| As a form of liver injury caused by long-term intake of a high fat diet,the incidence of nonalcoholic fatty liver disease(NAFLD)increased year by year,and had seriously threatened human health.However,pathogenesis and clinical drugs for the treatment of NAFLD have not been clarified so far.Therefore,exploring the pathogenesis of NAFLD is conducive to providing new ideas and means for the prevention and treatment of NAFLD.Stropharia rugoso-annulata,an edible mushroom with nutrient-rich,which was called the "meat among vegetables".S.rugoso-annulata polysaccharides(SRP)showed potential effects of hypoglycemic and lipid lowering,antioxidant and anti-inflammatory activities.However,acetylated S.rugoso-annulata polysaccharides(ASRP)has not been reported so far.In the present work,ASRP was prepared by acetylation of SRP,and the structure of ASRP was analyzed and its antioxidant activity in vitro was detected.Moreover,the NAFLD mice models induced by high-fat diet(HFD)were successfully established,the biological mechanism of ASRP alleviating liver injury of NAFLD was explored based on the biological functions of antioxidation,anti-inflammation and anti-hyperlipidemia,in order to provide a new direction for the development of future research on hepatoprotective drugs.The main results were as follows:(1)The optimum conditions for acetylation modification were acetic anhydride 3 m L,reaction time 3 h,reaction temperature 35℃,under these conditions,the highest acetyl substitution degree was 0.59±0.012.(2)The structural analysis results showed that ASRP was a homogeneous β-pyranose,rich in mannose and galactose,in a three-dimensional helical conformation with the characteristic absorption peak of acetyl group,and stable structural characterization.(3)In vitro test results showed that ASRP had good antioxidant activity in vitro.(4)ASRP could significantly alleviate liver injury in NAFLD mice.(1)ASRP could reduce body weight,liver weight and liver index of NAFLD mice,and regulate the activity of liver injury marker enzymes such as AST,ALT and ALP in serum.(2)ASRP could reduce the activity of cytochrome enzyme CYP2E1 and reduce the excessive accumulation of ROS.(3)Western blotting results showed that ASRP could activate Nrf2/HO-1 signaling pathway,promote the decoupling of Nrf2 and KEAP1,up-regulate the activities of downstream antioxidant enzymes such as HO-1,SOD,GSH-Px and CAT,inhibit the generation of lipid peroxidation product that is MDA,and alleviate liver injury caused by oxidative stress.(4)ASRP could mediate JNK1/AP-1 signaling pathway to play a highly effective anti-inflammatory role,down-regulate the expression of p-JNK1,p-c-Jun and p-c-Fos proteins,balance the levels of pro-inflammatory factors such as TNF-α,IL-1β and IL-6 and the anti-inflammatory factor IL-10,thereby regulate immune homeostasis and improve liver injury.(5)Real-time quantitative PCR and Western blotting results showed that ASRP could activate AMPK signaling pathway,promote AMPK phosphorylation,inhibit the expression of downstream kinases SREBP-1c,FASN and ACC1,reduce fatty acid synthesis,regulate serum levels of TC,TG,LDL-C and HDL-C,and effectively decrease liver lipid accumulation to relieve lipid metabolism disorders.(6)According to hepatic pathological observation of H&E and oil red O,ASRP could regulate cell arrangement disorder,repair cell rupture,alleviate inflammatory cell infiltration,reduce cellular fat accumulation,and improve steatosis.In conclusion,ASRP could activate functions of potential antioxidation,anti-inflammation and anti-hyperlipidemia by synergistically regulating Nrf2/HO-1,JNK1/AP-1,AMPK/SREBP-1c and other multi-signaling pathways,showing a good anti-NAFLD effect.This study provided theoretical support for the development of effective drugs for clinical NAFLD. |
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