| Objective:To investigate the mechanism of SERT involved in electroacupuncture regulating P2X3 receptor to relieve visceral pain in rats with irritable bowel syndrome.Methods:A rat model of visceral pain in irritable bowel syndrome was prepared with trinitrobenzene sulfonic acid（TNBS）enema.In the first part,SD rats with normal genotypes were divided into normal group,model group and electro-acupuncture group.In the electro-acupuncture group,bilateral"Tianshu"and"Shangjuxu"acpoints were selected for electroacupuncture treatment,the acupuncture depth was 5mm,the electroacupuncture parameters are 1m A,2/100Hz,30min/time,continuous treatment for 7 days.In the second part,SD rats with SERT-/-were divided into normal group,model group and electroacupuncture group,and they were treated for 7 days continuously.In the third part,SD rats with normal genotypes were divided into normal group,model group,electroacupuncture group,model+P2X3R agonist group,model+P2X3R agonist+electroacupuncture group,and model+P2X3R agonist group were given intrathecal injection ofα,β-me ATP10ul at a concentration of 10-5mmol/ul,10ul/time,continuous intervention for 7 days;model+P2X3R agonist+electroacupuncture group received intrathecal injection ofα,β-me ATP for 30minutes,followed by electroacupuncture for 7 days.Colon histopathological staining was used to observe the changes in colon morphology of rats in each group;Behavioral score was used to observe the changes of AWR score under different intensity of CRD stimulation.;The changes of 5-HT content in colon tissue of rats were detected by ELISA;Western Blot and real-time PCR were used to detect the protein and m RNA expression of SERT and P2X3 receptor in the colon tissue of rats in each group;The regulation of SERT-P2X3 receptor expression in colon tissue of rats with visceral pain of IBS was observed by immunofluorescence chemistry（double standard）.Results:Histopathological staining of the colon after 21 days of modeling showed that no obvious pathological manifestations were found in the colon tissue of the rats in the model group,which was consistent with the clinical pathological characteristics of IBS.AWR scores before intervention showed that the AWR scores of rats in the model group were significantly higher than those in the normal group under the CRD stimulation of various pressure levels（P<0.05）.According to the results of colon histopathological staining and behavioral scores,it was suggested that the visceral pain model of rats in IBS was successfully prepared.In the first part of the experiment,AWR score after intervention showed that the AWR score of the model group was higher than that of the normal group under the CRD stimulation of various pressure levels（P<0.01）,and the AWR score of the electroacupuncture group was significantly lower than that of the model group under the CRD stimulation of 20 and 80mm Hg pressure（P<0.05）.Under CRD stimulation of 40 and 60mm Hg,AWR score was not statistically significant compared with model group,but showed a downward trend.The content of 5-HT in colon tissue of IBS visceral pain rats was significantly higher than that of normal group（P<0.01）.The content of 5-HT in colon tissue of electroacupuncture group was significantly lower than that of model group（P<0.05）.The protein and m RNA expression of SERT in colon tissue of rats in model group were significantly lower than that in normal group（P<0.01）,while the protein and m RNA expression of P2X3 receptor were significantly higher than that in normal group（P<0.01）.The expression of SERT protein and m RNA in the colon tissue of the electroacupuncture group was significantly higher than that of the model group（P<0.05,P<0.01）,while the expression of P2X3 receptor protein and m RNA was significantly lower than that of the model group（P<0.01）.Immunofluorescence assay（double-standard）showed that electroacupuncture increased SERT expression and decreased P2X3 receptor expression in colon tissue of rats with IBS visceral pain.In the second part of the experiment,after intervention,the AWR scores of SERT-/-rats in the model group were significantly higher than those in the normal group under the CRD stimulation of 20,40 and 60mm Hg pressure（P<0.05,P<0.01）.The AWR scores in the model group under the CRD stimulation of 80mm Hg pressure showed no statistical significance compared with the normal group,but showed an upward trend.The AWR score of the electroacupuncture group was significantly lower than that of the model group under the CRD stimulation of 60mm Hg pressure level（P<0.05）.The AWR score of the electroacupuncture group was not statistically significant compared with that of the model group under the CRD stimulation of 20mm Hg and 80mm Hg pressure level,but showed a downward trend.The content of5-HT in colon tissue of rats in model group was significantly higher than that in normal group（P<0.01）.The content of 5-HT in colon tissue of rats in electroacupuncture group was significantly lower than that in model group（P<0.01）.The expression of P2X3 receptor protein and m RNA in colon tissue of SERT-/-rats in model group was significantly higher than that in normal group（P<0.01,P<0.05）.The expression of P2X3 receptor protein and m RNA in colon tissue of SERT-/-rats was significantly lower in the electroacupuncture group than in the model group（P<0.01,P<0.05）.In the third part of the experiment,after intervention,the AWR score of rats in the model group was significantly higher than that in the normal group under the CRD stimulation of 40 and 60mm Hg pressure level（P<0.05）.The AWR score in the model group under the CRD stimulation of 20 and 80mm Hg pressure level was not statistically significant compared with that in the normal group,but showed an upward trend.The AWR score of the electroacupuncture group was significantly lower than that of the model group under the CRD stimulation of 40 and 80mm Hg pressure level（P<0.05）.The AWR score of the electroacupuncture group under the CRD stimulation of 20 and 60mm Hg pressure level was not statistically significant compared with that of the model group,but showed a downward trend.The AWR scores of rats in model+P2X3 receptor agonist group were not statistically significant compared with those in model group under CRD pressure of 20 and 80mm Hg,but showed an upward trend.The AWR scores of rats in the model+P2X3 receptor agonist+electroacupuncture group were not statistically significant compared with those in the electroacupuncture group under various pressure levels of CRD stimulation,but showed an upward trend.The expression of SERT protein and m RNA in colon tissue of rats in model group was significantly lower than that in normal group（P<0.01）.Compared with the model group,the expression of SERT protein and m RNA in colon tissue of the electroacupuncture group was increased（P<0.05）.Compared with the model group,the expression of SERT and m RNA protein in the colon tissue of rats in the model group+P2X3 receptor agonist group was not statistically significant,but showed a downward trend.The expression of SERT and m RNA protein in the colon tissue of rats in the model+P2X3 receptor agonist+electroacupuncture group was not statistically significant compared with that in the electroacupuncture group,but it showed a decreasing trend.Conclusion:1.Electro-acupuncture on both sides of"Tianshu"and"Shangjuxu"points can effectively relieve visceral pain in rats with IBS;2.SERT can reduce the concentration of 5-HT through the conductive needle,thereby affecting the expression of P2X3 receptor in the colon tissue of rats with IBS visceral pain and improving the symptoms of IBS visceral pain;3.The P2X3 purinergic receptor may affect the expression of SERT and 5-HT,thereby exerting a regulatory effect on IBS visceral pain. |
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