| Objective:To investigate the therapeutic effect of Zhizhu Pill on STC model mice with spleen deficiency syndrome,to preliminarily investigate whether the therapeutic effect of Zhizhu Pill on STC is related to the regulation of colonic bile acids and TGR5 receptors to promote 5-HT release,and to preliminarily investigate whether the effect of Zhizhu Pill on bile acids is achieved through the regulation of intestinal flora.Method:The STC mice model with spleen deficiency was replicated by the compound factor of "administration of senna leaf decoction + low-fiber diet and water restriction on alternate days",and the treatment was carried out by gavage of low,medium and high doses of Zhizhu Pill decoction.The general condition and fecal condition of the mice were observed,and their body weight,fecal quality and fecal water content were statistically analyzed,and the intestinal propulsion rate of the mice was detected by the ink test;the 5-HT content in the serum and colon of each group of mice was detected by ELISA;the bile acid content in the colon contents of each group of mice was analyzed by Liquid Chromatography Mass Spectrometry;the expression of tgr5 mRNA in the colon of each group of mice was detected by q PCR.Western blot was used to detect the expression of TGR5 protein in the colon of each group of mice;Pearson correlation analysis was used to analyze the correlation between 5-HT and constipation efficacy evaluation index and bile acid changes;fluorescence co-localization method was used to analyze the correlation between 5-HT expression and TGR5 expression;16Sr DNA amplicon sequencing was used to analyze the changes of bacterial flora in the colon of each group of mice,Pearson Correlation analysis was performed to correlate the changes of bile acids with the changes of bacterial flora.Results:1.Zhizhu Pill improved the general status of STC mice model with spleen deficiency:mice in the spleen deficiency STC group showed signs of spleen deficiency and constipation,such as emaciation,reduced activity,weakness of limbs,unresponsiveness,hard stools,small quantity and small pellets;mice in the low,medium and high dose groups of Zhizhu Pill showed significantly better general status than mice in the spleen deficiency STC group,with shiny hair,fuller appearance,gradually increasing activity,gradually strong limbs,responsive,moister stools,larger pellets and more stools.The mice in the low,middle and high dose groups showed significant improvement in general condition compared with the mice in the spleen deficiency STC group.2.Zhizhu Pill alleviated the weight loss of STC mice model with spleen deficiency: after self-modeling,the weight of mice in control group increased gradually,and the weight of mice in model group decreased gradually.The weight of mice in Zhizhu Pill low-dose,medium-dose and high-dose groups decreased gradually 4 weeks before administration,and increased 4 weeks after administration,showing a trend of decreasing first and then increasing.After administration,compared with the control group,the body weight of model group decreased significantly(P<0.05),and Zhizhu Pill low-dose,medium-dose and high-dose groups could alleviate the body weight decline of mice in the spleen deficiency STC to different degrees(P>0.05).3.Zhizhu Pill significantly increased the fecal quality,fecal water content and intestinal propulsion rate of mice with spleen deficiency STC model: compared with the control group,the fecal quality,fecal water content and intestinal propulsion rate of mice with spleen deficiency STC group significantly decreased(P<0.01),compared with the spleen deficiency STC group,the fecal quality,fecal water content and intestinal propulsion rate of mice with Zhizhu Pill low dose group significantly increased(P<0.01),and the fecal water content and intestinal propulsion rate of mice with spleen deficiency STC group significantly increased(P<0.01).In the middle dose group of Zhizhu Pill,there was a significant increase in fecal mass,fecal water content and intestinal propulsion rate(P<0.05),and in the high dose group of Zhizhu Pill,there was an increase in fecal mass and fecal water content(P>0.05)and intestinal propulsion rate(P<0.01)4.Zhizhu Pill significantly increased the contents of 5-HT in colon and serum of STC model mice with spleen deficiency: compared with the control group,the contents of 5-HT in colon and serum of model group were significantly decreased(P<0.05);Compared with model group,5-HT content in colon and serum of mice in Zhizhu Pills low-dose,medium-dose and high-dose groups was significantly increased(P<0.05).5.Zhizhu Pill significantly increased the bile acid content in the colonic contents of mice with spleen deficiency STC model,and the increase of colonic and serum 5-HT content and improvement of constipation efficacy indexes were correlated with the increase of bile acid content: compared with the control group,the total bile acid content in the colonic contents of mice with spleen deficiency STC group was significantly reduced(P<0.01),and compared with the spleen deficiency STC group,the total bile acid content in the colonic contents of mice with Hovenia pill low dose group was significantly increased(P<0.05).The total bile acid content in the colon contents of mice in the low-dose group of Citrus aurantium pill was significantly increased compared with that in the STC group with spleen deficiency(P<0.05).There were significant differences in the contents of various bile acids between the control group,the spleen-deficiency STC group and the Zhizhu Pill low-dose group,and the most significant differences were found between the groups in the contents of HDCA,DCA,β-MCA,Apo CA,α-MCA,12-KLCA,7-KDCA and CA.HDCA,DCA,β-MCA,Apo CA,α-MCA in the colonic contents of mice in the spleen deficiency STC model group were significantly down-regulated compared with the control group(P<0.05),while HDCA,DCA,β-MCA,Apo CA,α-MCA in the colonic contents of mice in the Zhizhu Pill low-dose group were significantly up-regulated compared with the mice in the spleen deficiency STC group(P<0.05).β-MCA,α-MCA,12-KLCA,and CA changes were positively correlated.6.Zhizhu Pill significantly increased the expression of tgr5 mRNA and TGR5 protein in colon of STC model mice with spleen deficiency,and the increase of colon 5-HT was correlated with the increase of colon TGR5 protein: q PCR and WB results showed that the expression levels of tgr5 mRNA and TGR5 protein in colon tissue of model mice were significantly decreased compared with the control group(P<0.05).Compared with model group,the expression levels of tgr5 mRNA and TGR5 protein in the colon tissues of mice in Zhizhu Pill low-dose,medium-dose and high-dose groups were significantly increased(P<0.05).Fluorescence co-location results showed that 5-HT and TGR5 had a strong co-location relationship.7.The effect of Zhizhu Pill on bile acids was related to the regulation of intestinal flora:16Sr DNA sequencing results showed that the abundance of bile acid metabolism-related flora-Bacilli,Lactobacillales,Lactobacillaceae Lactobacillus were down-regulated in the model group and up-regulated after Zhizhu Pill gavage treatment,and were positively correlated with 12-KLCA,DCA,Apo CA,α-MCA,HDCA,β-MCA,and CA.Conclusion:1.Zhizhu Pill can improve the general status of spleen deficiency and constipation in STC model mice with spleen deficiency,alleviate the body weight reduction,and increase the stool quality,stool water content and intestinal propulsion rate in STC model mice with spleen deficiency,which has therapeutic effects on spleen-deficient STC mice.2.Colonic 5-HT was decreased in STC model mice with spleen deficiency,and Zhizhu Pill could improve the decreased colonic 5-HT in spleen-deficient STC mice,which was related to the regulation of colonic bile acids and TGR5 receptors.3.Bile acid metabolism-related flora-Bacilli,Lactobacillales,Lactobacillaceae Lactobacillus were down-regulated in the model group and up-regulated after Zhizhu Pill gavage treatment,and were positively correlated with 12-KLCA,DCA,Apo CA,α-MCA,HDCA,β-MCA,and CA. |
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