Protective Effect Of Aronia Melanocarpa Elliot Anthocyanin Combined With Aloe Polysaccharides On Alcoholic Liver Injury In Mice

Alcoholic liver disease（ALD）is one of the leading causes of chronic liver disease worldwide,and the burden of disease attributable to alcohol is rapidly increasing in Asian countries such as China,Korea,and India.General treatment measures for patients with ALD include antibiotic therapy,nutritional supplementation,and management of the alcohol withdrawal syndrome,and liver transplantation is the definitive treatment of choic for patients with advanced alcoholic hepatosclerosis.So far,the most effective treatment to alleviate ALD is long-term abstinence,and it is especially important to explore the prevention and treatment measures for ALD.The fruits of Aronia melanocarpa Elliot are very rich in polyphenolic bioactivities.Aornia mealnocarpa Elliot anthocyanin（AMA）has efficacy such an antioxidation,anti-aging,and modulation of the immune system.Aloe vera is an herbaceous plant that contains polysaccharides,anthrones,flavonoids,and other various biological activities,and Aloe polysaccharides（APs）have antitumor,anti-inflammatory,hypoglycemic,and wound healing promoting pharmacological effects.At present,the hepatoprotective effects of AMA and APs are mostly inclined to the study of protective effects pf carbon tetrachloride induced chemical liver injury,and have focused on a single study.Therefore,in this study,the possible targets of AMA and APs on ALD were predicted by network pharmacology and molecular docking,and animal experiments were conducted to explore the protective effect andmechanism of AMA and APs single administration group compared with AMA combined with APs administration on ALD.In this study,the PubChem database was used to obtain the 3D structures of the active ingredients of AMA and APs,and reverse docking using the Pharm Mapper platform was performed to obtain 395 regulatory targets of the active ingredients.There are 630 gene targets associated with alcoholic liver injury derived from Genecards database and Dis Ge NET database.The active ingredient regulatory targets of AMA and APs and disease-related targets of ALD were taken together to obtain 95core targets.The 95 core targets were used to contruct the PPI network by STRING database,and the results were imported into Cytoscape 3.9.1 software for mcode clustering analysis,and the core targets were subjected to GO enrichment analysis and KEGG pathway analysis using the Microscript database.Genes showing enrichment in biological processes such as cellular response to biological stimuli,response to nutrient levels,response to reactive oxygen species;Genes for cellular components such as caveolae,cytoplasmic vesicle lumen,and membrane area were enriched;Enriched in genes related to molecular fuctions such as protein tyrosine activated enzyme activity,protein kinase activity of transmembrane receptors,and drug binding;KEGG anslysis showed that genes in the PI3K-AKT signaling pathway,Ras signaling pathway and other genes were enriched.Molecular docking of the four active compounds including cyanidin-3-galactoside,cyanidin-3-arabinoside,mannose and glucose with the four core proteins of IL2,CASP3,AKT1 and ALB was performed using Autodock software and the docking score values were all lower than-4.25 kcal·mol^-1,indicating that the two had some binding activity.In animal experiments,42%（V/V）ethanol was used to induce alcoholic liver injury model in mice by gavage of single component high and low doses of AMA and APs,compared with gavage of AMA combined with APs,the mice body weight,liver index,serum levels of AST,ALT,TC,TG,HDL-C,LDL-C,the content of GSH-Px,ALDH in liver tissue and combined with HE staining,to explore the synergistic effects of AMA and APs on the alleviation of alcoholic liver injury.The results showed that the body weight of mice was 48.35±1.34g in the blank control group and only 37.20±1.89g in the model group.In the high dosage group of AMA combined with APs,the body weight increased to 46.40±2.65g,which was significantly higher compared with the model group.The levels of AST,ALT,TC and TG in the serum of mice in the model group were significantly increased,the levels of HDL-C and LDL-C were abnormal,and the abnormalities of related liver function indexes in the serum of mice treated with AMA combined with APs were greatly alleviated.The enzyme activity of GSH-Px in the liver tissue of the model mice was significantly lower than that in the blank control group,the enzyme activity of ALDH was significantly higher,and the enzyme activity of GSH-Px and ALDH in the liver tissue intervened by AMA and APs.The results of the appearance observation and he staining of the liver tissues showed that the livers of the mice in the blank control group were ruddy appearance,the hepatocytes were arranged in an orderly fashion,no phenomenon of cell degeneration,necrosis and inflammatory cell infiltration was seen,the livers of the mice in the model group were yellow white,the arrangement of the mouse liver cords was disordered,and the hepatocytes were turbid and swollen,and there was a significant improvement in liver histopathology after the intervention of AMA and APs.The above results indicated that AMA and APs could prevent alcohol induced liver injury in mice,and the combination was more effective than the single drug group.The mRNA expression of IL-1 and TNF-αinflammatory factors in mouse liver tissues was detected by RT-PCR technique,and the results showed that AMA and APs could decrease the inflammatory accumulation of IL-1 and TNF-αin mouse liver tissues,and the effect of AMA combined with APs was more significant.Western blot technology was used to detect the expression ofα7n ACh R,PI3K/AKT,Nrf2/HO-1 and other related signaling pathway proteins.The results showed that AMA and APs could alleviate alcohol induced liver injury in mice by activatingα7n ACh R,enhancing the phosphorylation levels of PI3K and AKT,organizing the overexpression of keap1,and reversing the low expression of Nrf2 and HO-1,increasing the expression of Bcl-2 protein,and inhibiting the expression of regulators IL-1βand p62.Taken together,the administration of AMA and APs could alleviate the symptoms of liver injury induced by alcohol in mice,and the intervention effect of AMA combined with APs was more obvious.