| Purpose:In this study,mast cell degranulation of duodenal mucosa in rats with functional dyspepsia,TLR9/MyD88 pathway related genes and inflammatory factors were observed,and based on TLR9/MyD88 pathway,the intervention mechanism of electroacupuncture at "Zusanli" and "Zhongwan" points was elucidated to relieve low-grade duodenal inflammation in rats with functional dyspepsia.The aim is to provide some experimental evidence for traditional Chinese medicine clinical treatment.Material and method:Forty-eight 10-day-old SPF SD rats,half male and half female,were randomly divided into normal group,model group,electroacupuncture group and non-meridian and non-acupoint group,with 12 rats in each group.Functional dyspepsia rat models were prepared by "iodoacetamide gavage combined with irregular diet".Rats in the normal group were given 2% sucrose solution by intragastric administration of iodoacetamide,and rats in the other three groups were given 2% sucrose solution by intragastric administration of 0.1%iodoacetamide,0.2m L each,once a day,for 6 days.After weaning,the rats were fed conventional diet.Except for the normal group,the other three groups were given alternating-day fasting without water.The rats were fed until they were adults(about 10 weeks of age),and the modeling was completed.Electroacupuncture group selected "Zusanli" and "Zhongwan" points for electroacupuncture intervention;In the non-meridian and non-acupoint group,"non-meridian and non-acupoint" was selected for electro-acupuncture intervention,and the frequency of electro-acupuncture density wave was set as 2Hz/15 Hz,and the current intensity was 0.5m A.The electro-acupuncture was performed once every morning,once for 20 min,for 14 days.The general condition and body weight of rats in each group were observed.HE staining was used to compare the duodenum morphology.The number of mast cells and degranulation of duodenal mucosa were compared by toluidine blue staining.The protein and mRNA expressions of toll-like receptor 9(TLR9)and myeloid differentiation factor 88(MyD88)in duodenum were determined by immunohistochemical method and real-time quantitative PCR.The expression levels of tumor necrosis factor α(TNF-α)and interleukin1β(IL-1β)in duodenum were determined by ELISA.Results:1.Observation of changes in general conditions of rats: After electroacupuncture intervention,compared with the model group,the body weight of rats in electroacupuncture group increased,the range of motion increased,the hair was relatively shiny,and the feces returned to normal.2.HE staining observation: the morphology and structure of duodenal tissue of rats in the normal group are clear and complete,the cells are arranged in order,and the thickness of muscular layer is uniform without abnormal changes;The duodenal mucosa of rats in model group and non-acupoint group was slightly broken,slightly disordered and irregular,with a small amount of inflammatory cell infiltration;In the electroacupuncture group,the morphology and structure of the duodenum of rats recovered,without obvious abnormality and obvious inflammatory cell infiltration.3.Observation with toluidine blue staining: the shape of mast cells in the duodenal mucosa of rats in the normal group was regular without degranulation;Compared with the normal group,the mast cells in the duodenal mucosa of rats in the model group and non-acupoint group increased and most of them were irregular.There were many floccules around the mast cells,and the degranulation phenomenon was obvious;Compared with the model group,the number of mast cells in the electroacupuncture group decreased,and the degranulation phenomenon was not obvious.4.Immunohistochemical observation: Compared with the normal group,the expressions of TLR9 and MyD88 in the model group were increased(P<0.01),while the expression of TLR9 and MyD88 in the non-acupoint group were increased(P<0.05)and significantly increased(P<0.01).Compared with model group,TLR9 expression level in electroacupuncture group was decreased(P<0.05),MyD88 expression level was significantly decreased(P< 0.01),and TLR9 expression level in non-acupuncture group was significantly decreased(P< 0.01).The expression level of MyD88 in electroacupuncture group was lower than that in non-acupuncture and non-acupoint group(P<0.05),while TLR9 expression in electroacupuncture group was lower than that in non-acupuncture and non-acupoint group(P> 0.05).5.PCR detection results: Compared with the normal group,the mRNA expressions of TLR9 and MyD88 in the model group were increased(P<0.01),while the mRNA expression of TLR9 and MyD88 in the non-acupoint group was increased(P<0.05)and significantly increased(P<0.01).Compared with model group,mRNA expressions of TLR9 and MyD88 in electroacupuncture group were decreased(P<0.05).The expression level of TLR9 mRNA in electroacupuncture group was decreased compared with that in non-acupuncture and non-acupoint group(P>0.05),and the expression level of MyD88 mRNA in electroacupuncture group was decreased compared with that in non-acupuncture and non-acupoint group(P<0.05).6.ELISA results: Compared with normal group,the expression of TNF-α was increased in model group and non-acupoint group(P<0.01),and the expression of IL-1β was increased in the other three groups(P<0.01).Compared with model group,the expressions of TNF-αand IL-1β in electroacupuncture group and non-acupoint group were decreased(P< 0.01).The expression levels of TNF-α and IL-1β in electroacupuncture group were lower than those in non-acupuncture group(P<0.01).Conclusion:1.Electroacupuncture at "Zusanli" and "Zhongwan" points can relieve low-grade duodenal inflammation,and has a certain intervention effect on functional dyspepsia model rats.2.Electroacupuncture at "Zusanli" and "Zhongwan" points can inhibit duodenal mast cell activation,reduce duodenal mast cell degranulation,down-regulate the expression of TLR9/MyD88 signaling pathway related genes and proteins,reduce the expression of inflammatory factors TNF-α and IL-1β,and relieve low-grade duodenal inflammation.It plays an intervention role in functional dyspepsia model rats. |
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