Effect Of Regulating Liver And Invigorating Spleen Decoction On The Function Of Antral Mucosa And Smooth Muscle In Rats With Functional Dyspepsia (Syndrome Of Liver-qi Stagnation And Spleen-qi Deficidency)

ObjectiveEstablish functional dyspepsia (FD) animal models with liver-qi stagnation and spleen-qi deficiency, used methods of chronic restraint stress(CRS), over fatigue, irregular feed. Observe the changes of general condition, and gastric emptying rate of animal models before and after the treatment of Regulating Liver and Invigorating Spleen Decoction (RLISD); detect the expression of gastrointestinal hormones in the plasma and receptors in the gastric body mucosa, and the expression level of the second messengers in the smooth muscle of gastric body in vitro derived from the animal models before and after the treatment of RLISD.Methods1. Establish animal model64SPF Sprague Dawley (SD) rats were selected, female, weight about200-220g, adaptive feeding for one weeks. Then all rats were randomly divided into four groups:normal group(NG), model group(MG), Regulating Liver and Invigorating Spleen Decoction group(RLIDG), Domperi-done group (DPG),16cases in each group. Adopting chronic astriction, excess fatigue and out of constant diet on rats to establish functional dyspepsia (syndrome of liver-qi stagnation and spleen-qi def icidency). The modeling time is14days. NG were fed wiht nornal diet, water supply; MG were giver sevral saline intragastric orally;8.54g/kg RLISD were administered to RLISDG,2.7mg/kg domperidone were administered to DPG for14days.2. Detection Observe the changes of general condition before and after the induction of FD animals, and the treatment of decoction; valuate the gastric motility through the experimental methods of gastric emptying. Plasma levels of vasoactive intestinal peptide(VIP) and substance P(SP) were measured by radioimmunoassay. Immunohistochemistry staining was used to detect the expressions of vasoactive intestinal polypeptide receptor2(VIP2R) and the substance P receptor2(SP2R) in the gastric mucosa, and the myosin light chain kinase (MLCK) in the mucosa layer and muscular layer of the gastric body. Smooth muscle cells separated from the muscular layer of the gastric body in the animal models were made to be single cell suspension, ELISA method was used to detect the expressions of inositol1,4,5-triphosphate(IP3), cAMP, and Ca2+in the smooth muscle cells.Results1. weight change Before modeling:there is no significant difference between each gruop (P>0.05). After modeling:compared with NG, the body weight in MG、RLISDG and DPG decreased significantly (P<0.001), while there is no significant difference in model group、RLISDG and DPG. After treating: Compared with MG, the body weight in RLISDG and GPG increased signinficantly (P<0.001), while there is no significant difference between RLISDG and DPG (P=0.07).2. Gastric emptying rate There is statistical significance difference among the four groups (F=60.643, P<0.001). Compared with NG, the rate of gastric emptying in MG decreased significantly (P<0.001). Campare with MG, the rate of gastric emptying in RLISDG and DPG increased ignificantly (P<0.001, P<0.001); between RLISDG and DPG, had no significant difference.3. Plasma levels of VIP/SP There is statistical significance difference among the four groups (VIP:F=4.650, P=0.008; SP:F=3.304, P=0.031). Compared with NG, the level of VIP in plasma of MG increased significantly (P=0.019), while level of SP decreased significantly (P=0.031). Compared with MG, the level of VIP in plasma of RLISDG and DPG decreased significantly (P-0.002, P=0.004); at the same time, the level of SP in plasma of RLISDG and DPG increased significantly (P=0.008, P=0.015). But compared with NG, the the level of VIP/SP in plasma of RLISDG and DPG had no significant differrence.4. The expressions of VIP2R、SP2R、MLCK in the mucosa layer(or muscular layer) of the gastric body4.1Expression of VIP2RUnder microscope, the positive expression of VIP2R were seen as brown color, mainly exist in cytoplasm, and nucleus are blue. The positive cell mainly exist in the basilar part of mucosa layer. There is statistical significance difference among the four groups (F=5.756, F=0.003). Compare with NG, the mean density of VIP2R in MG decreased, a significant difference between the two groups(P=0.001); Compare with the MG, the mean density of RLISDG and GPD both increased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (F=0.018, P=0.003); there was no statistical significance between RLISD group and Dompe-ridone group (P=0.3465).4.2Expression of SP2RUnder microscope, the positive expression of SP2R, which mainly exist in the basilar part of mucosa layer, were seen as brown color, mainly exist in cytoplasm, and nucleus are blue.There is statistical significance difference among the four groups CP=Z.294, P=0.035). Compare with NG, the mean density of SP2R in MG decreased, a significant difference between the two groups (P=0.008);Compare with the MG, the mean density of RLISDG and GPD both increased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (P=0.032, P=0.022); there was no statistical significance between RLISD group and Domperidone group (P=0.872).4.3Expression of MLCKUnder microscope, the positive expression of MLCK not only exist in the muscular layer and submucosa muscle, also exist in gland of mucosa layer. The positvie cell were seen as brown color, mainly exist in cytoplasm and nucleus are blue.The expression of MLCK in mucosa was statisted. There is statistical sig-nificance difference among the four groups (F=5.396,P=0.005).Compare with NG, the mean density of SP2R in MG decreased, a significant difference between the two groups(P=0.001);Compare with the MG, the mean density of RLISDG and GPD both increased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (P=0.005, P=0.021); there was no statistical significance between RLISD group and Domperidone group (P=0.546).As for the expression of MLCK in muscular layer, There is no statistical significance difference among the four groups (F=0.711, P=0.554)5. The content of IP3、cAMP、Ca2+in the smooth muscle cells of the gastric body.5.1The content of IP3 There is statistical significance difference among the four groups (F=5.027, P=0.007).Compare with NG, the content of IP3in MG decreased, a significant difference between the two groups(P=0.008);Compare with the MG, the content of IP3of RLISDG and GPD both increased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (P=0.002, P=0.003); there was no statistical significance between RLISD group and Domperidone group.5.2The content of cAMPThere is statistical significance difference among the four groups (F=4.901, P=0.007).Compare with NG, the content of cAMP in MG decreased, a significant difference between the two groups(P=0.016);Compare with the MG, the content of cAMP of RLISDG and GPD both increased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (P=0.005, P<0.002); there was no statistical significance between RLISD group and Domperidone group (P=0.652).5.3The content of Ca2+There is statistical significance difference among the four groups (F=9.413, P<0.001).Compare with NG, the content of Ca2+in MG increased, a significant difference between the two groups (P<0.001);Compare with the MG, the content of Ca2+of RLISDG and GPD both decreased, the differences between the MG and the RLISDG, the MG and the DPG are statistical significantly (P=0.003, P<0.001); there was no statistical significance between RLISD group and Domperidone group (P=0.159).Conclusion1. Gastric mucosa secretion dysfunction and gastric motility dysfunction might be one of the main causes of FD in rat. There was gastric mucosa secretion dysfunction in the model group rats, which behaved that the changs of the gastrointestinal hormones in the blood, and the decreases of expressions of VIP2R、SP2R、MLCK in the gastric mucosa. There was gastric motility dysfun-ction in the model group rats, which behaved that the increasing concentrations of Ca2+and the decreasing levels of IP3and cAMP in the resting smooth muscle cells.2. Regulating Liver and Invigorating Spleen Decoction is proved effec-tive in treating FD animals Syndrome of syndrome of liver-qi stagnation and spleen-qi deficidency. It can improve psychological behavior and promote gastric movement, the mechanisms of which are related to the improvement of gastric mucosa secretion function and gastric motility function.