The Effect And Mechanism Of Pueraria Lobata Starch On Alleviating Ulcerative Colitis And Non-alcoholic Fatty Liver Disease In Mice

Objective:The dried root of Pueraria lobata（Willd.）Ohwi（Family Leguminosae）is an edible food.In generally,isoflavones,including puerarin,daidzin,daidzein and their derivatives,are the main bioactive components of P.lobata root,which display activities of antioxidant,anti-inflammation,anti-tumor and anti-diabetes.However,P.lobata root is also enriched with starch constituents.Although P.lobata starches（PLS）as a potential functional food ingredient are also popularly consumed,however,the activity of PLS is scarcely investigated.Thus,our study aimed to investigate the structural and physiochemical characteristics of PLS,and determined the effect of PLS on gut microbiota.And dextran sulfate sodium（DSS）and high-fat diet（HFD）were used to induced the murine model of Ulcerative colitis（UC）and Non-alcoholic fatty liver disease（NAFLD）,respectively.Then,to explore the effect of Pueraria Lobata starch（PLS）on UC and NAFLD in mice,and the mechanism of gut microbiota.Methods:（1）The structural and physiochemical characteristics of PLS were determined by the Scanning electron microscopy（SEM）,¹H NMR and ¹³C NMR,Fourier transform infrared spectrometer（FT-IR）,High performance anion exchange chromatography（HPAEC）and other detective techniques.（2）The effects of PLS on the composition of gut bacteria and SCFAs metabolism were investigated by mouse fecal fermentation in vitro,via the methods of 16S r RNA sequencing and GC-MS,and the p H value of culture system after incubation was recorded.（3）The SPF C57BL/6J mice were randomly divided into two groups（n=10per group）,including control group and PLS group.The PLS group were gavaged with 200 mg/kg of gelatinized PLS daily for 21 days,and the control group mice were orally treated with distilled water accordingly.In order to determine the effect of PLS on gut microbial structure,the fresh mouse feces were collected to analysis the change of gut microbial composition.（4）A total of 40 mice were randomly divided into four groups:Ctrl group,DSS group,DSS+5′-ASA treatment group,DSS+PLS treatment group.The mice in PLS group were oral gavaged with gelatinized PLS（200 mg/kg）once daily for 21 days prior to DSS challenge.Subsequently,8-day DSS（3%w/v）challenge.Meanwhile,the 5′-ASA（200mg/kg）was given to the positive control group.The fresh feces,blood samples and colon tissues of mice were collected respectively.The colonic damages and the number of goblet cell were observed by H&E and Alcian blue/periodic acid-Schiff（AB/PAS）staining,the level of serum inflammatory factors and myeloperoxidase（MPO）were determined by ELISA,the pression of NF-κB p65 and IL-1βwere analyzed via Western Blot,the connected proteins of colon and colonic inflammatory cells were observed through immunohistochemistry（IHC）and immunofluorescence（IF）staining,and the gut microbial composition was analyzed via 16S r RNA sequencing.Next,to investigate whether the ameliorative effect of PLS on DSS-induced colitis was associated with modulation of intestinal microbiota,mice were administered with broad-spectrum antibiotic treatment to deplete gut microbiome.（5）The mice were randomly divided into four groups（n=6 per group）,including ND group as a control group,HFD group,silymarin treatment group as a positive control,and PLS treatment group.The high-fat high-cholesterol diet（HFD）was used to induce the NAFLD mice for 8 weeks.The mice in silymarin group were gavaged with 100 mg/kg of the silymarin every other day for 8 consecutive weeks.PLS group were orally treated with 400 mg/kg of gelatinized PLS every day for 8 consecutive weeks.On day 56,the samples were further collected.The biochemical indices in serum were determined by corresponding kits via ELISA,H&E,Masson and oil red O staining to analyze the degree of inflammation,hepatic steatosis and dyslipidemia in colon.（6）To examine the main active ingredients of PLS,the amylose and amylopectin fractions of Pueraria lobata were isolated and purified by Schoch’s method,and then their protective effect was evaluated in colitis mice and compared with PLS.Results:（1）PLS consisted of mixed population of granules with polyhedral or spherical surface,displaying a bimodal size distribution within 0.6-30μm.It showed a trimodal distribution of different molecular weight peaks.The apparent content of resistant starch was 23.14%,with amylose fraction of 18.2%.And the molecular weight was 1.93×10⁷ Da.PLS showed a branching degree and an average polymerization rate of 2.06%and 20.74%,respectively,with fairly high proportion of B₁ short chains.PLS had a high crystallinity degree of 37.76%and consisted of C-type starch,and the solubility and swelling power of PLS were 38.51%and28.10 g/g,respectively.PLS showing high hot stability of the viscosity which gelatinized at 64.46-79.61 ^oC,with a high range of gelatinization（15.15 ^oC）and high enthalpy（13.98 J/g）.（2）In vitro fermentation of PLS resulted in specifically altered composition of gut microbiota and increased production of SCFAs.Fermentation with 5%PLS significantly promoted the growth of Lactobacillus while significantly inhibited Escherichia-Shigella,Enterococcus and Desulfovibrio.PLS increased the contents of acetic acid,butyric acid,valeric acid,isovaleric acid and hexanoic acid were significantly promoted,and the p H value of the 5%PLS group was significantly lower than the control group.Moreover,at the genus level,a21-day supplementation of PLS was significantly increased the abundance of beneficial bacteria in normal mice,such as Lactobacillus,Dubosiella and Lachnospiraceae＿NK4A136＿group.（3）Compare to the DSS group,PLS treatment significantly alleviated DSS-induced murine weight loss,decreased DAI scores inhibited colon lengths,reduced histological scores of colon,increased the number of goblet cells,reversed the level of serum IL-1β,IL-6,TNF-αand MPO,decreased the expression of the NF-κB p65and IL-1βat protein level,maintaining barrier function via recovering ZO-1 and occludin expressions,regulated CD8⁺T cells,CD86⁺macrophages and Ly6G⁺neutrophils in colitis mice.Moreover,PLS regulated relative abundance of norank＿f＿＿Muribaculaceae,Bifidobacterium,Lactobacillus and Dubosiella etc.,and increased the production of acetic acid,propionic acid,butyric acid and valeric acid.The protective effect of PLS against colitis was in a gut microbiota-dependent manner.（4）PLS remarkably reduced body weight,reduced the level of serum IL-6 and TNF-α,alleviated histological damages,inhibited serum ALT,AST,TC,TG and LDL-C while up-regulated serum HDL-C in mice with high-fat high-cholesterol diet induced NAFLD.Besides,PLS significantly regulate gut microbial structure,which increased the abundance of beneficial bacteria,such as Lactobacillus,Bifidobacterium and Turicibacter,while inhibited potentially harmful ones,Desulfovibrio.（5）PLA significantly reduced the weight changes,increased DAI scores,higher survival rates,shorter colon lengths,histological changes and the increased the level of serum IL-6 and TNF-αin DSS-induced colitis,and the protective effect of PLA was comparable compared to the PLS.It was demonstrated that amylose fraction but not amylopectin was responsible for the prebiotic effect of PLS.Conclusion:（1）PLS had a regulate effect on gut microbiota.And PLS attenuated DSS-induced colitis in a manner dependent on gut microbiota.（2）PLS could alleviate inflammation,hepatic steatosis and dyslipidemia in NAFLD mice,and ameliorate HFD-mediated gut dysbiosis.（3）Notably,the amylose fraction of PLS was responsible for the prebiotic effect of PLS,our study suggested a potentially new application of PLS and its amylose as new functional foods for the prevention and treatment of UC or NAFLD.