| Non-alcoholic steatosis(NAFLD)is a kind of metabolic disease with complex pathogenesis,which has caused a serious impact on human health.However,no drugs can be used to treat NAFLD directly.It is known that endoplasmic reticulum stress-mediated inflammation can further aggravate non-alcoholic fatty liver disease.Polysaccharides are proven to have multiple biological activities,especially for their prominent efficiency in antiinflammatory.Tangerine peel is one of the most important medicinal resources in our country,but the therapeutic effect of Tangerine peel polysaccharide(DTPP)on NAFLD has not been reported.In this thesis,DTPP was extracted and characterized,and the efficiency of DTPP on anti-inflammatory and anti-endoplasmic reticulum stress was explored both in vitro and in vivo.First,mouse macrophages RAW264.7 was stimulated by LPS+IFN-γ as the inflammatory cell model,pro-inflammatory specific markers such as TLR4,CD11 c,and i NOS were analyzed by flow cytometry,and the secretion of NO,IL-6,and TNF-α in culture medium was also determined.The expression levels of Chop,GRP78,and xbp1 s genes were analyzed by realtime fluorescence quantitative PCR.THLE-2 cells induced by sodium oleate were used as a lipid accumulation cell model to explore the alleviating effect of DTPP on cellular lipid accumulation and the regulatory effect on endoplasmic reticulum stress-related genes.A highfat feed-induced NAFLD zebrafish model was constructed to evaluate the alleviating effect of DTPP on hepatic steatosis,and the liver of NAFLD zebrafish was extracted for gene transcriptome analysis to explore its potential pharmacological mechanism.Results:(1)The total sugar content of DTPP was 89.04%,the total protein content was 0.193%,and the molecular weight was 98.001 k Da.The polysaccharide linking mode of DTPP were[Araf-(1→],[Arap-(1→],[→3)-Araf-(1→],[→5)-Araf-(1→],[Galp-(1→],[→3,5)-Araf-(1→],[→4)-Galp-(1→],[→4)-Glcp-(1→],[→3)-Galp-(1→],[→6-Glcp-(1→],[→6)-Galp-(1→],[→4,6)-Galp-(1→] and [→3,6)-Galp-(1→].DTPP monosaccharide composition was rhamnose,arabinose,galactose,glucose,and galacturonic acid,the corresponding molar ratio was0.029:0.168:0.137:0.019:0.647.(2)According to the cell proliferation assay,DTPP significantly inhibited the survival rate of macrophages at the concentration of 80 μg/m L but had no toxic effect on macrophages in the concentration range of 10 to 40 μg/m L.DTPP could significantly reduce the content of macrophage pro-inflammatory cytokines such as NO,TNF-α and IL-6,and inhibit the expression of TLR4,CD11 c and i NOS.The expression of genes Chop,xbp1 s,GRP78,and inflammatory factor-related genes TNF-α,IL-6 and i NOS was down-regulated,and the expression of NF-κB and IRE1α-related proteins was attenuated.(3)DTPP had no obvious toxic effect on human normal hepatic THLE-2 cells in the concentration range of 10-40 μg/m L.DTPP can effectively reduce the production of lipids in THLE-2 cells.At the same time,the gene expression of IRE1α,GRP78 and xbp1 s was decreased.(4)DTPP could effectively inhibit liver lipid accumulation and liver macrophage aggregation in zebrafish with high-fat diet-induced NAFLD.The zebrafish liver transcriptome analysis showed that DTPP inhibited liver lipid accumulation and macrophage accumulation,which was related to inflammation and endoplasmic reticulum stress-related pathways.Conclusion:(1)The monosaccharide composition of DTPP was rhamnose,arabinose,galactose,glucose,and galacturonic acid.The monosaccharide component with the largest proportion was arabinose.(2)DTPP could inhibit ER stress-related inflammatory response in macrophages by interfering with LPS/TLR4 signaling pathway,and reduce endoplasmic reticulum stress and lipid accumulation in hepatocytes.(3)DTPP could reduce lipid accumulation and macrophage aggregation in the liver of zebrafish,and finally alleviate liver steatosis. |
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