| Objective Through the metabolomics analysis of serum and urine of rats with constipation induced by loperamide hydrochloride after administration of geixigune-3,combined with the effect of geixigune-3 on c-kit and SCF in colon tissue of rats with constipation,the mechanism of the drug in treating constipation was discussed.Methods SPF male SD rats were randomly divided into blank group,model group,geixigune-3 granules group,geixigune-3 decoction group and positive group.Except the blank group,rats in other groups were injected subcutaneously with loperamide hydrochloride to establish constipation model.After successful modeling,corresponding drugs were given for treatment,saline was given to the blank and model groups.The body weight,24h food and water consumption,24h urine volume,number,weight and water content of feces were observed.Urine and serum of rats were collected.The metabolomics of urine and serum was studied by ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry to find the related metabolites and metabolic pathways of geixigune-3.Western blot was used to detect the expression of c-kit and SCF protein in colon tissue of rats in each group;RT-PCR was used to detect the expression of c-kit and SCF m RNA in colon tissue of rats in each group.Results Weight analysis:Compared with the blank group,the body weight of rats in the model group decreased significantly(P<0.01);Compared with the model group,the weight of rats in each treatment group had no significant difference(P>0.05).Analysis of 24h food and water consumption:Compared with the blank group,the 24h food intake of model group rats decreased(P<0.01);Compared with the model group,the 24h food intake of rats in geixigune-3 granules group and positive administration group increased(P<0.05).Compared with the blank group,there was no significant difference in the amount of 24h drinking water in each group(P>0.05);Compared with the model group,the 24h drinking water volume of geixigune-3 granules group increased(P<0.05).Analysis of 24h urine volume,fecal weight,grain number and moisture content:Compared with the blank group,the 24h urine volume of the model group increased significantly(P<0.01);Compared with the model group,the 24h urine volume of rats in geixigune-3 granules and positive administration group decreased(P<0.05).Compared with the blank group,the 24-hour fecal weight of the model group decreased significantly(P<0.01);Compared with the model group,the 24-hour fecal weight of rats in each treatment group increased significantly(P<0.05or P<0.01).Compared with the blank group,the number of 24h fecal particles in the model group decreased significantly(P<0.01);Compared with the model group,the number of 24h fecal particles in each treatment group increased(P<0.05).Compared with the blank group,there was no significant difference in 24h fecal moisture content in the model group(P>0.05);Compared with the model group,the 24h fecal moisture content of rats in each treatment group increased significantly(P<0.01).Analysis of serum metabolomics of rats with constipation treated with Geixigune-3:Under the positive and negative ion modes,the PCA scores of serum samples from rats in the particle group and decoction group were,QC aggregation was good,indicating that the method established in the experiment had good repeatability and stability.R~2X=0.544 in the positive ion mode and R~2X=0.442 in the negative ion mode of the sample.It can be seen from the OPLS-DA score chart that the sample points of the particle group and the decoction group are both within the confidence interval,showing a trend of symmetrical distribution,good aggregation within the group,and significant separation between groups.8h sample positive ion mode R~2X=0.770,R~2Y=0.980,Q~2=0.943;Negative ion mode R~2X=0.717,R~2Y=0.989,Q~2=0.953.12h sample positive ion mode R~2X=0.882,R~2Y=0.969,Q~2=0.910;Negative ion mode R~2X=0.789,R~2Y=0.994,Q~2=0.953.Combining VIP>1,P<0.05,FC≥2 as the standard,the final screening identified 73differential metabolites at 8 hours and 55 differential metabolites at 12 hours.The metabolic pathways are glycerol phospholipid metabolism and sphingolipid metabolism.Analysis of urine metabolomics of rats with constipation treated with Geixigune-3:The PCA scores of urine samples of rats in each group were in positive and negative ion mode,and the QC aggregation was good,indicating that the method established in the experiment had good repeatability and stability.R~2X=0.893 in the positive ion mode and R~2X=0.745 in the negative ion mode of the sample.It can be seen from the OPLS-DA score chart that the sample points of the two groups of urine samples are located within the confidence interval,showing a symmetrical distribution trend,with good aggregation within the group and significant separation between groups.Comparing the blank group with the model group,the positive ion mode R~2X=0.822,R~2Y=0.990,Q~2=0.942;negative ion mode R~2X=0.549,R~2Y=0.961,Q~2=0.909.Comparing the particle group with the model group,the positive ion mode R~2X=0.804,R~2Y=0.985,Q~2=0.884;negative ion mode R~2X=0.488,R2Y=0.995,Q~2=0.823.Comparing the decoction group with the model group,the positive ion mode R~2X=0.836,R~2Y=0.988,Q~2=0.650;negative ion mode R~2X=0.961,R~2Y=1.000,Q~2=0.816.Comparing the particle group with the decoction group,positive ion mode R~2X=0.908,R~2Y=0.999,Q~2=0.753;negative ion mode R~2X=0.695,R~2Y=0.849,Q~2=0.612.With VIP>1,P<0.05,FC≥2as the criteria,the final screening identified 35 differential metabolites between the blank group and the model group,22 differential metabolites between the particle group and the model group,22 differential metabolites between the decoction group and the model group,and 520 differential metabolites between the particle group and the decoction group.and the metabolic pathway was tryptophan metabolism,steroid biosynthesis,ubiquinone and other terpenoid quinones biosynthesis.Western blot was used to detect the expression of c-kit and SCF protein in colon tissue of rats in each group:Compared with the blank group,the expression of c-kit protein in colon tissue of model group was significantly lower(P<0.01);Compared with the model group,the expression of c-kit protein in the groups treated with geixigune-3 granules and geixigune-3 decoction increased(P<0.05),and there was no significant difference in the positive treatment group(P>0.05).Compared with the blank group,the expression of SCF protein in the model group decreased significantly(P<0.01);Compared with the model group,the expression of SCF protein in the groups treated with geixigune-3 granules,geixigune-3 decoction and positive administration was significantly higher(P<0.01).RT-PCR was used to detect the expression of c-kit and SCF m RNA in colon tissue of rats in each group:Compared with the blank group,the expression of c-kit m RNA in colon tissue of the model group was significantly lower(P<0.01);Compared with the model group,the expression of c-kit m RNA was significantly increased in the group treated with geixigune-3 granules(P<0.01),and increased in the group treated with geixigune-3 decoction(P<0.05),but there was no significant difference in the positive treatment group(P>0.05).Compared with the blank group,the expression of SCF m RNA in the model group decreased significantly(P<0.01);Compared with the model group,the expression of SCF m RNA was significantly increased in the groups given geixigune-3granules and geixigune-3 decoction(P<0.01),and the expression of SCF m RNA was increased in the positive group(P<0.05).Conclusion:Administering Geixigune-3 can improve the general condition of constipation rats,and it can alleviate constipation through multiple pathways through serum and urine metabolism.The function of ICC may be regulated through the c-kit/SCF signaling pathway,thereby treating constipation. 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