| Objective: Inflammatory bowel disease(IBD)is a chronic,nonspecific and recurrent intestinal inflammatory disease.The incidence of inflammatory bowel disease is rising,which has become a global disease in the newly industrialized countries,including Asia,and has caused great burden to the society and economy.EA is effective in the treatment of inflammatory bowel disease.It is simple,safe and easy to operate.It is widely used in the treatment of inflammatory bowel disease.By establishing a visceral inflammatory pain model,this study screened the TNBS solubility of the best model and the internal target of treating inflammatory bowel disease by EA.The aim of this study was to explore the peripheral immune mechanism of EA in the treatment of inflammatory pain and provide a scientific basis for the treatment of inflammatory pain.The IBD model was established with the aqueous ethanol solution of different volume gradient TNBS solution +50%.The DAI score,colon length,propulsive rate,histology score,survival rate,Elisa detection serum and the content of IL-beta in colon tissue were tested,and the optimal TNBS model volume for the establishment of inflammatory bowel disease model was selected.In the selection of acupoint,the Evans blue tail vein was injected to observe the exudation point of Evans blue on the body surface of the rat.The sensitized and non-sensitized acupoints were selected according to the location of the exudation point,and the Bristol score,mechanical pain threshold and pathological injury of the colon were compared by EA and non-sensitized acupoints.The length of intestine is to provide scientific evidence for studying the mechanism of EA treatment of IBD and the basis of selecting points.The previous study of our group showed that EA could inhibit the expression of inflammatory factors by activating endogenous cannabinoid receptor type II receptor(CB2R),thus exerting analgesic effect.Therefore,this study would provide a theoretical basis for the treatment ofinflammatory bowel disease by EA as the starting target of EA in the treatment of inflammatory bowel disease.The clinical application provides the experimental basis.Method : 1.40 male SPF grade C57BL/6(20-25g)mice were randomly divided into four groups: normal group(solvent control group),80mg/kg TNBS+50% anhydrous ethanol group,100mg/kg TNBS+50%anhydrous ethanol group and 120mg/kg TNBS+50% anhydrous ethanol group with 10 rats in each group.The DAI score,the length of the rectum,the HS score and the survival rate of the mice were measured.2.Adult male SPF grade SD rats,except the normal group(10)were enema through the anus saline,and the rest were carried out with TNBS solution enema to establish the inflammatory bowel disease model.10 model rats were randomly selected through the tail injection of Evan blue to observe the body surface exudation point and determine the sensitized and non-sensitized acupoints.The other 30 rats were randomly divided into three groups: model group,electro acupuncture acupoint group,and electro acupuncture nonsensitization acupoint group,10 rats in each group.The treatment points for the treatment of inflammatory bowel disease were determined by comparing the Bristol score,the mechanical pain threshold,the histological score of the colonic pathological injury,the propulsive rate and the length of the rectum.3.There were 90 SPF male C57BL/6(20-25g)mice.The experimental A:40mice were randomly divided into the normal group,the model group,the EA group and the false EA group according to the random number table.Each group was divided into 10 rats in each group.DAI score,mechanical pain threshold,HS score,colon length and propulsive rate were measured.The protein expression levels of CB1 R,CB2R,IL-1β and SP in colon tissue were measured by DAI score,mechanical pain threshold,HS score,colon length and propulsive rate,and Western blot,and the expression of CB2 R coexpression and CB1 R was detected by immunofluorescence histochemical method,and the morphological structure of colon tissue was observed by HE staining.Experiment B:50 C57BL/6 male mice were randomly divided into 5groups: normal group,model group,EA group,EA +CB1R receptor antagonist AM251 group(EA+AM251),EA +CB2 receptor antagonist AM630group(EA+AM630),10 rats in each group.CB1 R and CB2 R antagonists were intraperitoneally injected with 30 min before EA.DAI score,mechanical pain threshold,HS score,colon length,propulsive rate,and Western blot were used to detect the expression of IL-1β and SP protein.Result:1.Effects of different volume gradient TNBS on DAI,HS,colon length,propelling rate,survival rate and related biochemical indexes in colitis mice1)DAI score: 7 days after model building,120mg/kg TNBS>100mg/kg TNBS group>80mg/kg TNBS group>normal group;2)HS score: 7 days after model building,80mg/kg TNBS group and 120mg/kg TNBS group were higher than those after making models.There was no statistical significance(P>0.05);3)colon length: after 7 days of modeling,the propulsive rate of 120mg/kg TNBS<100mg/kg TNBS group<80mg/kg TNBS group<normal group;4)after modeling,the propulsion rate of 100mg/kg TNBS group and 120mg/kg TNBS group was significantly higher than that of 80mg/kg TNBS group.10 There was no significant difference in 0mg/kg TNBS group(P>0.05);5)biochemical indexes: the IL-1β content in the colon tissue of the 120mg/kg TNBS group was the highest,and there was no significant difference between the 120mg/kg TNBS group and the 100mg/kg TNBS group in the serum tissue but higher than the 80mg/kg TNBS group,and 6)the survival rate: 120mg/kg TNBS<100mg/kg TNBS group<80mg/kg TNBS group< normal group.2.The exudation point of EB is highly correlated with BL25 of bilateral acupoints.In the control group,the number of Evans blue exudation point was very few.There was a significant EB exudation point in the back and abdomen skin of IBD rats induced by TNBS model group.The number of Evans blue exudation point in the model group was significantly different from that of the control group(P<0.05).The EB exudation point of IBD rats showed a neurogenic segmental distribution and concentrated in the T10-L6 segment,and the exudation point of L4-6 increased significantly than that of T10-12 and L1-3segments(P<0.05),showing a symmetrical distribution.3.The effect of electroacupuncture sensitization point on BSS score,mechanical pain threshold,colon length,propelling rate and HS score.1)There was no statistical difference between the 4 groups before the model BSS score(0d),and there was a comparability(P>0.05).The BSS score index of the model group,the EA sensitized acupoint group and the EA non sensitized acupoint group was significantly higher than that of the normal group(P<0.05).Compared with the model group,the EA point group was 4days after the model(5d),BSS The score was lower than that of the 5 day model group(P<0.05).Compared with the EA point group,the EA non sensitized acupoint group was higher than the EA point group(P<0.05)in the fourth days(5d)and the model seventh days(8d),and 2)the mechanical pain threshold of the 4 groups(0d,basic value)was not statistically poor.The PWT model group,EA point group and EA non sensitized acupoint group were significantly lower than the normal group(P<0.05).Compared with the model group,the PWT value of the acupoint group was higher than that of the model group(P<0.05)and the EA non-sensitized acupoint group was not statistically significant compared with the model group(P>0.05);compared with the EA point group,the PWT of the EA non sensitized acupoint group was lower than that of the EA sensitized acupoint group(P<0.05)after fourth days.Compared with the normal group,the colon length and the HS score of the model group,the EA point group and the EA non sensitized acupoint group were significantly different(P<0.05).There was a significant difference between the group and the EA non sensitized acupoint group(P<0.05),but there was no significant difference between the EA sensitized acupoint group and the normal group(P>0.05).Compared with the model group,the colon length,the propulsive rate and the HS score of the EA sensitized acupoint group were significantly different(P<0.05);Compared with the control group,there was a significant difference in the colon length,propelling rate and HS score between EA and non-sensitized acupoint group(P<0.05).4.The effect of EA on DAI score,mechanical pain threshold,colon length,propelling rate and HS score1)Compared with the normal group,the HS score,the propelling rate and the length of the rectum in the model group and the false EA group were statistically significant(P<0.05).There was no significant difference between the HS score and the propelling rate in the EA group compared with the normal group(P>0.05).Compared with the model group,the HS score,the propulsive rate,the length of the rectum and the sham acupuncture group were compared with the model group.There was significant difference in the length of rectum(P<0.05).There was no significant difference in the HS score and propelling rate in the sham EA group(P>0.05).Compared with the EA group,the HS score,the propelling rate and the length of the rectum were statistically significant(P< 0.05).2)compared with the normal group,the DAI score and the mechanical pain threshold of the model group and EA group were significantly changed(P<0.05).Compared with the model group,the DAI score of the EA group was significantly reduced from third days to the end of the experiment,and the mechanical pain threshold was significantly increased(P<0.05),and the DAI score of the sham EA group and the machine were mechanical.There was no significant difference in pain thresholdbetween the model group and the model group(P>0.05).Compared with the EA group,there was no significant difference in DAI score and mechanical pain threshold between the two groups(P>0.05).3)compared with the normal group,the expression level of CB1 R and CB2 R protein in the model group was statistically significant(P<0.05);the expression level of CB1 R and CB2 R protein in the EA group was significantly higher than that in the model group(P<0.05),while the expression level of CB1 R and CB2 R protein in the sham-EA group was significantly different from that in the EA group(P<0.05);The expression level of IL-1 β and SP protein in the intestinal tissue was significantly different from that in the model group(P<0.05),but the expression level of IL-1β and SP protein in the sham acupuncture group was significantly different from that in the EA group(P<0.05).4)compared with the normal group,the percentage of the macrophage single cell in the colon tissue of the TNBS group,the EA group and the sham EA group increased significantly(P<0.05),and the percentage of the CB2 R single cell in the colon tissue of the TNBS group,the EA group and the sham EA group increased significantly(P<0.05).The percentage of CB2 R single standard cells in the area of visual field was significantly higher than that in the TNBS group and the sham EA group(P<0.05).The percentage of CB2 R and macrophage double standard cells in the cell area of the macrophage was significantly increased in the EA group(P<0.05).Compared with the normal group,the percentage of CB1 R cells in the colon tissue in the colonic tissue of the model group increased,but there was no significant difference(P > 0.05).Compared with the model group,the percentage of CB1 R cells in the colon tissue in the EA group increased significantly(P<0.05).Compared with the EA group,The percentage of total cell area in the colon tissue of the sham EA group was decreased significantly(P<0.05).The colon of the model group had obvious lesion structure,such as lost epithelial cells,goblet cell loss,inflammatory cellinfiltration,recess disappearance,or EA group,showing less epithelial abscission,ulceration and leukocyte infiltration,and thickening of the musculus muscularis.In addition to relieving the symptoms of upper dermatitis,the EA group observed better and less vascular proliferation from the form of colonic crypt.5.Effects of cannabinoid receptor antagonists on HS score,propelling rate,rectal length,mechanical pain,DAI score,SP and IL-1β protein expression levels1)compared with the normal group,the HS score,the propelling rate and the length of the rectum in the model group and the antagonist group were statistically significant(P<0.05).There was no significant difference between the EA group and the normal group(P>0.05).Compared with the model group,the difference of HS score,propelling rate and rectal length in the EA group was statistically significant(P<0.05)Compared with EA group,there was a significant difference in HS score,propelling rate and rectal length between the two groups(P<0.05).2)compared with the normal group,the DAI score and the mechanical pain threshold of the model group and EA group were significantly changed(P<0.05).Compared with the model group,the DAI score of the EA group was significantly reduced from third days to the end of the experiment,and the mechanical pain threshold was significantly increased(P<0.05),and the analgesic effect of the EA could be reversed by the antagonist,so that the DAI score was scored.There was no significant difference between mechanical pain threshold and model group(P>0.05).3)compared with the normal group,the protein expression level of SP and IL-1β in the colon tissue of the model group was significantly increased(P<0.05),while the EA could significantly reduce the protein expression level of SP and IL-1 β in the colon tissue of IBD(P<0.05),and CB1 and CB2 receptor antagonists could significantly reverse the expression of IL-1 β and SP inIBD colon tissue induced by TNBS.Inhibitory effect(P<0.05).Conclusion: 1.According to DAI score,colon length and evaluation of IL-1βin colon tissue,120mg/kg TNBS group was better than group 100mg/kg TNBS.According to propelling rate,serum IL-1β,HS score and survival rate analysis,both doses can be used for model..If the sample size was large and the survival rate was considered,the 100mg/kg TNBS group could be selected;and this study was comprehensive.In view of the above factors,the dosage of80mg/kg TNBS was chosen when TNBS established the IBD model.2.The exudation point of EB in inflammatory bowel disease is mainly distributed in the back and is symmetrical near the midline,which is highly correlated with the distribution of back Shu points.The area of exudation on the back of rat is highly correlated with the area of bilateral "Da Chang Shu" area.Very few EB exudates are distributed in the thoracic segment.Therefore,combining acupuncture and moxibustion with acupuncture and moxibustion for treatment of diarrhea and acupoints,we chose bilateral "Da Chang Shu"(BL25)as the treatment acupoint of sensitized acupoint group.As the exudation point is less in the thoracic area,the bilateral "Xinshu" point(BL15)in the thoracic segment is used as the treatment point for the non-sensitized acupoint group.EA sensitized acupoints can reduce the score of BSS,HS score,propulsive rate,length of rectum,and improve mechanical pain threshold to improve the regulation mechanism of intestinal dysfunction and visceral pain sensitivity in IBD rats.3.EA can regulate the HS score,propulsive rate,length of rectum,DAI score and mechanical pain threshold induced by TNBS in mice with inflammatory bowel disease.4.EA can regulate the HS score,propulsive rate,length of rectum,DAI score and mechanical pain threshold induced by TNBS induced IBD in mice,and promote the activation of CB1 R and CB2 R in the colon tissue of IBD modelmice,and inhibit the expression of inflammatory factor IL-1 β and pain induced SP,and improve the structure of colon.CB1 and CB2 receptor antagonists can significantly reverse the inhibitory effect of EA on the expression of IL-1β and SP protein in the colon tissue induced by TNBS and the regulation of HS score,propulsive rate,rectal length,DAI score and mechanical pain threshold in mice with inflammatory bowel disease,indicating that CB1 R and CB2 R can be activated by EA in inflammatory bowel disease and play the role of the town.The effect of pain and regulation of intestinal dysfunction. |
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