Relationship Between CNP And BDNF In Brain And Gastrointestinal Tract Of FD Rats With Liver Stagnation And Spleen Deficiency Syndrome And Changes Of Intestinal Flora

Objective:To observe the expression of natriuretic peptide CNP and brain-derived neurotrophic factor BDNF in the gastrointestinal tract and hippocampus of rats with liver stagnation and spleen deficiency,and to explore their mutual use and Xiaoyao San intervention.At the same time,the diversity and distribution of intestinal flora in rats with liver stagnation and spleen deficiency were observed,and the intervention effect of Xiaoyao San was discussed.Methods:1.The model of liver stagnation and spleen deficiency was established by the solitary cage maintenance method and chronic unpredictable mild stimulation,and different doses of Xiaoyao suspension were administered by gavage.Through the behavioral performance and weight gain of the rats in the open field experiment,the sucrose preference experiment reflected the change of pleasure in rats after stress;the absorption function of the small intestine showed the urinary D-xylose excretion rate;the gastric residual rate and the small intestine propulsion experiment showed Identification of liver stagnation and spleen deficiency syndrome by changes in gastric emptying function.2.Western-blotting method was used to detect the expression of natriuretic peptide B receptor（NPRB）and BDNF protein in gastrointestinal tract and brain of FD rats with liver stagnation and spleen deficiency syndrome.Real-time PCR was used to detect the changes of gene expression and immunohistochemistry.The expressions of NP receptor and BDNF in gastric antrum,hippocampus and colon of FD rats were detected.The primary hippocampal neurons of normal SD rats were cultured in vitro within 24hours after birth.The cells were identified by immunofluorescence,hippocampal neurons were treated with CNP and BDNF,and the optimal effect of CNP and BDNF was detected by CCKit8 kit.Based on the concentration,the possible interaction mechanism between CNP and BDNF was explored.The expression of CNP and BDNF gene was detected by real-time polymerase chain reaction to further verify the interaction between CNP and BDNF,and whether CNP participates in the secretion of BDNF on hippocampal neurons.3.The feces of the model rats were taken and DNA was extracted.Primers amplify the variable V4 region of 16S rDNA.The amplified products were sequenced using Illumina technology and further analyzed for species diversity and distribution of fecal flora.Results:After the completion of the rat model test,the rats in the normal group were free-spirited,the hair was soft and moist,and the reaction was as usual.Compared with normal rats,the results of the model group showed that the body was thin,the hair was dry and dull,and there were stains around.The eyes were dark red,the stool was thin,and the arch back was curled into the corner,indicating that the modeling affected the big The general behavior of the rat.Compared with the weight of the rat model group,the high,middle and low groups of Xiaoyao San increased rapidly.The other groups of rats decreased in general in the open field test,and the preference for sugar and water decreased,indicating the rats’ability to explore behavior.Decrease,the responsiveness to rewards declines,the lack of pleasure is a typical characteristic of liver stagnation and spleen deficiency syndrome,and the D-xylose excretion rate rises,suggesting that the intestinal absorption of nutrient function is improved.The gastric residual rate was higher than that of the normal group,and the small intestine pushing rate was exactly the opposite,indicating that the gastric emptying ability and the pushing power of the small intestine decreased.Immunohistochemistry of paraffin sections showed that BDNF and NPR-B had brown granules in the hippocampal C3,C4 and apical processes,and positive staining was present in the cell membrane.It is also present in the cytoplasm and nucleus of basal cells of gastrointestinal smooth muscle.Western-blotting results showed that compared with the model of liver stagnation and spleen deficiency,there were significant differences in the expression of natriuretic peptide receptor B protein（NPR-B）and brain-derived neurotrophic factor in gastric antrum,colon and hippocampus of each group.（n=9,P<0.01）.The results of RT-PCR showed that the expression of the gene was consistent with that of Western.The expression of NPR-B gene in the tissues of the treatment group was adjusted to different degrees,and the expression level of BDNF was down-regulated.Xiaoyao San has a dose-dependent improvement effect on liver stagnation and spleen deficiency in FD rats.The mature rat primary cell technology was used to successfully extract the hippocampal neurons from the hippocampus,and the cells were identified by immunofluorescence chemistry.The positive cells were highlighted by fluorescent green and nucleus blue staining,that MAP-2 expression was positive.After treatment of mature hippocampal neurons with differential concentrations of CNP factors(10^-8,10^-7,10^-6mol/L),CNP promotion at concentrations of 10^-7mol/L Apoptosis of cells,while BDNF increases cell survival.RT-PCR results showed that after concentration of BDNF and CNP(10^-8,10^-7,10^-6M/ng/mL),hippocampal neurons were treated at concentrations of 10^-7mol/L.CNP can down-regulate the expression of BDNF.Sequencing results indicated that all samples included approximately 20 gates.Proteobacteria,thick-walled bacteria,Bacteroides,cyanobacteria and soft-walled bacteria are the five most abundant abundances.The relative abundance of the thick-walled bacteria in the feces of the model group was higher than that of the normal group,and the results were the same as those of the Proteobacteria;while the relative abundance of the cyanobacteria and the Bacteroides was lower,and the model group was found after the intervention of Xiaoyao Powder.The bacteria whose relative abundance changed were restored to the level of normal rats.The abundance heat map of the cluster analysis also showed the same results as the abundance analysis of the flora.Conclusions:CNP and BDNF were expressed in different degrees in the brain and gastrointestinal tract of FD rats with liver stagnation and spleen deficiency syndrome.CNP down-regulated the expression of BDNF,and Xiaoyao Powder could improve the expression of BDNF in a dose-dependent manner.Xiaoyao San has an intervention effect on the structural composition and distribution disorder of the intestinal flora of FD rats with liver stagnation and spleen deficiency.