| Objective: In this study,streptozotocin(STZ)combined with high-fat and high-sugar diet was used to establish the rat model of type 2 diabetes mellitus(T2DM),and the active Pseudostellariae polysaccharide(PF40)was given ingavage To study the mechanism of pseudostellariae polysaccharide on improving intestinal immunity in T2 DM rats.This study provides a theoretical basis for the clinical application of Pseudostellariae polysaccharide in the anti-T2 DM prescription,and provides a new perspective of "intestinal immune system regulation" for the mechanism of TCM prevention and treatment of T2 DM.Methods: 1.The PF40 was prepared by hot water extraction and alcohol precipitation classification method.The monosaccharide content of PF40 polysaccharide was determined by PMP-HPLC derivatization.The physicochemical properties of the Pseudostellariae polysaccharide were preliminaries studied by infrared spectroscopy,nuclear magnetic resonance,ultraviolet spectroscopy,Congo red experiment,thermogravimetric analysis,Zeta-potential and Sem analysis.2.Type 2 diabetic rat model was established by intraperitoneal injection of STZ combined with high-fat and high-sugar diet,The effects of Pseudostellariae polysaccharide on fasting blood glucose,serum insulin,oral glucose tolerance and insulin resistance index(HOMA-IR)of rats were studied by gavage.3.The pathological structure of intestinal tissue was observed by HE staining and the changes of innate immune cells were counted to study the effects of PF40 on the structural structure of intestinal tissue and innate immune cells of rats.4.The apoptosis of jejunum cells was detected by TUNEL method,the ultrastructure of jejunum cells was observed by transmission electron microscopy,the proportion of Th17 and Treg cells in JEjunum T cell subsets was determined by flow cytometry,and the expression of RORγt and Foxp3 proteins,key transcription factors of Th17/Treg cell differentiation,were detected by Western blot.The contents of SIg A and Ig A in jejunum were measured by elisa,and the effect of PF40 on the proportion balance of Th17/Treg cells in JEjunum of T2 DM rats and its mechanism was studied.Results: 1.PF40 was prepared by water extraction and alcohol precipitation classification method,and the yield was 18.63%.The purity of PF40 was 79.85% The average content of glucose and galactose in PF40 polysaccharide was 84.06% and 2.50% respectively.by phenol sulfuric acid method.PF40 has no obvious absorption peak at 260 nm and 280 nm in uv spectrum,indicating that PF40 contains no protein and nucleic acid.Ir and NMR analysis showed that α-pyranose was present in the monosaccharide residues of PF40.Congo red experiment results show that the maximum absorption wavelength of PF40-Congo red mixed solution in the concentration range of 0 ～ 0.5 mol/L Na OH occurs red shift,the concentration reaches 0.04 mol/L after blue shift,the concentration is greater than 0.2 mol/L,indicating that PF40 contains three helix structure.The Zeta potential of PF40 was-31.61 m V.Thermogrgravity analysis showed that PF40 experienced two times of weight loss.The first time was between 30 °C and 168°C with a weight loss rate of 6.52%,and the second time was between 168°C and 450°C with a weight loss rate of 57.58%.PF40 experienced one endothermic reaction and two exothermic reactions with an endothermic reaction of 276.5°C.The peaks of the two exothermic reactions were 96.6°C and 217.7°C,respectively.At low magnification,the surface of PF40 was rough and flaked,while at high magnification,the surface of polysaccharide was smooth.2.After 45 days of PF40 intervention with an effective dose of 1.5g/kg,PF40 polysaccharide significantly reduced fasting blood glucose,serum insulin and HOMA-IR compared with the model group;The glucose tolerance of rats was increased(P < 0.05).3.Compared with model group,PF40 significantly increased the goblet cell and epithelial lymphocyte numbers in duodenum,jejunum,ileum and colon,and decreased the lymphocyte density in lamina proper(P < 0.05).In addition,PF40 significantly increased the length and V/C value of jejunum in T2 DM rats(P < 0.01).4.The detection results of jejunum cell apoptosis showed that PF40 could inhibit the apoptosis of jejunum cells induced by STZ.The ultrastructure of jejunum cells was further observed by transmission electron microscopy.Flow cytometry results showed that PF40 could significantly reduce the proportion of Th17 cells and Th17/Treg value in jejunum of model rats,increase Foxp3 protein expression,and increase the content of SIg A and Ig A in jejunum(P < 0.01).Conclusion: 1.PF40 polysaccharide is mainly composed of glucose,which is a kind of α-pyranose with three-strand spiral structure and has good stability,providing theoretical reference for the further development and utilization of Pseudostellariae polysaccharide.2.PF40 is involved in the regulation of glucose metabolism in T2 DM model rats,and can significantly improve STZ-induced pathological injury of intestinal tissue,restore the density of natural immune cells in the small intestine,and reduce jejunum cell apoptosis.PF40 is beneficial to the balance restoration of Th17/Treg cells in jejunum,and its mechanism may be related to Foxp3 protein. |
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