Protective Effects And Mechanism Of Tongxieyaofang Polysaccharides On Liver Depression And Spleen Deficiency Mice

Post-weaning diarrhea is common in clinical breeding of piglets and rabbits,and it is the first major challenge that breeders face.Due to its high morbidity and mortality,and the conflicting views on its pathogenicity.And there is no consensus on its treatment,which has resulted in significant economic losses for breeders.In the past,antibiotics were widely used for prevention and treatment,but with the current restrictions on the use of antibiotics,there is an urgent need for new alternative drugs.In this study,post-weaning diarrhea in piglets and rabbits was diagnosed as liver depression and spleen deficiency syndrome through traditional Chinese veterinary medicine,and based on the clinical manifestations,the classic formula Tongxieyaofang was deemed the most suitable treatment for this syndrome.Polysaccharides are biologically active macromolecules that are widely used in clinical practice for disease prevention.Therefore,this study extracted,purified and characterized the polysaccharides from Tongxieyaofang,and established a model of liver depression and spleen deficiency in post-weaning mice through chronic unpredictable stress.The mechanism of the development of liver depression and spleen deficiency syndrome in mice and the protective effect of polysaccharides from Tongxieyaofang were explored through the brain-gut-microbiota axis,with the aim of providing a theoretical basis for the clinical prevention and treatment of post-weaning diarrhea.1.Separation,purification,and structural characterization of Tongxieyaofang polysaccharidesIn this chapter,crude polysaccharides were obtained from Tongxieyaofang using a water-alcohol precipitation method.The polysaccharides were then separated and purified using DEAE-cellulose and Sepharose 6FF,resulting in the neutral polysaccharide(TXYF-NP)and acidic polysaccharide(TXYF-AP).The sugar content of TXYF-NP and TXYF-AP was 73.5% and 83.4%,respectively,and the polyphenol content of TXYF-AP was 1.6%.The structures of the two polysaccharides were characterized using size exclusion chromatography combined with multi-angle laser light scattering,high-performance liquid chromatography,gas chromatography-mass spectrometry,and nuclear magnetic resonance(NMR).The results showed that TXYFNP was mainly composed of fructose and had a typical inulin structure,with an α-1,4-Glc chain and a weight-average molecular weight of 3.56 k Da.TXYF-AP was a pectin polysaccharide that contained a main chain of homogalacturonan,side chains of rhamnogalacturonan and arabinogalactan(AG-I or II),with a β-1,4-Glc chain.And the weight-average molecular weight was 64.541 k Da.2.Effects of Tongxieyaofang polysaccharides on 5-HT metabolism and Glu-GABA receptors in liver depression and spleen deficiency miceThis chapter aimed to investigate the effects of Tongxieyaofang polysaccharides on depressive-like behavior in liver-depressed spleen-deficient mice induced by chronic unpredictable stress.Ninety-six 3-week-old male C57BL/6 mice were randomly divided into eight groups(n=12): Control group,Model group,low-,medium-,and high-dose TXYF-NP groups(50,100,and 200 mg/kg/day),TXYF-AP group(100mg/kg/day),NP+AP group(100 mg/kg/day,NP:AP=5:2),and FOS+GOS group(100mg/kg/day,FOS:GOS=4:3).Polysaccharide solutions were administered to each group daily.After 2 h of treatment,except for the control group,all other groups underwent 4weeks of chronic unpredictable stress to induce a liver-depressed spleen-deficient model.Real-time fluorescence quantitative PCR,western blotting,and ELISA were used to investigate the effects of Tongxieyaofang polysaccharides on 5-HT metabolism and Glu-GABA receptors.The results showed that chronic unpredictable stress induced slow growth,lack of pleasure,and depressive-like behavior in the model group.Serum levels of adrenocorticotropic hormone and corticosterone were significantly increased in the model group,and the expression of Crhr1,Nr3c1,and Nr3c2 genes in the hypothalamus was upregulated,while their expression in the hippocampus was downregulated,and histopathological changes were observed in the hippocampus.That indicating chronic unpredictable stress activated the hypothalamic-pituitary-adrenal axis and impaired the hippocampal negative feedback regulation.Except for Gria1,the expression of Glu/GABA-related receptors in the hippocampus of the model group was downregulated,and serotonin metabolism disorder was mainly manifested by an increase in colonic serotonin content and a decrease in hippocampal serotonin content.These findings suggest that chronic unpredictable stress may induce depressive-like behavior in liver-depressed spleen-deficient mice by damaging hippocampal synaptic transmission and serotonin metabolism.Tongxieyaofang polysaccharides showed protective effects against stress-induced damage,with the most significant effect observed in the NP-M group.3.Effects of Tongxieyaofang polysaccharides on gut microbiota and metabolites in in liver depression and spleen deficiency miceIn this chapter,we investigated the pharmacological mechanism of Tongxieyaofang polysaccharides on gut microbiota and metabolites in in liverdepressed spleen-deficient mice using 16 S r DNA sequencing and untargeted metabolomics.Results showed that: CUS caused gut microbiota dysbiosis in mice,with a decrease in relative abundance of Firmicutes and an increase in Bacteroidetes,a significant decrease in the dominant genus Lactobacillus,and a significant increase in the family Muribaculaceae.And CUS caused disturbance in gut metabolites of mice,with enriched metabolic pathways mainly involved in protein digestion and absorption,amino acid biosynthesis,bile metabolism,and steroid hormone biosynthesis.Ppolysaccharides from different groups had varying effects on gut microbiota in mice with liver depression and spleen deficiency,with high-dose neutral polysaccharides showing the best effect in restoring the relative abundance of Lactobacillus,while medium-dose neutral polysaccharides could increase the relative abundance of Lachnospiraceae and Ruminococcaceae,and all neutral polysaccharides could increase the relative abundance of Bacteroides.And TXYF-NP could protect against disturbance in bile metabolism and steroid hormone biosynthesis caused by CUS,and correlation analysis showed that this effect may be related to the increase in relative abundance of Lactobacillus and Bacteroides.4.Effects of Tongxieyaofang Polysaccharides on Intestinal Mechanical Barrier in liver depression and spleen deficiency miceIncreased intestinal permeability can lead to diarrhea and depression.This chapter investigated the effects of chronic unpredictable stress on the mechanical barrier of the colon in mice and the protective effects of Tongxieyaofang polysaccharides on it,using real-time fluorescent quantitative PCR and protein imprinting technology.The results of the study indicate that CUS led to an increase in serum LPS levels in mice.Moreover,it caused apoptosis and necrosis in the colonic epithelial cells of mice,which suggests a potential increase in colon permeability.This effect could be attributed to the activation of the TLR4/NF-κB signaling pathway.Tongxieyaofang polysaccharides could protect against the increase in colon permeability induced by CUS,with the most significant effect observed in the NP-M group.5.Effects of Tongxieyaofang polysaccharides on reproductive function in in liver depression and spleen deficiency miceDue to the homology of liver and kidney,abnormal liver drainage function will inevitably lead to problems in kidney storage function.In this chapter,the effects of Tongxieyaofang polysaccharides on reproductive function of mice with liver depression and spleen deficiency induced by CUS were explored through ELISA,RT-q PCR and other methods.According to the results,CUS resulted in a decrease in testicular weight in mice but an increase in the testicular organ index,sperm concentration,and survival rate.Additionally,CUS led to an increase in serum levels of follicle-stimulating hormone,luteinizing hormone,and testosterone in mice.The mechanism of action may be linked to the hypothalamic-pituitary-gonadal axis.TXYF-NP could prevent the hyperfunction induced by CUS in mice,and the optimal effect was achieved with a moderate dose of neutral polysaccharides.In conclusion,CUS caused hyperfunction of the HPA axis in mice,disorder of tryptophan metabolism,and disorder of gut microbiota and its metabolites.Disordered gut microbiota led to an increase in the number of gram-negative bacteria and excessive production of LPS,which damaged the mechanical barrier of the colon.Hyperfunction of the HPA axis interfered with the HPG axis,leading to hyperfunction of reproductive function in mice.TXYF-NP mainly consist of inulin,which can improve the depressive-like behavior,damage to the colon mechanical barrier,and hyperfunction of reproductive function induced by CUS by improving the gut microbiota and increasing the relative abundance of potential probiotics.TXYF-AP mainly consist of pectin polysaccharides,which can improve damage to the colon mechanical barrier by their anti-inflammatory and antioxidant activity,and may improve depressive-like behavior.