 28 days.There was no difference between the groups in each pressure level（P<0.05）.2)By comparing the results of macroscopic pathological inflammation between groups,the inflammation scores in the 3 days and 7 days groups were significantly higher than those in the 0 days and 28 days groups（P<0.05）;there was no difference in the inflammation scores between the 0 and 28 days groups.3)HE staining observation of histopathological results:0 days group of colonic mucosal epithelial local damage and shedding,a small amount of inflammatory cells infiltrated around the gland,part of the submucosal congestion and edema;3days group of multiple small ulcers,interstitial inflammatory cell infiltration The mucosal glands were of different sizes and arranged disorderly;in the 7-day group,the glandular epithelial cells were slightly hyperplasia,the mucosal epithelial hyperplasia was repaired,and a small amount of inflammatory cells infiltrated around the gland;in the 28-day group,the colonic tissue was intact and the glands were arranged neatly.Significant inflammatory cell infiltration.Experiment 2:1)AWR score results:the pressure group of the model group was significantly higher than the normal group at 30,45,60 mm Hg（P<0.05）,and the pressure level of the electroacupuncture group at 45,60 mm Hg was significantly lower than that of the model group（P<0.05）.2)Immunofluorescence（double-labeled）results showed that the intestinal chromaffin-specific markers TPH1 and CaMKⅡ were highly co-expressed at colonic epithelial cells.3)Western Blot results showed that compared with the normal group,the expression of TRPV1,p CaMKⅡ and TPH1 protein in the colon tissue of the model group and electroacupuncture group increased significantly（P<0.01,P<0.05,P<0.01）;compared with the model group The expression of TRPV1 and TPH1 protein in colon tissue of electroacupuncture group was significantly decreased（P<0.05,P<0.01）.4)Real-time PCR results showed that compared with the normal group,the expression of TRPV1 and TPH1 mRNA in the colon tissue of the model group increased significantly（P<0.01,P<0.05）;compared with the model group,TRPV1 in the colon tissue of the electroacupuncture group The mRNA expression was significantly decreased（P<0.01）.There was no difference in CaMKⅡ mRNA expression between the three groups.5)ELISA results showed that compared with the normal group,the 5-HT content in the colon tissue of the model group and the electroacupuncture group was significantly increased（P<0.05）.Experiment 31)Western Blot results showed that there was no difference in CaMKⅡ and TPH1 protein expression between the groups.2)The results of Real-time PCR showed that there was no difference in the expression of CaMKⅡ and TPH1 mRNA between the blank group,the model group and the electroacupuncture group.Compared with the blank+electroacupuncture group,the expression of CaMKⅡ mRNA in the colon tissue of the electroacupuncture group was significantly increased（P<0.05）.Conclusion1)Electroacupuncture at Zusanli acupoint（ST36）can effectively alleviate visceral pain induced by TNBS in IBS model mice2)TRPV1 may affect the hapenning of visceral hypersensitivity by mediating the phosphorylation of CaMKⅡ and regulating the activity of TPH1,thereby affecting the synthesis and release of 5-HT.3)Electroacupuncture may regulate the expression of TPH1 in colon tissue of IBS visceral allodynia mice by TRPV1. |
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