Effect Of Banxia Xiexin Decoction On Intercellular Communication Of SiRNA Interfering With Cx43 Gene Silencing In GIST-882 Cell

[Objective]To further explore the regulation mechanism of Banxia Xiexin Tang on gastrointestinal motility disorders,we took the gap junctional intercellular communication as the core,from the perspective of the relationship between Cx43 and gastrointestinal motility disorders,revealing the cellular and the molecular mechanism of Banxia Xiexin Tang in regulating gastrointestinal motility by Cx43,which reveals the connotation of"Xinxia Pi" from the perspective of intercellular communication.This not only could provide scientific support for the therapeutic effect of Banxia Xiexin Tang,but also help to improve the effectiveness of Banxia Xiexin Tang in regulating gastrointestinal motility disorder based on the mechanism,to promote the modernization of Banxia Xiexin Tang in the treatment of digestive system diseases.[Methods]Male SD rats were divided into Banxia Xiexin Tang group(the whole recipe group)to prepare BT-containing serum and control group(the normal deionized group)to prepare control serum.Gastrointestinal stromal tumor cell line(GIST-882)was purchased from Shanghai Valley Biotechnology Co.,Ltd.(91310114662486089Y).Stable cultured and passaged,they were randomly divided into control group,model group with Cx43-siRNA interference,and Cx43-siRNA interference of Banxia Xiexin Tang containing serum group(the whole recipe group).Cell viability and survival rate were examined by CCK-8 kits.The cell cycle and apoptosis were analyzed by flow cytometry.Intracellular calcium concentration was detected by calcium ion fluorescence probe(Fluo-3 AM).RT-PCR method was used to detect the expression of Cx43 mRNA,MAPK mRNA,PKA mRNA and PKC mRNA in GIST-882 cells.The expression levels of connexin 43 and its phosphorylated MAPK,PKA,PKC and PKG in GIST-882 cells were detected by Western blot.[Results](1)The cell viability and cell survival rate:Compared with the control group,cell viability and cell survival rate was significantly decreased in the model group(P<0.05);compared with the model group,cell viability and cell survival rate of Banxia XiexinTang group was significantly increased(P<0.05).(2)The cell apoptosis and cycle:Apoptosis,there was no significant difference between the groups(P>0.05).Cell cycle,in the period of G1 phase,the model group increased and the whole group decreased,while there was no significant difference between the groups(P>0.05).Compared with the control group,the cell cycle of the model group was significantly lower(P<0.01)in the period of G2.Compared with the model group,the cell cycle of the whole group was higher,but there was no statistical significance(P>0.05).In the period of S,compared with the control group,the cell cycle of model group is lower,but no statistical significance(P>0.05);compared with the model group,the cycle of whole group was higher(P>0.05),and it just had the trend,but no statistical significance.(3)The level of calcium in cells:Compared with the control group,the calcium ion concentration of the model group was significantly increased(P<0.05);compared with the model group,the concentration of calcium ion in the whole group cells was significantly decreased(P<0.05).(4)The expression of related proteins:compared with the control group,the expression of Cx43,MAPK protein in the model groups was significantly decreased(P<0.05),and the expression of P-Cx43,PKA,PKC,PKG protein in the model groups was no significant difference(P>0.05);compared with the model group,the expression of Cx43 and MAPK protein in the whole groups was significantly higher(P<0.01),and the expression of P-Cx43,PKA,PKC,PKG protein compared with the model group had no significant difference(P>0.05).(5)RT-PCR:compared with the control group,the expression of Cx43 mRNA,MAPK mRNA,PKA mRNA in the model group was significantly decreased(P<0.05);compared with the model group,the expression of Cx43 mRNA,PKA mRNA,MAPK mRNA in the whole group increased significantly(P<0.05);the expression of PKC mRNA in model group increased than the control’s,and decreased than the whole group,but the difference was not statistically significant.[Conclusion](1)Banxia Xiexin Tang can promote the proliferation of GIST-882 cell line transfected with Cx43-siRNA.Moreover,it can increase the survival of the cells through the growth of the cell cycle and not inhibit the apoptosis.(2)Banxia Xiexin Tang can reduce the concentration of intracellular calcium,which can regulate and protect the second messenger Ca2+ channel.(3)Banxia Xiexin Tang can increase the expression of Cx43 and MAPK protein and enhance the expression of Cx43 mRNA,MAPK mRNA,PKA mRNA.It is suggested that Banxia Xiexin Tang could regulate intercellular communication and promote the recovery of gastrointestinal dysfunction.Based on the above conclusions,from the view of experimental study of modern molecular mechanism,we can conclude that Banxia Xiexin Tang could improve the gap junction and enhance the function of gap junctional intercellular communication,and regulate the intracellular second messenger Ca2+ channel,MAPK signaling pathway and PKA protein kinase,thereby promoting the recovery of GIST-882 cells with Cx43-siRNA transfection,and finally to promote the recovery of gastrointestinal dysfunction.This provides the possibility for the treatment of "Xinxia Pi" by Banxia Xiexin Tang,and lays the foundation for the study of the mechanism of modern molecular biology.