| Background:IBS is a common gastrointestinal disorder and one of the predominant diseases in acupuncture and moxibustion treatment,but the available data can not fully elucidate its therapeutic mechanism.Recent studies have shown that mast cell activation plays a key role in abnormal immune-nerve interaction in IBS intestinal tract.Tryptase is one of the important markers of intestinal mast cell activation and degranulation.It can cleave and activate intestinal neuronal protease receptor 2(PAR2),promote the release of CGRP and SP,and induce visceral hypersensitivity.Based on previous studies,it has been found that MC activation,PAR2 and CGRP interact,among which PAR2 is a critical mediator.Methods:The PI-IBS visceral hypersensitivity rat model was established by TNBS,and the intestinal sensitivity was evaluated by AWR score.Background:IBS is a common disease of digestive system,and is one of the dominant diseases in acupuncture treatment,but the existing data do not fully elucidated its therapeutic mechanism.Four weeks after TNBS enema,model+PAR2-AP group(M + PAR2-AP),model + PAR2-AP + electro-acupuncture group(M +PAR2-AP+EA)were treated with PAR2-AP enema.Two groups receiving electro-acupuncture treatment were treated with electro-acupuncture at Zusanli and Tianshu points for two weeks.The model group and the control group did not receive any other special treatment.At the end of the course of treatment,each group underwent another AWR score to evaluate the change of intestinal sensitivity.The number and morphological changes of mast cells were observed under electron microscopy by toluidine blue staining,and the expression of PAR2,CGRP,SP and TPSP in colon mucosa and dorsal root ganglion were detected by immunoprotein imprinting,real-time fluorescence quantitative PCT and immunofluorescence.Results:1.AWR scores of rats in each group:Compared with the control group,AWR scores of each group increased;AWR scores of model+electro-acupuncture group decreased significantly compared with model + PAR2-AP group;AWR scores of model + PAR2-AP +electro-acupuncture group decreased significantly compared with model+electro-acupuncture group;AWR scores of model+PAR2-AP+electro-acupuncture group increased compared with model+electro-acupuncture group.2.Morphology of mast cells and detection of degranulation markers:Microscopic observation showed that MC in the control group was regular,round-like,with intact membrane and no degranulation;Compared with the control group,MC in the model group was irregular,with many flocs around the cells and degranulation;Compared with the model group,MC in the model+electro-acupuncture group was regular,elliptic,with few flocs around it.Compared with the model+ PAR2-AP group,the model +PAR2-AP+electro-acupuncture group had regular morphology,elliptical shape,few flocs around,and no obvious degree of degranulation.Compared with the control group and the model+EA group,the expression level of tryptase(TPSP),a marker of mast cell degranulation,was significantly increased in the model group.See picture 6A.Compared with the model group,the expression of TPSP mRNA and protein in the model+EA group was significantly decreased;compared with the model+PAR2-AP group,the expression of TPSP mRNA and protein in the model+PAR2-AP+EA group was significantly decreased.3.The expression of PAR2,CGRP and SP in colonic mucosa:Compared with the control group,the expression level of PAR2 in the model group was significantly higher;compared with the model group,the expression level of PAR2 in the model+EA group was significantly lower;compared with the model+PAR2-AP group,the expression level of PAR2-AP+EA group was lower,but still higher than that in the control group and the model+EA group.Compared with the model group,the expression of the above proteins and mRNAs in the model+EA group was significantly lower,and the expression of the above proteins and mRNAs in the model +PAR2-AP+EA group was significantly lower than that in the model+PAR2-AP group.4.Expression of PAR2,CGRP and SP in dorsal root ganglion:Compared with the control group,the expression level of the above-mentioned proteins in the model group was significantly higher;compared with the model group,the expression level of the above-mentioned proteins in the model+EA group was significantly lower;compared with the model+PAR2-AP group,the expression level of the above-mentioned proteins in the model + PAR2-AP+ EA group was lower,but still higher than that in the control group and the model+EA group.Compared with model group,the expression of protein and mRNA in model + EA group was significantly decreased,and compared with model+PAR2-AP group,the expression of protein and mRNA in model+PAR2-AP+EA group was significantly decreased.Conclusion:1.Under the pathological condition of PI-IBS caused by TNBS enema,the threshold of visceral pain in rats was significantly decreased.The mast cells had activated degranulation and excited adjacent nerve endings,up-regulated the expression of PAR2 and promoted the neurotransmitters CGRP and SP,induced viscera.Highly sensitive.2.Electroacupuncture can significantly improve the visceral pain threshold of PI-IBS model rats,inhibit the activation of colon MC,down-regulate the expression of PAR2,TPSP,CGRP,SP protein and mRNA in colon tissue and dorsal root ganglia,and promote the recovery of visceral sensory conduction pathway.3.Under the condition of exogenous application of PAR2 agonist,electroacupuncture relieves visceral hypersensitivity and weakens the down-regulation effect on PAR2,TPSP,CGRP,SP and other biological active substances,and reversely proves that electroacupuncture regulates the expression of PAR2 to MC.The activation-PAR2-CGRP loop forms a benign regulation.To sum up:electroacupuncture can reduce the visceral hypersensitivity state by down-regulating the level of MC activation-PAR2-SP/CGRP signal axis in the colon tissue and dorsal root ganglia of PI-IBS rats,where PAR2 is electroacupuncture regulating MC activation-An important target for the PAR2-CGRP loop.Electroacupuncture at Zusanli and Tianshu can strengthen spleen and tonic,regulate qi and relieve pain,and regulate immune-neural dialogue is a potential important mechanism.This paper preliminarily clarifies that electroacupuncture regulates PAR2-mediated immune-neural interaction for the treatment of visceral pain.One of the mechanisms,but due to the complexity of the mechanism of visceral pain,further research is needed to elaborate. |
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