Natriuretic Peptide Signaling Pathway Up-regulated May Be Involved In Loss Of Gastric ICC Of Rats With Liver-Stagnation And Spleen-Deficiency Syndrome

Objective: The aim of this study was to investigate the relationship between the natriuretic peptide signaling pathway and the loss of gastric ICC in rats with GYPX.Methods:1.At first,all rats were divided into two groups.Rats in model group were singly housed and assigned to one of the chronic unpredictable mild stress（CUMS）randomly.Conditions such as spirits,vitality and the color of ear mucosa were observed every day.The measure of weight,open-field test,sucrose preference test and urine D-xylose excretion rate test were performed before modeling and at the end of the experiment.At the end of the experiment,the gastric residual rate was tested.The above methods were used to identity whether the model was successful.2.Western blotting analyses were performed to detect the protein expression of NPRs,c-kit and SCF in the gastric of rats.Real-Time PCR analyses were used to detect the gene expression of NPRs,c-kit and SCF in the same place of rats.3.The gastric smooth muscle cells（GSMCs）were extracted from normal SD rats and identified using immunocytochemistry.Different concentrations of drugs were used to treat GSMCs,while control GSMCs were cultured with culture medium in the absence of any drugs.The difference of SCF expression in cells was detected by Western-blotting method.MTT assay was performed to detect the proliferation of GSMCs treated with different concentrations of cANF.Results:1.The general state of rats in model group was poor compared with rats in normal group.Rats in model group grew slowly.The number of crossed-grids,standing and grooming and the rate of sucrose preference were reduced,which indicated that the locomotor activity of rats in model group was decreased significantly compared with normal group.Gastric residual rate was higher than that of the normal group.Urinary D-xylose excretion rate was decreased,which showed that the absorption function of small intestine was poor.2.These results of western-blotting and RT-PCR showed that the expression of ckit and stem cell factor（SCF）in stomach were down-regulated in model rats at the protein and mRNA levels.The expression of natriuretic peptide receptor（NPR）-A,B and C were up-regulated in the stomach of model rats at the protein and mRNA levels.3.Immunohistochemistry showed that cells extracted from SD rats were α-actinpositive,such that the cells were confirmed to be smooth muscle cells.NPR-A,B can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells（GSMCs）with different concentrations of C-type natriuretic peptide（CNP）.8-BrcGMP had a similar effect as CNP on the expression of SCF,but treatment with cANF did not.The results of the methyl thiazolyl tetrazolium bromide（MTT）assay indicated that high concentrations of cANF(10^-6 mol/L)restrained the proliferation of cultured GSMCs.Conclusions: 1.It was successful to make the animal model of GYPX using the method of chronic unpredictable mild stress combined with single cage feeding.2.NPs signaling pathway was up-regulated in the stomach of model rats,while SCF/c-kit signaling pathway was down-regulated.3.CNP/NPR-A,B/cGMP signaling pathway affected the expression of SCF.4.cANF/NPR-C signaling pathway inhibited the proliferation of GSMCs.The up-regulation of the NPs/NPR-C and NPs/NPR-A,B/cGMP signaling pathways may be involved in the loss of gastric ICC via an indirect decrease in the production of SCF.