| Objective:In this study,the effects of electroacupuncture on the BDNF/TrKB-PLC-Ca2+ signaling pathway and EGCs cell activation in IBS-D rats were analyzed using immunofluorescence double-label,western blot,transmission electron microscopy,real-time fluorescence quantitative PCR and flow cytometry,and the mechanism of electroacupuncture alleviating IBS-D visceral hypersensitivity was further analyzed.Methods:(1)Modeling and grouping:40 adult female SD rats were randomly divided into the Control group(n=8)and the Preparation model group(n=32).The IBS-D rat model was prepared by combining chronic unpredictable mild stress(CUMS)and Senna decoction.After the model was verified successfully,32 rats in the preparation model group were randomly divided into Model group,EA group,EA+TrkB group,and EA+DMSO group,with 8 rats in each group.(2)Electroacupuncture intervention:the next day after the successful model replication,the rats in the EA group,the EA+TrkB group,and the EA+DMSO group were bound,and EA was performed at unilateral ST 25,LR3 and ST 36 points once a day,20min each time,for 14 consecutive days,and acupuncture was performed alternately at the left and right acupoints.The Control group and Model group were only bound(same as the EA group),but no acupuncture.(3)General behavior test of IBS-D rats:Body weight,food intake,and water intake of rats in each experimental stage were recorded and analyzed to evaluate the effect of EA on the behavior of rats in each group;Elevated cross maze test(OT%)was used to evaluate the depression and anxiety state of IBS-D rats.The gastrointestinal dysfunction and diarrhea of IBS-D rats were determined by Fecal water content and Bristol score.(4)Detection of visceral hypersensitivity in IBS-D rats:After EA treatment,abdominal withdrawal reflex(AWR)and Visceral pain threshold were used to evaluate the changes of visceral hypersensitivity in rats in each group,and to observe the regulating effect of EA on visceral hypersensitivity in IBS-D rats.(5)Activation status of EGCs cells:Ultrastructural changes of EGCs cells were observed under transmission electron microscopy.The Immunofluorescence double-label technique was used to observe the number of EGCs cells and the co-expression of GFAP/Cx43 in IBS-D rats’ colons.The Ca2+ content of IBS-D rats’ colon was further analyzed by flow cytometry,and the changes of SP content in the colon after EGCs overactivation were analyzed by WB technique.(6)Detection of BDNF/TRKB-PLC-Ca2+ signaling pathway:RT-PCR and Immunofluorescence techniques were used to observe the number of EGCs in the colon of IBS-D rats,the co-location expression of GFAP/BDNF and GFAP/TrkB,as well as the expression changes of BDNF mRNA and TrkB mRNA.It is speculated whether BDNF can specifically bind the TrkB receptor on EGCs cells.In addition,the expression of PLC-γ protein and Ca2+ in the colon was detected by WB and flow cytometry,and the regulation of key molecules in the BDNF/TrKB-PLC-Ca2+ signaling pathway during EA treatment of IBS-D was comprehensively evaluated.Results:Experiment 1:Alleviating effect of EA on visceral hypersensitivity in IBS-D rats(1)Experimental results of weight,eating,and drinking volume of rats:After modeling,compared with the Control group,rats in the Model group lost weight due to severe diarrhea(P<0.05);After the intervention,compared with the Model group,the body weight of the EA group increased significantly(P<0.05);After modeling,compared with Control group,food intake of the model group was decreased(P<0.05);After the intervention,the feed intake of the EA group was significantly increased(P<0.05).In addition,EA had no regulating effect on water intake before and after intervention(P>0.05).(2)Feces situation of rats in each group:After modeling,compared with the Control group,the Fecal water content of IBS-D model rats was significantly increased(P<0.05),and the Bristol score was continuously significantly increased(P<0.05).IBS-D rats in each group had severe diarrhea,and fecal characteristics were loose stools.After the intervention,Fecal water content in the EA group and EA+DMSO group was significantly reduced,and Bristol score was decreased(P<0.05).Compared with the EA group,the EA+TrkB group had severe diarrhea and increased Fecal water content(P<0.05).(3)Experimental results of rat elevated cross maze in each group:After modeling,compared with the Control group,IBS-D rats’ elevated cross maze 0T%continued to decrease(0T%<20%,P<0.05),IBS-D rats showed significant anxiety state;After the intervention,0T%has increased in the EA group and EA+DMSO group(0T%>20%,P<0.05);Compared with EA group,the elevated cross maze(0T%)in EA+TrkB group was significantly decreased(P<0.05).(4)Changes of visceral hypersensitivity of rats in each group:when the rectal dilatation pressure was 20mmHg,40mmHg,60mmHg,compared with the Control group,the AWR score of IBS-D rats was significantly increased,showing obvious visceral hypersensitivity(P<0.05).After the intervention,the EA group and EA+DMSO group could significantly relieve visceral hypersensitivity of IBS-D rats(P<0.05).Compared with the EA group,diarrhea was more serious and AWR score was higher in the EA+TrkB group(P<0.05).When the rectal dilation pressure was 80mmHg,the AWR score of the the EA group and the EA+DMSO group increased after modeling compared with the Control group(P<0.05).After the intervention,there was no statistical difference in all groups compared with the Control group(P>0.05).After modeling,compared with the Control group,the visceral pain threshold of IBS-D rats continued to decrease significantly,and the hypersensitivity status of rats in each group was stable during modeling(P<0.05).After the intervention,EA could significantly adjust the visceral pain threshold and improve visceral hypersensitivity of IBS-D rats(P<0.05).Compared with the EA group,the visceral pain threshold of the EA+TrkB group was lower(P<0.05).(5)Pathological section results of rats in each group:The mucosa,submucosa,muscularity,and adventitia of the colon tissues of rats in each group were stratified significantly,with complete structure,and no significant inflammatory cell infiltration or pathological changes,no obvious degeneration and necrosis,which was consistent with the pathological characteristics of functional gastrointestinal diseases.Experiment 2:Inhibition of intestinal EGCs activation by EA in IBS-D rats(1)Regulating effect of EA on the morphology and structure of EGCs cellsUnder Transmission Electron Microscopy,the morphology and structure of EGCs cells in the colonic submucosa of the Control group were normal,and all organelles in the cytoplasm were intact and clear.In IBS-D rats,EGCs had chromatin margins,and the perinuclear space was significantly widened.There was abundant Golgi apparatus in the cytoplasm,and most rough endoplasmic reticulum was expanded and cystic.In contrast,only a few mitochondria in the cytoplasm of the EA group and the EA+DMSO group showed mild swelling after EA intervention.However,most mitochondria in the EGCs cytoplasm of the EA+TrkB group were swollen,and crisis were broken,dissolved or even disappeared.(2)Changes in the number of EGCs cells before and after EA interventionCompared with the Control group,GFAP(EGCs cell number)in colon tissue of IBS-D rats in model group was significantly increased,with statistical significance(P<0.01).Compared with the Model group,GFAP in colon tissue of rats in EA group and EA+DMSO group was significantly decreased,with statistical significance(P<0.01).Compared with the EA group,the colon tissue GFAP in the EA+TrkB group was significantly increased(P<0.05).(3)Co-located expression of GFAP and Cx43WB results showed that Cx43 protein expression was significantly increased in the Model group compared with the Control group(P<0.01).Compared with the Model group,Cx43 protein expression in the colon tissue of rats in the EA group and the EA+DMSO group was significantly decreased(P<0.01).Fluorescence double standard results showed that compared with the Control group,the co-expression of GFAP/Cx43 protein was significantly increased in the Model group(P<0.01).Compared with Model group,the protein content of GFAP/Cx43 in colon tissue of rats in the EA group,the EA+TrkB group,and EA+DMSO group was significantly decreased(P<0.01).(4)Expression of Cx43 and SP in IBS-D ratsWB results showed that compared with the Control group,Cx43 and SP expressions were significantly increased in the Model group(P<0.01).Compared with the Model group,the expressions of Cx43 and SP in colon tissue of the EA group and the EA+DMSO group were significantly decreased(P<0.01);Compared with the EA group,Cx43 and SP expression were increased in the EA+TrkB group after blocking the effect of EA with TrkB agonist(P<0.01).Experiment 3:The regulation effect of EA on BDNF/TrkB signaling pathway in IBS-D rats(1)mRNA expression of BDNF and TrkB in IBS-D rats’ colonCompared with the Control group,the expression of BDNF mRNA and TrkB mRNA in the Model group were significantly increased(P<0.01).Compared with the Model group,the expressions of BDNF mRNA and TrkB mRNA in colon tissue of rats in the EA group and EA+DMSO group were decreased(P<0.05);After blocking the effect of EA,there was no significant difference in the expression of BDNF mRNA and TrkB mRNA in the colon tissue of rats in the EA+TrkB group(P>0.05).(2)Changes in the number of EGCs and co-location of GFAP/TrkB and GFAP/BDNF in each groupCompared with the Control group,GFAP(EGCs cell number)in the colon tissue of the Model group was significantly increased(P<0.01).Compared with Model group,GFAP(EGCs cell number)in colon tissue of rats in EA group and EA+DMSO group was significantly decreased,with statistical significance(P<0.01);There was no significant change in GFAP(EGCs cell number)in colon tissue of rats in EA+TrkB group(P>0.05).Compared with the Control group,the contents of GFAP/TrkB and GFAP/BDNF in colon of the Model group were increased(P<0.05).Compared with Model group,the protein contents of GFAP/TrkB and GFAP/BDNF in the colon of the EA group were decreased(P<0.05);Compared with the Model group,the protein contents of GFAP/TrkB and GFAP/BDNF in colon of rats in the EA+TrkB group were not significantly changed after the effect of EA was blocked by the injection of TrkB receptor agonist(P>0.05).Compared with the EA+DMSO group,the protein content of GFAP/TrkB in the colon was increased in the EA+TrkB group(P<0.05).(3)Changes of PLC-y protein content in the colon of rats in each groupCompared with the Control group,the expression of PLC-yin colon of the Model group was significantly increased,with extremely significant statistical significance(P<0.01).Compared with the Model group,the expression of PLC-γ protein in the colon of EA group was significantly decreased(P<0.05),but no difference was found in EA+TrkB group(P>0.05).Compared with the EA+DMSO group,the expression of PLC-γ protein in the colon of rats in the EA+TrkB group was increased.(4)Changes of colonic Ca2+ in rats in each groupFlow cytometry results showed that compared with Control group,Ca2+ content in colon of IBS-D rats in the Model group was significantly increased(P<0.01).Compared with the model,the colonic Ca2+ in the EA group and EA+DMSO group was significantly decreased(P<0.01);Compared with the EA group,the colonic Ca2+ in the EA+TrkB group was increased(P>0.05).Conclusions:1.EA can regulate visceral hypersensitivity and gastrointestinal dysfunction in IBS-D rats,and improve their anxiety.2.EA can reduce the number of EGCs cells and reshape the ultrastructure of EGCs cells,and inhibit the excessive activation of EGCs cells by reducing the content of Cx43 protein and SP in IBS-D rats,thus playing a role in the treatment of IBS-D visceral hypersensitivity.3.EA can down-regulate the expression of key molecules in the BDNF/TrKB-PLC-Ca2+signaling pathway,thus inhibiting the over-activation state of EGCs cells,and when the TrkB receptor is activated,the effect of EA is blocked,suggesting that EA may alleviate visceral hypersensitivity of IBS-D rats through the BDNF/TrKB-PLC-Ca2+ pathway. |
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