| Objective:To observe the changes of fecal metabolites in chronic unpredictable mild stress(CUMS)rats with liver depression and spleen deficiency,and the intervention and study on the regulating mechanism of Xiaoyaosan decoction(Taiping Huimin Heji Ju Prescription).Method:Forty-five male SD rats weighing 180-210g were randomly divided into three groups:the control group,the stress group and the stress+XYS group,with 15 rats in each group.The rats of the stress group and the stress+XYS group were subjected to 6 weeks of stress by 9 kinds of stress factors,including restraint for 3 hours,tail clamping for 2 minutes,hot water swimming for 5 minutes,tilt for 12 hours,day and night reversal for 24 hours,wet packing for 24 hours,fasting water for 24 hours,noise for 3 hours and ice water swimming for 5 minutes.1 to 2 kinds of stress stimuli were randomly given per day,and the same stimuli were not repeated within 48 hours.Rats in the stress+XYS group were given Xiaoyaosan decotion(3.854 g/kg/d)30minutes before the establishment of the model.Rats in the control group and the model group were given the same volume of saline.The rats in each group were observed on day 0,day 7,day 14,day 21,day 28,day 35 and day 42 for general state,food intake and body weight,sucrose consumption test and open field test.At the end of the experiment,fresh rat feces were collected and weighed within 4 hours,then dried and weighed to calculate the water content of rat feces;5%D-xylose was administered one hour before sampling,2%sodium pentobarbital(4 mg/100 g body weight)was injected intraperitoneally to anesthetize rats,blood was taken off and Detection of D-xylose content in rat serum was measured by resorcinol method;carbon powder suspension was administered 30 minutes before sampling,and the whole small bowel was taken out after anesthesia.The carbon powder propulsion distance and the total length of the small bowel were measured,and calculation small bowel transit.Fresh rat feces(>500mg/grain)were collected and stored in EP tube at-80?.The metabolites of rat feces were detected by ~1H Nuclear Magnetic Resonance(~1H NMR)metabonomics.Partial least square-discriminant analysis(PLS-DA)was used for multivariate statistical analysis to compare the different metabolites.Results:1.General state of rats in each groupBefore the beginning of the experiment,the rats in each group had glossy fur,ruddy ears,flexible movement and moderate dry and wet stool formation.In the first week of modeling,rats in the stress group showed strong resistance,struggle and hoarse when restrained and stimulated by swimming stress.They were easy to break away,and their hair was erect,and their stool increased.In the second week of the experiment,the rats in the stress group were more vigilant,but the phenomenon of struggle resistance,hoarse and break-away during restraint was reduced;in the third week,the rats in the stress group were less resistant to stress,the color of ear and claw became lighter,the gloss of fur became worse,and occasionally feces were soft.At the 4th week,the resistance of rats in the stress group was weakened,while the struggle,resistance and jump of rats in swimming were reduced,and they hardly struggled when they were restrained,so they were easy to be grasped.At the fifth week,the rats in the stress group showed delayed reaction,tiredness,laziness,inactivity,light color of ear and claw,low gloss,yellowish hair and soft stool.At week 6,the rats in the catching of the stress group showed no resistance,laziness,crouching in corners and soft stool.At the beginning of the experiment,there was no significant difference in appearance and response to external stimuli between the stress+XYS group and the stress group.At the 3rd-5th week,the stress response and fur gloss of the rats in the stress+XYS group were improved compared with the stress group.Until the 6th week,the mental state and activity of the rats in the stress+XYS group were improved significantly compared with the stress group,and the hair was slightly dim,but tidy and not easy to fall off,and the stool was mostly moderate dry and wet.2.Changes of body weight and food intake of rats in each groupBefore the experiment,there was no significant difference in body weight and food intake of rats in each group(P>0.05);after the stress factors were applied,compared with the control group,the weight of rats in the stress group increased slowly,and their food intake decreased(P<0.01 or P<0.05);compared with the stress group,the weight and food intake of rats in stress+XYS group increased,especially in the last two weeks(day35,day42)of the experiment(P<0.05).3.Open-field testOn day 0,there were no significant differences in the total 5-minute moving distance,the total 5-minute moving distance in the central area,the total numbers of 5-minute cross in the central area and the 5-minute residence time in the central area between the groups(P>0.05).Compared with the control group,the total 5-minute moving distance and the total 5-minute crossing numbers in the central area of the stress group decreased significantly on the 7th day(P<0.01 or P<0.05);the 5-minute residence time in the central area of the stress group decreased significantly on the 14th day(P<0.05);and the total 5-minute moving distance,5-minute moving distance,5-minute crossing numbers and 5-minute residence time in the central area of the stress group on the 21st,28th,35th and 42nd days,all decreased significantly(P<0.01 or P<0.05).Compared with the stress group,on the 21st day,the total number of5-minute passes in the central area of the stress+XYS group increased significantly(P<0.05);on the 28th day,the 5-minute residence time in the central area of the stress+XYS group increased significantly(P<0.05);on the35th day,the total number of 5-minute passes in the central area of stress+XYS group increased significantly(P<0.01),while on the 42nd day,the total 5-minute moving distance,the total number of 5-minute crossing numbers and moving distance in the central area increased significantly(P<0.05).4.Sucrose consumption testOn day 0,7 and 14,there was no significant difference in sucrose consumption among groups(P>0.05).Compared with the control group,on the 21st,28th,35th and 42nd day,the sucrose consumption in the stress group decreased significantly(P<0.01);compared with the stress group,on the 28th,35th and 42nd day,the sucrose consumption in the stress+XYS group increased significantly(P<0.05 or P<0.01).5.Fecal water contentAt the end of the experiment,compared with the control group,the fecal water content in the stress group increased significantly(P<0.05);compared with the stress group,the fecal water content in the stress+XYS group decreased significantly(P<0.05).6.Serum D-xylose content in ratsCompared with the control group,the serum D-xylose content in the stress group decreased significantly(P<0.01);compared with the stress group,the serum D-xylose content in the stress+XYS group increased significantly(P<0.01).7.Small bowel transitCompared with the control group,the small bowel transit in the stress group was significantly lower(P<0.05);compared with the stress group,the small bowel transit in the stress+XYS group had an upward trend,but there was no significant difference(P>0.05).8.Detection of fecal metabolitesA total of 48 metabolites were obtained from rat feces by metabolomic analysis,mainly including of amino acids and the derivatives,amines and ammoniums compounds,organic acids,nucleic acid components,sugars,alcohols.PLS-DA multivariate statistical analysis was used to obtain 13different metabolites of acetate,propionate,butyrate,glucose,ribose,maltose,xylose,alanine,leucine,phenylalanine,tyrosine,valine,isoleucine.Compared with the control group,the concentrations of acetate,butyrate,glucose,ribose,alanine,leucine,xylose,phenylalanine,tyrosine,valine,isoleucine in the stress group were significantly lower(P<0.05 or P<0.01);compared with the stress group,the concentration of ribose,alanine,valine,isoleucine in stress+XYS group increased significantly(P<0.05).Conclusion:1.With chronic unpredictable mild stress for 6 weeks,the weight and food intake of rats decreased,the ability of autonomous behaviors in open-field experiments decreased,the sucrose consumption decreased,the propulsive rate of small intestinal carbon powder decreased,the content of D-xylose in serum decreased,the water content in feces increased,and the regulating effect on above indexes of Xiaoyaosan decoction,showed that the rat models appear liver depression and spleen deficiency syndrome.2.Chronic unpredictable mild stress can lead to the decrease or disorder of amino acid metabolism,sugar metabolism and short-chain fatty acid metabolism in rats with liver depression and spleen deficiency.The 13metabolites of acetate,propionate,butyrate,glucose,ribose,maltose,xylose,alanine,leucine,phenylalanine,tyrosine,valine and isoleucine in rat feces can be used as the small molecular compounds closely related to chronic unpredictable mild stress with liver depression and spleen deficiency.3.Xiaoyaosan decoction can regulate the concentration of ribose,alanine,valine and isoleucine in feces of chronic unpredictable mild stress model rats with liver depression and spleen deficiency.It is suggested that the anti-stress effect of Xiaoyaosan decoction on relieving liver depression may be related to the regulation of amino acid and sugar metabolism in vivo,and its specific metabolic pathway needs further study. |
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