Mechanism Of EA At ST36 On In The Treatment Of Liver-stagnation And Spleen Deficiency-type FD Rats Through Cx43 And SCF/c-kit Regulating ICC Network Structure

ObjectiveFunctional dyspepsia is one of the most common functional gastrointestinal diseases in the clinic.Gastric motility disorder is the main pathological mechanism that induces the symptoms of functional dyspepsia.The interstitial cells of Cajal(ICC Interstitial cells of Cajal)can produce and transmit slow waves.When the supplementation of ICC is reduced or lost,gastrointestinal motility disorders are associated.ICC specific receptor c-kit,SCF(Stem cell factor)can bind and stimulate SCF/c-kit signal pathway,whice is a major signaling pathway to complement and refreshement of ICC.Connexin 43(Cx43)plays an important role in maintaining the structural integrity of the Cajal interstitial cell network.In this experiment,the most common type of liver stagnation and spleen deficiency in TCM classification was established,and the rat model of FD was established,in order to observe the specificity change of ICC in rats under the condition of FD and treatment of electroacupuncture.The relationship between FD and ICC was explored by various expression changes of c-kit,SCF and Cx43.In order to further clarify the mechanism of EA at ST36 on ICC,kit neutralizing antibody(ACK2)was used as a blocker of SCF/c-kit signaling pathway before therapeutic intervention?To exploer whether EA at ST36 mediates c-kit,SCF regulate SCF/c-kit signaling pathway and up-regulate Cx43,promote the ICC of dynamic number balance and complete network structure.In order to provide further experimental basis and new ideas for effective prevention and treatment of functional dyspepsia.MethodSixty male SD rats of SPF grade were randomly divided into blank control group,model group,EA group,SEA group,ACK2 + EA group.Except for the blank control group,the other 4 groups were treated with multi-factor intervention of Guo's tail-stimulation method + irregular feeding + ice saline gavage.On the fifth day of modeling,rats were given intraperitoneal injection of kit neutralizing antibody(ACK2)at a dose of 0.2 mg/Kg for 10 days,to prepare for FD rats of blocking SCF/c-kit.After the modeling was completed,the electric stimulation of EA ST36 intervention treatment was performed on the 3 groups other than the blank group and model group,and the SEA group was treated for 10 days on sham acupoint(5mm lateral to ST36).After eletroacupuncture treatment,the expressions of c-kit,SCF and Cx43 in the gastric body of each group were detected by Western blot.The expression levels of c-kit and SCF in the gastric body of each group were detected by Real-time RT-qPCR.The ultrastructural changes of the network of ICC�IM of each group of rats were observed by transmission electron microscopy(TEM).Result1.Weight of each group of rats: the weight of each group of rats given method of multi-factor intervention modeling was significantly lower than that of the normal control group(P<0.01),indicating that modeling method of multi-factor intervention can significantly suppress weight gain.After treament of electroacupuncture at ST36,the weight of the rats in the EA and the ACK2 + EA group increased compared with the model group,the difference was statistically significant(P<0.05).Compared with the SEA group,the weight gain of the EA group was faster,and the difference was statistically significant(P<0.05).2.Comparison of SCF and c-kit in gastric body tissue: compared with the blank control group,the expressions of SCF and c-kit protein in the model group showed significant decrease(P<0.01).The expression of SCF and c-kit in the model group decreased and was lower than that in the EA group;the expression of SCF and c-kit in the SEA group was also lower than EA group,both the difference was statistically significant(P < 0.05).After the application of ACK2 blocker,the expression of SCF and c-kit in the ACK2+ EA group was higher than that in the model group(P<0.05).3.Comparison of SCF and c-kit mRNA in gastric body tissue: the expression of SCF and c-kit mRNA in the model group was lower than that in the blank control group,which was statistically significant(P<0.01),consistent with the results of WB.Compared with the EA group,the levels of SCF and c-kit mRNA in the model group decreased(P<0.05);the level of mRNA of the target molecule of the SEA also decreased(P<0.05).Compared with the model group,the mRNA levels of SCF and c-kit in the ACK2 + EA group were up-regulated(P<0.05),and the results of WB were verified.4.Comparison of Cx43 protein in the gastric body tissue: compared with the blank control group,the expression of Cx43 in the model group decreased significantly,and there was significant difference(P<0.01).Compared with the model group,the expression of Cx43 protein in the EA group and the ACK2+EA group increased,the difference was statistically significant(P<0.05).Compared with the electroacupuncture group,the expression of Cx43 protein in the SEA group was decreased(P<0.05).5.Comparison of ultrastructure of ICC-IM network in gastric body tissue: the ICC-IM in the normal control group showed a spindle-shaped,dense cytoplasm,abundant mitochondria in the endoplasmic reticulum,and extended bipolar processes,which was closely related to the nearby nerve fiber and smooth muscle cell.The ICC-IM of the model group exhibited a spindle-shape and parallel to the smooth muscle cells,but the extension of processes was shortened and did not form a gap junction with the nearby smooth muscle cell.The shape of ICC-IM in the EA group changed,but the shape of the bipolar spindle did not change,and it formed a gap junction with nearby nerve and smooth muscle cells.The biggest difference between the ICC-IM of SEA group and the EA group is that processes are sharply shortened and does not form a complete jap junction network structure,but the distance between the processes and the smooth muscles is shortened.The ICC-IM in the ACK2 + EA group showed a spindle shape,and the processes extension was longer than the model group and formed a gap junction.Conclusion1.This study found that in the FD state,the expression levels of SCF,c-kit and Cx43 molecules decreased,indicating the loss of Cajal interstitial cells and the destruction of network structure,suggesting that there is a close relationship between the type of liver-stagnation and spleen-deficiency FD and Cajal interstitial cells.2.The treatment of electroacupuncture at ST36 can improve the molecules expression levels of SCF,c-kit and Cx43 to a certain extent,which is beneficial to the increase of the number of ICC and gap junctions network structure of ICC,and to some extent to improve the damage of ICC network function.Further repairing gastric motility.3.Specific blocking of SCF/c-kit signaling pathway inICC.The expression of SCF and c-kit in the ACK2 + EA group did not decrease,but was up-regulated compared with the model group.This suggests that the electroacupuncture ST36 can mediate the expression of SCF and c-kit and promote the increase of interstitial cells of Cajal,which may be related to the regulation of SCF/c-kit signaling pathway.