Study On The Cholinergic Anti-inflammatory Pathway Mechanism Of Electroacupuncture In Postoperative Intestinal Paralysis Model Mic

BackgroundPostoperative ileus(POI)is a common gastrointestinal motility disorder caused by surgical trauma.The clinical symptoms of POI may include abdominal distension,abdominal pain,nausea,vomiting,loss of bowel sounds,inability to tolerate food,and delay in exhaust.POI increases the suffering of the pain,causes other complications,prolongs hospital stays,increases medical costs,and puts heavy burdens on the society.So far,there is still lack of effective targeted agents for treating POI due to the complexity of its pathogenesis.POI is considered as a disorder that belongs to the category of "abdominal distension" in traditional Chinese medicine.The basic pathogenesis of POI is "obstruction causes the pain".Acupuncture is a traditional non-pharmacological therapy,and it has the dual characteristics of specificity and wholeness.Moreover,the side effects produced by acupuncture are few.Electroacupuncture(EA)is a novel therapy which was developed on the basis of traditional acupuncture therapy.A number of clinical studies have shown that acupuncture can significantly improve the symptoms of patients with POI.However,its mechanism is still unclear.It is very important to clarify the biological basis of acupuncture through the modern technology,which is also the key for acupuncture to be recognized internationally.The local aseptic inflammatory response caused by intestinal tube exposure and surgical operation is an important pathologic mechanism of POI.Cholinergic anti-inflammatory pathway is a recently discovered neuroimmune regulation pathway,which can control the occurrence and development of inflammation.This anti-inflammatory pathway is based on vagus nerve,acetylcholine,and α7nAChR.Compared with the traditional humoral anti-inflammatory pathway,the cholinergic anti-inflammatory pathway is characterized by rapid,direct,and efficient regulation to inflammation.Based on previous studies,we put forward the hypothesis that "EA at acupoint ST36 could attenuate POI by activating the vagus nerve mediated cholinergic anti-inflammatory pathway".Therefore,we plan to systematically research the roles of α7nAChR,macrophage,and JAK2-STAT3 signaling pathway in the beneficial effect caused by EA.ObjectiveThe molecular biological techniques were employed to explore the roles of α7nAChR,macrophage,and JAK2-STAT3 signaling pathway in the beneficial effect of EA for treating POI.The mechanism of EA for treating POI will be systematically investigated in this study.This study also aims to provide modern scientific basis for the treatment of POI by EA.Methods1.C57BL/6 mice underwent laparotomy followed by standardized small bowel manipulation to induce POI.2.EA at ST36 or a non-traditional acupuncture point on the tail in a mouse model of POI.The depth of stainless steel needle insertion was 3mm.The current strength of EA was 1mA and the EA duration was 20 minutes.3.C57BL/6 mice were randomly divided into six groups,namely:sham operation group,POI model group,ST3 6 EA group(POI+2Hz EA group,POI+10Hz EA group,POI+30Hz EA group,POI+100Hz EA group).The optimum EA frequency filtered by this experiment was used in subsequent experiments.Then another group of C57BL/6 mice were randomly divided into six groups,namely:POI+ST37 group,POI+ST39 group,POI+ST25 group,POI+CV4 group,POI+CV12 group,POI+Non-acupoint group.Mice were treated by EA immediately after the operation and mice were executed 24 hours after the operation.The optimum acupoint filtered by this experiment was used in subsequent studies.The intestinal motility of mice was determined by the intestinal fluorescence detection method.The levels of inflammatory cytokines TNF-α and IL-6 in serum was determined by enzyme linked immunosorbent assay.4.Immunofluorescence was employed to identify the activity of macrophage,and α7nAChR.Western blot was used to evaluate the expression of α7nAChR.This part of study aimed to explore the effect of EA on macrophage activation and α7nAChR in a mouse model of POI.5.Immunofluorescence was employed to identify the P-JAK2 and P-STAT3.Western blot was used to evaluate the expression of P-JAK2 and P-STAT3,This part of study aimed to explore the effect of EA on JAK2-STAT3 signaling pathway in a mouse model of POI.6.The inhibitors of α7nAChR、JAK2,and STAT3 were used to verify the experimental results.The intestinal motility of mice was determined by the intestinal fluorescence detection method.The levels of inflammatory cytokines in serum was evaluated by enzyme linked immunosorbent assay.7.Results are presented as mean ± standard error of the mean.Data were statistically evaluated using the one-way analysis of variance(ANOVA).Aprobability value(P)of less than 0.05 was considered significant.SPSS 20.0 software was used to perform statistical analysis.Results1.The effect of EA on intestinal motility and inflammatory cytokines at different frequencies in a mouse model of POI.Compared with sham group,the intestinal motility of POI group decreased significantly,and the serum concentrations of TNF-α and IL-6 increased significantly.The intestinal motility was improved by EA at 10Hz or 30Hz.The serum concentrations of TNF-α and IL-6 were decreased by EA at 2Hz,10Hz,or 30Hz.2.The effect of EA at 10Hz on intestinal motility and inflammatory cytokines at different acupoints in a mouse model of POI.The intestinal motility of model mice was improved by EA at ST36,ST37,ST39,or CV4.The serum concentration of TNF-α was decreased by EA at ST36,ST37,or ST39.In addition,the serum concentration of IL-6 was decreased significantly by EA at ST36,ST37,ST39,CV4,or CV12.3.The effect of EA on the activation of macrophage and the expression of α7nAChR in a mouse model of POI.Compared with the sham group,the expression of macrophage marker F4/80 was significantly increased in the model group.Compared with the sham group,the expression of α7nAChR was significantly increased in the model group.EA enhanced the expression of α7nAChR significantly.4.The effect of EA on the activity of JAK2-STAT3 signaling pathway in a mouse model of POI.Compared with the sham group,the activity of P-JAK2 and P-STAT3 was significantly increased in the model group.EA at ST36 significantly increased the activation of P-JAK2 and P-STAT3.EA at non-acupoint did not notably affected the activity of JAK2-STAT3 signaling pathway.5.The effect of EA on intestinal motility and inflammatory cytokines after treatment with inhibitors in a mouse model of POI.The effect of EA on intestinal motility and inflammatory cytokines was partly suppressed after treatment with inhibitors MLA,α-BGT,WP1066,orα7nAChR.Conclusions1.In our study,the optimum EA frequency for treating POI was 10Hz or 30Hz,the optimum EA acupoint was ST36,ST37,ST39,or CV4.2.The activation of macrophages in the intestinal muscle layer of the POI models was reduced by EA at ST36.3.The expression of α7nAChR in the intestinal muscle layer of the POI models was increased by EA at ST36.4.The activity of the JAK2-STAT3 signaling pathway in the intestinal muscle layer of the POI models was enhanced by EA at ST36.In conclusion,EA at ST36 ameliorates postoperative ileus in mice via a α7AChR-dependent JAK2-STAT3 signaling pathway.