Study On The Cellular Molecular Mechanism Of Banxia Xiexin Decoction In The Treatment Of Functional Dyspepsia

objectiveTo explore the cellular and molecular mechanism of Banxia Xiexin Decoction in the treatment of functional dyspepsia from miRNA.methodData mining:CiteSpace was used to conduct visual analysis on the literature of clinical studies related to Banxia Xiexin Decoction on CNKI,cluster analysis and emersion analysis were conducted on the key words of the literature,and co-occurrence analysis was conducted on the authors and publishing institutions.The target and mechanism of action of Banxia Xiexin Decoction in the treatment of functional dyspepsia were predicted by network pharmacology.Authoritative online database was used to predict the target genes and mechanism of action of miR-451-5p.Experimental part:In vitro cell experiments to verify the core targets and related pathways of network pharmacological prediction.The inhibition/overexpression model of miR-451-5p was established.ICC proliferation was detected by cck8 method,transfection was detected by qPCR method,cell differentiation was detected by immunofluorescence double staining method,and cell cycle and apoptosis were detected by flow cytometry.WB method was used to detect the effects of cyclic-related proteins and JAK1/STAT3/ERK signaling pathway proteins.The drug serum of Banxia Xiexin Decoction was used to intervene the ICC with the inhibition of miR451-5p expression.The effect of Banxia Xiexin Decoction on the survival rate of ICC with the inhibition of miR-451-5p expression was detected by cck8 method.The effect of Banxia Xiexin Decoction on the expression of miR-451-5p in ICC with the inhibition of miR-451-5p expression was detected by qPCR method.Flow cytometry was used to detect the effects of Banxia Xiexin Decoction on the inhibition of miR-451-5p expression on ICC cycle and apoptosis.WB method was used to detect the effects of Banxia Xiexin Decoction on the inhibition of miR-451-5p expression on ICC cycle related proteins and JAK1/STAT3/ERK signaling pathway.resultsCiteSpace results show that the common diseases treated by Banxia Xiexin Decoction are chronic atrophic gastritis and functional dyspepsia,while the current research focus is diabetic gastroparesis.The network pharmacological results showed that the mechanism of action of Banxia Xiexin Decoction in the treatment of FD may be related to the ICC PI3K-Akt signaling pathway,MAPK signaling pathway,JAK-STAT signaling pathway,etc.,but whether there is a regulatory relationship remains to be further verified.The prediction results of miR-451-5p showed that miR-451-5p might cause changes in the expression levels of target genes such as MYC,STAT3,IL15,PDGFB,HSPA5 and TNFRSF1A.These target genes may affect the proliferation and development of ICC through acting on the JAK-STAT signaling pathway,MAPK signaling pathway and PI3K-Akt signaling pathway,so as to affect the occurrence and development of FD disease.The network pharmacological experiment showed that compared with the blank group,the protein expressions of JAK1,STAT3,Cyclin D1,RAF1,c-myc and MAPK1 in the normal group had no statistical significance.Compared with the normal group,the protein expressions of JAK1,STAT3,Cyclin D1,RAF1 and MAPK1 in TCM group were significantly increased(P<0.01,P<0.05),while the protein expression of c-myc was decreased,but the difference was not statistically significant.The effect of miR-451-5p on ICC differentiation:compared with the blank group,there was no statistically significant difference in average gray values between the inhibitor NC group and the mimics NC group.Compared with the inhibitor NC group,the average gray values of the inhibitor group decreased,but the difference was not statistically significant.Compared with the Mimics NC group,the average gray values of the Mimics group all increased,but the difference was not statistically significant.The effect of miR-451-5p on ICC cycle:Compared with the blank group,there was no statistically significant difference in cell cycle between the inhibitor NC group and the mimics NC group.Compared with inhibitor NC group,the increase in G1 phase was significantly higher in inhibitor NC group(P<0.01),and the decrease in S phase and G2 phase was significantly higher(P<0.05,P<0.01).Compared with the Mimics NC group,the Mimics group significantly decreased in the S stage(P<0.01),increased significantly in the G2 stage(P<0.01),and decreased somewhat in the G1 stage,but the difference was not statistically significant.The effect of miR-451-5p on ICC apoptosis:Compared with the blank group,there was no statistically significant difference in cell cycle between the inhibitor NC group and the mimics NC group.Compared with inhibitor NC group,the apoptosis rate in inhibitor NC group was significantly increased(P<0.01).Compared with the mimics NC group,the apoptosis rate of the mimics group was significantly decreased(P<0.01).Effects of miR-451-5p on the JAK1/STAT3/ERK signaling path way:Compared with the blank group,the protein expressions of JAK1,STAT3,p-STAT3,c-MYC,Cyclin D1,RAF1,MAPK1,p-ERK and MEK in the inhibitor NC group and the mimics NC group were not statistically significant.Compared with the inhibitor NC group,the protein expressions of JAK1,STAT3,pSTAT3,Cyclin D1,MAPK1,p-ERK and MEK in the inhibitor group were significantly decreased(P<0.01,P<0.05),while the protein expressions of c-MYC and RAF1 in the inhibitor NC group were not statistically significant.Compared with the Mimics NC group,the protein expressions of JAK1,STAT3,p-STAT3,Cyclin D1,MAPK1 and MEK in the Mimics group were significantly increased(P<0.01,P<0.05),while the protein expressions of c-MYC,RAF1 and p-ERK in the Mimics group were not statistically significant.The effect of Banxia Xiexin Decoction on the ICC cycle of miR-451-5p expression inhibition:Compared with the blank group,the difference of cell cycle in the inhibitor NC group was not statistically significant.Compared with inhibitor NC group,the increase in G1 phase was significantly higher in inhibitor NC group(P<0.01),and the decrease in S phase and G2 phase was significantly higher(P<0.05,P<0.01).Compared with the inhibitor group,there was no significant difference in cell cycle between the normal group and the inhibitor group.It was significantly decreased in G1 stage(P<0.01)and increased in S and G2 stage(P<0.01)in TCM group.Effect of Banxia Xiexin Decoction on ICC apoptosis inhibited by miR-451-5p expression:Compared with blank group,apoptosis rate of inhibitor NC showed no statistical significance.Compared with inhibitor NC group,the apoptosis rate in inhibitor NC group was significantly increased(P<0.01).Compared with inhibitor group,there was no significant difference in apoptosis rate in normal group.The apoptosis rate of TCM group was significantly decreased(P<0.01).Effects of Banxia Xiexin Decoction on the ICC JAK1/STAT3/ERK signaling pathway inhibiting miR-451-5p expression:Compared with the blank group,the expression of JAK1,STAT3,p-STAT3,Cyclin D1,RAF1,MAPK1,p-ERK and MEK protein in the inhibitor NC group had no statistical significance.Compared with inhibitorNC group,the protein expressions of JAK1,STAT3,p-STAT3,Cyclin D1,MAPK1,p-ERK and MEK in inhibitor NC group were significantly decreased(P<0.01,P<0.05),and the expression of RAF 1 was not statistically significant.Compared with inhibitor group,the expression of JAK1,STAT3,p-STAT3,Cyclin D1,RAF1,MAPK1,p-ERK and MEK protein in normal group had no statistical significance.The protein expressions of JAK1,STAT3,p-STAT3,Cyclin D1,RAF1,MAPK1,p-ERK and MEK in TCM group were significantly increased(P<0.01,P<0.05).conclusion1.The mechanism of action of Banxia Xiexin Decoction in the treatment of FD is related to the JAK-STat and MAPK signaling pathways of ICC.The experimental results of using Banxia Xiexin Decoction containing serum to interfere with normal ICC cells indicate the correctness of the network pharmacological prediction.2.Inhibition of miR-451-5p can lead to the stagnation of ICC cell cycle and inhibit ICC proliferation.MiR-451-5p can increase the expression of Cyclin D1 protein through the positive regulation of JAK-STAT and ERK signaling pathways,and affect the progress of cell cycle,which also demonstrates the correctness of the prediction of miRNA target genes.3.Banxia Xiexin Decoction can inhibit the apoptosis of ICC by up-regulating the expression of miR-451-5p,promote the proliferation of ICC,activate the JAK1/STAT3/ERK signaling pathway,increase the expression of Cyclin D1 protein,and drive the process of ICC from G1 phase to S phase.