Structural Analysis Of Polysaccharides From Atractylodis Rhizoma And Its Protective Mechanism On Alcoholic Bowel-liver Tissue Diseases

Objective: Alcohol is one of the top 10 risk factors for the global burden of disease.Excessive long-term alcohol consumption disrupts the normal functioning of the intestines,liver,and other organs of the digestive system,leading to the development of Alcoholic bowel disease(ABD)and Alcoholic liver disease(ALD).Plant polysaccharides have attracted the attention of researchers due to their pharmacological activities such as anti-inflammatory,antioxidant and bidirectional immunomodulation.Atractylodis Rhizoma is a traditional bulk medicinal herb in China,which has the efficacy of drying dampness,strengthening the spleen,dispelling wind and dispersing cold,and is widely used in the treatment of various digestive disorders.Atractylodis Rhizoma polysaccharide(AP),as the main medicinal component,its structural characteristics and protective effects and mechanisms on ABD and ALD are rarely reported.Therefore,in this study,the extraction,isolation,purification and structural analysis of polysaccharides from Atractylodis Rhizoma were carried out.The antioxidant and anti-inflammatory activities of AP were investigated in vitro.Based on Nrf2/HO-1,TLR4/MyD88/NF-κB and other signaling pathways,combined with microbiome and metabolomics techniques,the improvement effect and potential mechanism of AP on alcoholic intestinal and liver injury were discussed by using alcohol-induced intestinal and liver injury rat model with "gut-liver axis" metabolism as the main line.The aim was to elucidate the structural features and biological activities of AP and to provide a theoretical basis for the development of drug candidates for the prevention and treatment of ABD and ALD.Methods:1.Atractylodis Rhizoma crude polysaccharide was prepared using water extraction and alcohol precipitation and Sevag method,and the crude polysaccharide was purified by ion exchange and gel permeation chromatography to obtain Atractylodis Rhizoma polysaccharide(AP).AP was structurally characterized by ion chromatography(IC),ultraviolet spectroscopy(UV),Fourier transform infrared spectroscopy(FT-IR),gas chromatography-mass spectrometry(GC-MS),nuclear magnetic resonance(NMR)and scanning electron microscopy(SEM)techniques.2.The scavenging ability of AP against DPPH radicals,ABTS radicals,hydroxyl radicals and iron-reducing power were detected by using the kit method to verify its antioxidant activity in vitro.A macrophage RAW264.7 inflammation model induced by lipopolysaccharide(LPS)was established,and the effects of AP on the expression of TLR4/MyD88/NF-κB signaling pathway,inflammatory enzymes(iNOS and COX2),and the release of inflammatory cytokines(TNF-α,IL-1β,and IL-6)were examined by real-time fluorescence quantitative PCR(RT-qPCR),Western blot(WB),and kit assay to determine its in vitro Anti-inflammatory activity.3.Rat intestinal and liver injury models were constructed using alcohol,and the protective mechanisms of AP against alcoholic intestinal and liver injury were explored by kit,RT-qPCR,WB,immunofluorescence(IF),immunohistochemistry(IHC)with histopathological structures,inflammatory cytokines(TNF-α,IL-1β and IL-6),antioxidant enzymes(SOD,CAT and GSH-Px),liver injury marker enzymes(ALT and AST),alcohol metabolizing enzymes(ADH and ALDH),tight junction proteins(ZO-1 and Occludin),inflammatory pathway genes and proteins(TLR4,MyD88,TNFR1,NF-κB p-p65 and p-IkBα),oxidative stress pathway genes and proteins(Nrf2 and HO-1),and lipid metabolism pathway genes and proteins(AMPK,SREBP-1c,ACC,FAS,PPARα,and CPT-1)as the detection indexes,combined with 16 S rRNA high-throughput sequencing and non-target metabolomics.Results:1.Structural analysis of AP:(1)AP is a heteropolysaccharide with a heavy average molecular weight of 60.61 k Da,consisting mainly of arabinose and galactose.(2)Methylation and NMR analyses indicate that the probable repeating unit of AP consists of→3,6)-α-D-Galp-(1→ residues constituting the main chain,with a side chain ofα-L-Araf-(1→ attached to C-6 of the main chain.2.In vitro antioxidant and anti-inflammatory activities of AP:(1)AP has good antioxidant activity,with strong scavenging ability of DPPH radicals,ABTS radicals,hydroxyl radicals,as well as iron-reducing power.(2)AP has strong anti-inflammatory activity and is able to reduce the release of inflammatory enzymes(iNOS and COX2)and inflammatory cytokines(TNF-α,IL-1β and IL-6)and decrease the inflammatory response by down-regulating the expression of TLR4/MyD88/NF-κB signaling pathway in LPS-activated macrophages RAW264.7.3.Protective effect of AP against alcoholic intestinal injury:(1)AP can reduce alcohol-induced intestinal damage in rats and improve pathological changes such as epithelial cell atrophy,crypt disruption,mucosal muscular layer disruption,submucosal edema,and inflammatory cell infiltration in colonic tissues.(2)AP can reduce oxidative stress injury in rat intestine by activating Nrf2/HO-1 signaling pathway and maintaining the balance between ROS and antioxidant enzymes(SOD,CAT and GSH-Px).(3)AP reduces inflammatory cytokine release and decreases the inflammatory response in the rat intestine by inhibiting the TLR4/MyD88/NF-κB signaling pathway.(4)AP promotes the expression of tight junction proteins ZO-1 and Occludin,repairs intestinal mucosal damage,and reduces intestinal leakage of endotoxins such as LPS.4.Effects of AP on gut flora and metabolites:(1)AP ameliorates alcohol-induced intestinal microecological dysregulation and promotes the growth of potentially beneficial bacteria Lactobacillus＿taiwanensis,Limosilactobacillus＿reuteri and Akkermansia＿muciniphila.(2)AP promotes the production of potentially beneficial metabolites such as SCFAs,Indole-3-propionic acid,and N-Eicosapentaenoyl Tryptophan,and inhibits the production of potentially harmful metabolites such as Hexadecanedioic acid,Mephentermine,and Alectrol,etc.,by regulating amino acid metabolism,lipid metabolism and carbohydrate metabolism.5.Protective effect of AP on alcoholic liver injury:(1)AP was able to reduce alcohol-induced liver swelling,improve hepatocyte arrangement disorders,and reduce pathological changes such as fat vacuolization and inflammatory cell infiltration in rats.(2)AP may ameliorate alcoholic liver injury by decreasing the activity of liver injury marker enzymes(ALT and AST)and modulating the alcohol metabolizing enzyme(ADH and ALDH)systems.(3)AP may protect against oxidative stress damage by increasing the activities of SOD,CAT and GSH-Px in the liver,decreasing the level of ROS,and promoting the nuclear translocation of Nrf2 and the expression of HO-1.(4)AP reduces the release of TNF-α,IL-1β and IL-6 and attenuates the inflammatory response in the liver by inhibiting the TLR4/MyD88/NF-κB signaling pathway.(5)AP can regulate fatty acid synthesis or oxidation and reduce fat accumulation in the liver through the AMPK/SREBP-1c/ACC/FAS and AMPK/PPARα/CPT-1 signaling pathways.(6)Correlation analysis showed that the improvement of intestinal microecological imbalance and metabolic profile disorders by AP further repaired liver damage through the "gut-liver axis".Conclusion:1.AP is a novel arabinogalactan with a possible repeating unit structure main chain consisting of →3,6)-α-D-Galp-(1→,with a branched α-L-Araf-(1→ attached at the C-6position of the residue.2.AP has strong in vitro antioxidant and anti-inflammatory activity.3.AP could not only improve alcohol-induced oxidative stress injury and inflammatory response in rat intestine induced by alcohol by promoting Nrf2/HO-1 signaling and inhibiting the activation of TLR4/MyD88/NF-κB pathway,but also was able to repair intestinal mucosal injury and reduce intestinal leakage of LPS by promoting the expression of tight junction proteins ZO-1 and Occludin.4.AP can shape the structure of good gut flora by increasing the abundance of potential probiotics and improve the metabolic profile of rats through the regulation of amino acid metabolism,lipid metabolism and carbohydrate metabolism.5.AP can regulate Nrf2/HO-1,TLR4/MyD88/NF-κB,AMPK/SREBP-1c/ACC/FAS,and AMPK/PPARα/CPT-1 signaling pathways via the "gut-liver axis",and ameliorate alcohol-induced oxidative stress,inflammation,and lipid metabolism disorders in rat liver.In general,this study shows that AP has a good effect on reducing alcoholic "gut-liver axis" tissue damage and is a promising prebiotic.