Effect Of Tangerine Peel And Fructus Aurantii With Bupleurum On Functional Dyspepsia Mice Induced By Multiple Stress

According to TCM syndrome differentiation,functional dyspepsia?FD?is mostly caused by liver-stomach disharmony.Chaihu Shugan Powder?CSP?is one of the classical representation of liver-spleen harmonizing prescription,and is used to treat functional dyspepsia in clinical practice.In this prescription,Pericarpium citri reticulatae and Fructus aurantii?CZ?play an important role in the treatment of functional dyspepsia because of its effect of regulating qi-flowing for strengthening spleen.To reconcile the liver and spleen,it is supplemented with Bupleurum which is coursing liver qi and resolving depression Chinese medicines.How to use modern language to explain the scientific connotation and mechanism of compatibility?This is a starting point of this research.Objective?1?Establishment and evaluation of FD Model mice which was similar with clinical symptoms.?2?To compare the different effect of Pericarpium citri reticulatae,Fructus aurantii?CZ?and Pericarpium citri reticulatae,Fructus aurantii,Bupleurum?CCZ?on the FD mice.?3?To investigate relationship bewteen gastric motility enhancement which is caused by Pericarpium citri reticulatae,Fructus aurantii?CZ?is supplemented with Bupleurum and interstitial cells of Cajal?ICC?.?4?To investigate relationship bewteen gastric motility enhancement which is caused by Pericarpium citri reticulatae,Fructus aurantii?CZ?is supplemented with Bupleurum and the 5-HT system.?5?To investigate the mechanism of promoting gastric motility of Pericarpium citri reticulatae,Fructus aurantii?CZ?in compatible with Bupleurum through 5-HT system.Method?1?The FD mouse model is established by triple-one multiple stress method,The specific method is restraint stress?6h per day?,swimming,shock and chronic unpredictable stimulation during 30 days.During the modeling period,the body weight,food intake and water intake,percentage of sugar preference and scores of open-field behavioral activities were recorded regularly.For evaluating the overall gastrointestinal function,the gastric residual rate and intestial driving rate in normal group and model group were measured by phenol red method after treatment.MTL and SP concentration in Serum were detected by ELISA.For evaluating model comprehensively,the liver,stomach and colon tissues of normal group and model group were collected for pathological analysis.?2?After 30 days of modeling,FD mice were intragastrically administered for 12days.The medicinal groups were divided into Pericarpium citri reticulatae,Fructus aurantii group?CZ?,Bupleurum,Pericarpium citri reticulatae and Fructus aurantii group?CCZ?.After administration,the body weight,food intake and water intake,percentage of sugar preference,scores of open-field behavioral activities and the gastric residual rate were measured,substance P?SP?and motilin?MTL?in serum were determined by ELISA.?3?The GAPDH was used as the reference,the relative expression levels of mRNA and protein of c-kit in gastric antrum tissue were determined by real-time fluorescence quantitative PCR and Westren Blot.?4?The GAPDH was used as the reference,5-HT concentration in Serum and 5-HT concentration in brain were detected by ELISA,the relative expression levels of mRNA and protein of 5-HT₄R in duodenal tissue were determined by real-time fluorescence quantitative PCR and Westren Blot.?5?CZ and CCZ containing serum was prepared according to serum pharmacological methods.The enzymolysis method was used to extract and purify rat gastric antral smooth muscle cells,and the cell immunofluorescence technique was used to identify the smooth muscle cells,the growth curve was drawn by CCK-8 method and the activity of DSP containing serum on smooth muscle cells was determined.Fluo-3-am was used as Ca²⁺fluorescent probe,and the change of Ca²⁺fluorescence intensity in smooth muscle cells of each group was measured by laser scanning confocal microscope.Results?1?Compared with mice in the normal group,FD mice weight was reduced,the average daily intake and water intake decreased,and the percentage of sugar water preference and voluntary activity were reduced,Loose stools and depression-like symptoms appear.Pathological section shows that organic disease of gastrointestinal tissue isn't exist.Those symptoms were similar to clinical patients with functional dyspepsia caused by liver-stomach disharmony,which indicating that FD mouse model was successfully established.?2?After treatment by TCM,CZ and CCZ could counteract the decrease of body weight,of the percentage of sugar water preference and the score of spontaneous activity?P<0.05,P<0.05?,also could improve the gastric residual rate?P<0.01,P<0.01?and the contene of MTL and SP?P<0.01,P<0.01?in serum of model mice.The gastric residual rate and level of serum SP in CCZ is higher than that of CZ?P<0.05,P<0.05?,the effect of improving is evident.?3?Compared with the normal group,the relative expression levels of mRNA and protein of c-kit in gastric antrum tissue of model group were down-regulated?P<0.01?.CZ and CCZ can increase the expression level of c-kit protein?P<0.01,P<0.01?,CCZ has no advantage over CZ.?4?Compared with the normal group,5-HT content in Serum and 5-HT content in brain tissue of FD model mice decreased?P<0.01?.The relative expression levels of mRNA and protein of 5-HT₄R in duodenal tissue were down-regulated?P<0.01?.Compared with the model group,CZ and CCZ can increase the content of 5-HT and the expression level of5-HT₄R protein?P<0.01,P<0.05?,and CCZ is higher than CZ?P<0.05?.?5?After successfully isolated,purified and cultured rat gastric antrum smooth muscle cells,the logarithmic growth period was 3-4 days by CCK-8.By comparing the serum effect of each group,compared with the normal serum group,10%perridone,10%CZ and10%CCZ can make the smooth muscle cells more activited?P<0.01,P<0.01,P<0.01?and increase intracellular Ca²⁺fluorescence,10%CCZ is higher than 10%CZ?P<0.01?.ConclusionThe FD mice symptoms induced by triple-one multiple stress method are similar with clinical functional dyspepsia patients caused by liver-stomach disharmony,which indicated that psychological stress is an important pathogenic factor of functional dyspepsia.In Chaihu Shugan Powder,Pericarpium citri reticulatae and Fructus aurantii?CZ?can reduce the gastric residual rate,increase gastric motility,and improve symptoms of FD.Its became more effective aftercompatible with Bupleurum,the enhanced mechanism has nothing to do with gastrointestinal factors?MTL,ICC?,gastric motility enhancement is achieved by enhancing the 5-HT system which could increase GSMC intracellular Ca²⁺fluorescence(5-HT,5-HT₄R,Substance P,Ca²⁺).