Changes Of NPs Signal Pathway In Intestinal Tract Of Rats With Syndrome Of Liver-qi Stagnation And Spleen-qi Deficiency And The Interventional Effect Of Xiaoyaosan

Objective: Liver-gi stagnation(GY) and Spleen-qi deficiency(PX) are two relatively independent syndromes. There is a definite relationship in pathogenesis between them. Liver-gi stagnation and Spleen-qi deficiency(GYPX) is a Compound symptom of GY and PX. Clinical observation showed that the common symptoms of patient suffering from GYPX are gastrointestinal motility dysfunction. Research of combining traditional Chinese and Western medicine indicated that GYPX-induced gastrointestinal motility dysfunction is closely related to inappropriate secretion of neurotransmitter, hormone and cytokines. Natriuretic peptide is polypeptide hormone existing both in the nervous system and the gastrointestinal tract. NPs regulate a variety of physiological function, including natriuresis, dieresis, vasodilation, lower blood pressure and control electrolyte homeostasis. Many studies show that NPs and NPRs are widely distributed in the gastrointestinal tract, playing inhibiting role in gastrointestinal motility in autocrine and paracrine manner. NPs inhibit contraction activity of gastrointestinal smooth muscle mainly through NPs-NPRs-p GC-c GMP-PKG signaling pathway. However, it is still unclear whether NPs-NPRs-p GC-c GMP signaling pathway is involved in gastrointestinal disorders caused by GYPX and the relationship between NPs signaling pathway and the therapeutic effect of Xiaoyao Powder(XYS) to gastrointestinal disorders caused byGYPX. Therefore, we establish the rat model of GYPX with method of chronic unpredictable mild stress(CUMS) and treat rats with different doses of XYS or mirtazapine. The aim of this study is to clarify changes of NPs-NPRs-p GC-c GMP signaling pathway in rats’ gut of GYPX and intervention effect of Liver-soothing and spleen-strengthening decoction. This study is performed from the following four parts:Part I: Establish the rats model of GYPX and treat with Xiaoyao Powder and mirtazapineMethods: A total of 54 rats were divided into sixth groups: normal control group, model group, Low, Middle, High dose XYS groups and mirtazapine group. The rats were singly housed except the normal control group of three rats per cage. Primary controls were fed normal chow, ad lib. The rats were exposed to alternative stress each day for four weeks except the normal control group. XYS intervention groups and mirtazapine group were perfused intragastrically with different doses XYS and mirtazapine solution, respectively, one hour before stimulation. Normal group and model group were perfused intragastrically with normal saline at the same time. Modeling experiment lasted for four weeks; intragastric administration lasted for sixth weeks. The gavage doses were: low dose group: 7.65 g / kg · d, middle dose group: 15.3 g/ kg · d, and high dose group: 30.6 g/kg · d, mirtazapine group: 10mg/kg· d. Observe and record rats’ general condition, weight, open-field test results, urine D-xylose excretory rate weekly.Results:(1) The general condition of the model rats are as followings: exhausted demeanor, puffed hair, raspy voice with crouching together in the corner, loose stool, the slow response with no struggle when seized.(2)The weight of model group increase slowest, the mirtazapine group increases fastest. XYS improve the slow growth of rats’ weight in a dose-dependent manner. The difference between normal group、mirtazapine groups、XYS high dose group 、XYS middle dose group and model group were significant after four weeks(n = 9, ##P < 0.01).(3) After four weeks, the numbers of crossed-grids, standing and grooming times in open-field test were compared. The number of model group decrease mostly, and the mirtazapine group decreased slightly. XYS improve the reduction of the numbers in a dose-dependent manner. The difference between normal group, treatment groups and model group were significant after four weeks(n = 9, ##P < 0.01).(4) The result of urine D-xylose excretory rate showed that the decreasing degree of model group is greatest, the normal group is the same essentially and the mirtazapine group decreased slightly. XYS improve the reduction of urine D-xylose excretory rate in a dose-dependent manner. The difference between normal group, treatment groups and model group were significant after four weeks(n = 9, ##P < 0.01).Part II: The changes of NPs in rats’ gut of GYPX and intervention effect of XYS and mirtazapineMethods: In this section, we observed the distribution and expression of ANP, CNP in jejunum, colon and rectum using immunohistochemistry techniques. Detect the m RNA expression differences of ANP, CNP though Real-Time PCR.Results:(1) The results of rats’ intraperitoneal dissection showed: the rats of model group have a lot of contents and gas in gastrointestinal tract, intraperitoneal secretion of exudates, intestines expand extremely, and cecum is serious particularly. hyperemia and swelling of intestinal mucosal, Intestinal wall thinning, sour smell、poor intestinal motility. The rats of normal group has little content in gastrointestinal tract, intestinal wall is moist and pink, normal gastrointestinal morphology, good intestinal motility.(2) ANP and CNP immunopositive brown granules were detected in mucosa、muscle layer of rats’ gut. ANP and CNP positive-staining was evidenced in the nucleus and cytoplasm of smooth muscle cells, basal granular cells and stromal cells. The results showed that mucosa and muscl e layer of rats’ gut in sixth groups all expressed NPs, but differentially. The expression of ANP and CNP in model group were significantlyhigher than that in other groups(n = 9; ##P < 0.01). The expression of ANP and CNP between XYS high dose group and mirtazapine group has no significant difference. XYS inhibit the expression of ANP and CNP in a dose- dependent manner. The expression of ANP in jejunum is most, while CNP in colon is most.(3) The results of Real-Time PCR showed that the m RNA of ANP and CNP were expressed in rats’ jejunum, colon and rectum, but the expression level in each group is different. The m RNA level trend of ANP and CNP is consistent with its immunohistochemical results.Part III: The changes of NPRs in rats’ gut of GYPX and interv ention effect of XYS and mirtazapineMethods: In this section, we observed the distribution and expression of NPR-A, NPR-B and NPR-C in jejunum, colon and rectum using immunohistochemistry techniques. Detect the m RNA and protein expression differences of NPR-A, NPR-B and NPR-C though Real-Time PCR and Western Blot.Results:(1) NPR-A, NPR-B and NPR-C immunopositive brown granules were detected in mucosa, muscle layer of rats’ gut. NPR-A, NPR-B and NPR-C positive-staining was evidenced in the membrane of smooth muscle cells, basal granular cells and stromal cells. The results showed that mucosa and muscle layer of rats’ gut in sixth groups all expressed NPRs, but differentially. The expression of NPR-A, NPR-B and NPR-C in model group were significantly higher than that in other groups(n = 9; ##P < 0.01). The expression of NPR-A, NPR-B and NPR-C between XYS high dose group and XYS middle dose group, XYS high dose group and mirtazapine group has no significant difference. XYS inhibit the expression of NPR-A, NPR-B and NPR-C in a dose-dependent manner. The expression of NPR-A and NPR-C in jejunum is most, while NPR-B in colon is most.(2) The results of Real-Time PCR showed that the m RNA of NPR-A, NPR-B and NPR-C were expressed in rats’ jejunum, colon and rectum, but the expression level in each group is different. The m RNA level trend of NPR-A, NPR-B and NPR-C isconsistent with its immunohistochemical results.(3) The results of Western Blot showed that NPR-A and NPR-C were expressed in rats’ jejunum, colon and rectum, but the expression level in each group is different. The protein level trend of NPR-A and NPR-C is consistent with its immunohistochemical results.Part IV: changes of gastrointestinal hormones and c GMP content in rats of GYPX.Methods: In this part, we detect changes of gastrin and gastric inhibitory peptide in serum and changes of c GMP in serum and colon tissue homogenates using ELISA.Results:(1) The content of gastrin in serum of model group were significantly lower than that in other groups(n = 9; ##P < 0.01). The content of gastrin between XYS high dose group and mirtazapine group has no significant difference. XYS promote the synthesis and secretion of gastrin in a dose- dependent manner.(2) The content of gastric inhibitory peptide in serum of model group were significantly higher than that in other groups(n = 9; ##P < 0.01). The content of gastric inhibitory peptide between XYS high dose group and mirtazapine group has no significant difference. XYS inhibit the synthesis and secretion of gastric inhibitory peptide in a dose-dependent manner.(3) The content of c GMP in serum of model group were significantly higher than that in other groups(n = 9; ##P < 0.01).The content of c GMP between XYS high dose group and XYS middle dose group has no significant difference. XYS inhibit the synthesis and secretion of c GMP in a dose- dependent manner.(4) The content of c GMP in colon of model group were significantly higher than that in other groups(n = 9; ##P < 0.01). The content of c GMP in colon between XYS high dose group and mirtazapine group, XYS middle dose group has no significant difference. XYS inhibit the synthesis and secretion of c GMP in a dose- dependent manner.Conclusions:(1) ANP and CNP exist in the nucleus and cytoplasm of smooth muscle cells, basal granular cells and stromal cells in mucosa, muscle layer of rats’ gut. NPR-A, NPR-B and NPR-C exist in the Membrane of smooth muscle cells, basal granular cells and stromal cells in mucosa, muscle layer of rats’ gut.(2) The up-regulation of NPs-NPRs-p GC-c GMP signaling pathway may be one of the reasons of gastrointestinal motility disorders induced by GYPX. XYS in a dosedependent manner and mirtazapine play a role of therapeutic effect by down-regulating this signaling pathway. The high dose of Xiaoyaosan is the optimal dose in each dose. The therapeutic effect of mirtazapine is Similar with high-dose XYS.(3)The GYPX-induced gastrointestinal motility disorders may have a relationship with changes of gastrin and gastric inhibitory peptide content. XYS may play a therapeutic role by regulating synthesis and secretion of gastrin and gastric inhibitory peptide. This is the first study confirming the fact that NPs-NPRs-p GC-c GMP signaling pathway is upregulated in rats’ gut of GYPX and clarifies intervention effect of XYS which is the typical prescription of Liver-soothing and spleen-strengthening decoction. This study has theoretical significance in pathogenesis of GYPX-induced gastrointestinal motility disorders, and provides some experimental evidence for further research.