| Background and Aims:Irritable bowel syndrome(IBS)is a frequent functional gastrointestinal disorder,patients often have chronic abdominal pain and diarrhea,constipation,emergency,abdominal distention,and often associated with the occurrence of inflammation and damage of intestinal mucosa barrier,existing data is not fully explain the mechanism of IBS patients with injury of intestinal mucosa,based on the existing data,the study found Akt-related pathways aggregate inflammatory and metabolic signals,and NF-κB,as a key signaling molecule regulating intestinal permeability,is activated to release inflammatory factors in the immune barrier,which can lead to damage of the intestinal mucosal barrier.Therefore,this study established a mouse model of irritable bowel syndrome to observe the effects of electroacupuncture "Tianshu" and"Zusanli" on the miR-29a/Akt/NF-κB pathways in IBS murine model,to research and discussion the key role of electroacupuncture through what in repairing the damaged intestinal mucosal barrier from the perspective of inflammatory response.Methods:1.Experiment 1:Electroacupuncture reduced the inflammatory response induced by Akt-NF-κB and regulated intestinal mucosal barrier proteinC57 mice were randomly assigned to blank control group,model group,model+electroacupuncture group,Aktl inhibition+ model group,and Aktl inhibition+model group+electroacupuncture group.Water avoidance stress(WAS)WAS used to construct models,and Aktl inhibitor gavage WAS performed simultaneously in Aktl inhibition+model group and Akt1 inhibition+ model group with electroacupuncture.Intestinal sensitivity was assessed with AWR score after modeling.Model+electroacupuncture group,Aktl inhibition+model group+electroacupuncture group were treated with bilateral Tianshu Zusanli electroacupuncture for 10 days.The control group did not do special treatment.After treatment,the AWR was evaluated again.The whole blood and colon tissue were collected,and the integrity of colon epithelial tissue was observed under hematoxylin staining microscope(X400).The relative expression levels of protein kinase B(Akt),P(phosphorylated)-Akt,NF-κ B,P-NF κ B,IKKα,IKKβ,Claudin1,Occludin and ZO-1 in colon tissues were detected by Westen blot.The mRNA relative expression levels of protein kinase B(Akt),NF-κB,IKK α,IKK β,Claudin1,Occludin and ZO-1 in colon tissues were detected by fluorescence quantitative PCR.The expression levels of interleukin-1β(IL1β),interleukin-6(IL6)and tumor necrosis factor-α(TNF α)in serum were determined by ELISA.2.Experiment 2:The mechanism of electroacupuncture to repair intestinal mucosal barrier by inhibiting Akt phosphorylation through miR-29aC57 mice were randomly assigned to blank control group,water avoidance stress model group,model+miR-29a inhibition group,and model +electroacupuncture group.After intraperitoneal injection of miR-29a inhibition+model group for 28 days,water avoidance stress(WAS)WAS used to construct the model.Intestinal sensitivity was assessed with AWR score after modeling.Model+electroacupuncture group,bilateral Tianshu Zusanli electroacupuncture treatment for 10 days.The control group did not do special treatment.After treatment,the AWR was evaluated again.The whole blood and colon tissue were collected,and the integrity of colon epithelial tissue was observed under hematoxylin staining microscope(X400).Westen blot and immunofluorescence were used to detect the relative expression levels of protein kinase B(Akt),Claudinl and Occludin in colon tissues.PCR was used to detect the expression level of miR-29a and the mRNA expression levels of protein kinase B(Akt),Claudinl and Occludin.Results:1.Changes of abdominal wall evacuation reflex score(AWR score)in each group:①Experiment 1:Compared with the control group,AWR score of 20mmHg and 40mmHg were increased in the model group(P<0.001),and AWR score of 20mmHg and 40mmHg were increased in the Aktl inhibitor+model group(P<0.001)and 40mmHg(P<0.05).Compared with model group,AWR score of 20mmHg and 40mmHg in model+electroacupuncture group were decreased(P<0.001).Compared with Aktl inhibition+model group,the AWR score of Aktl inhibition+model+electroacupuncture group was decreased at 20mmHg(P<0.001),and the AWR score of 40mmHg was decreased(P<0.05).②Experiment 2:Compared with the control group,the AWR scores of 15mmHg and 30mmHg in model group were showed an upward trend(P<0.01).Based on the model group,The AWR score of the model+miR-29a inhibition group was decreased at 15mmHg,and the AWR score of 30mmHg was decreased with statistical significance(P<0.05).There was the AWR scores of model+electroacupuncture group at 15mmHg and 30mmHg an obvious downward trend(P<0.01).Compared with model+miR-29a inhibition group,AWR score of 15mmHg in model+electroacupuncture group was decreased,with statistical significance(P<0.05).2.The expression of inflammatory factors:①Experiment 1:Each group was compared with the blank control group to determine the relative expression level of the modified factor:Akt,p-Akt,NF-κB,p-NF-κB,IKKa and IKKβ in the model group were varying degrees of increase(P<0.001,P<0.01).The mRNA relative expression levels of Akt,NF-κB,IKKa and IKKβ were increased(P<0.001).The expression of IL1β,IL6α and TNFa was increased by ELISA,(P<0.001).There was the expressions of Akt,p-Akt,NF-κB,p-NF-κB,IKKα and IKKβ in the Aktl inhibitor+model group an increase,(P<0.01).The mRNA relative expressions of Akt,NF-κB,IKKa and IKKβ were increased(P<0.001,P<0.01,P<0.05);The expression of IL1β,IL6α and TNFα was increased by ELISA(P<0.001).Compared with the model group,the relative expression levels of Akt,p-Akt,NF-κB,p-NF-κB,IKKα and IKKβshowed an obvious upward trend in model+electroacupuncture group were decreased,with statistical significance(P<0.05,P<0.01,P<0.001).The mRNA relative expression levels of Akt,NF-κB and IKKβ were increased(P<0.01,P<0.001).ELISA expression of IL-1β,IL-6αand TNFα was decreased,and the difference was statistically significant(P<0.001).The relative protein expression levels of Akt,p-Akt,NF-κB,p-NF-κB and IKKα in the Aktl inhibitor+model group were decreased(P<0.05).The mRNA relative expression levels of Akt,NF-κB and IKKα were increased(P<0.05,P<0.01).The expression of IL-1β,IL-6α and TNFα was decreased by ELISA(P<0.05,P<0.01,P<0.001).Compared with the Aktl inhibitor+model group,the relative expression levels of Akt,p-Akt,NF-κB,p-NF-κB,IKα and IKKβin the Aktl inhibitor+model+electroacupuncture group decreased(P<0.01,P<0.05).The mRNA relative expression levels of Akt,NF-κB,IKKα and IKKβ were decreased(P<0.01,P<0.001).The expression of IL-1β,IL-6α and TNFα was decreased by ELISA(P<0.05,P<0.001).②Experiment 2:Compared with the control group,p-Akt/Akt expression was significantly increased in the model group(P<0.001).Compared with the normal control group,the expression of miR-29a was down-regulated in the model+inhibition group(P<0.001).The expression of miR-29a was down-regulated in the model+electroacupuncture group(P<0.01).The expression of miR-29a in the model+electroacupuncture group was slightly increased compared with the model+miR-29a inhibition group(P<0.05).3.The expression of colon mucosa protein Claudinl,Occludin:①Experiment 1:Compared with the control group,the expression of Claudinl,Occludin in other groups decreased to different degrees(P<0.05);Compared with model group,the expression of Claudinl and Occludin increased in model+electric acupuncture group(P<0.05).Compared with Aktl inhibition+model group,the expression of Claudinl and Occludin increased in Aktl inhibition+model+electroacupuncture group(P<0.05).②Experiment 2:Compared with the control group,the expression of Claudinl and Occludin in the model group was significantly decreased(P<0.05);compared with the model group,the expression of Claudinl and Occludin in the model+miR-29a inhibition group was significantly increased(P<0.05).The expression of Claudinl and Occludin in model+electric acupuncture group was significantly increased(P<0.05).4.Relative expression of miR-29a:Experiment 2:Compared with the control group,the expression of miR-29a was decreased in the model+miR-29a inhibition group and the model +electroacupuncture group(P<0.05).Compared with the model +miR-29a inhibition group,the expression of miR-29a was down-regulated in the model+electroacupuncture group(P<0.05).Conclusions:This study shows that(1)In IBS model mice caused by WAS chronic stress,the visceral pain threshold showed a significant decrease.The increased phosphorylation of Akt induces the release of inflammatory factors NF-κB,IKK α,and IKK β,and the activated NF-κB induces the decrease of colonic epithelial mucosal proteins NF-κB,IKK α,and IKK β,resulting in the damage of colonic mechanical barrier.(2)Electroacupuncture can significantly increase the visceral pain threshold of IBS model mice,inhibit the phosphorylation of Akt,and increase the expression of intestinal barrier tight junction proteins Claudin1 and Occludin.(3)Electroacupuncture may inhibit the phosphorylation of Akt through the expression of miR-29a,increase the expressions of tight junction proteins Claudin-1 and Occludin,and decrease intestinal mucosal permeability. |
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