Preventive Effect And Mechanism Of Soybean Meal-derived Antioxidant Peptides On Acute Alcoholic Liver Injury

This work was part of the Scientific Research Project of Education Department of Jilin Province(JJKH20211129KJ).In recent years,the incidence of alcoholic liver disease in China has shown an upward trend year by year with the continuous increase in alcohol consumption,which has seriously endangered the lives and health of Chinese people.Using food-derived antioxidant active substances to intervene and regulate the body's oxidative stress state caused by alcohol is an important way to alleviate and improve alcoholic liver injury.Soybean meal is rich in high-quality soybean protein.Soybean meal-derived antioxidant peptides(SAPs)obtained by hydrolysis have the potential to prevent alcoholic liver damage,but there are no relevant research reports.This article takes SAPs as the research object.First,the ultrasonic-assisted enzymatic hydrolysis preparation process parameters were optimized through single factor and response surface experiments,and then the ultrafiltration technology was used for separation and purification.At the same time,the components with the best antioxidant activity were screened out and the sequence was identified,then the antioxidant activity of the obtained peptides was characterized.Finally,a mouse model was constructed to explore the preventive effect of SAPs on acute alcoholic liver injury from oxidative stress,lipid metabolism and inflammatory factors,and transcriptomics technology was used to clarify the preventive mechanism of SAPs.This research can increase the added value of the soybean industry,and which is of great significance to the development of safe and effective food-derived hepatoprotective peptide products.The main research contents and results of the thesis are as follows:(1)Using soybean meal as raw material,research on the preparation,separation and purification,sequence identification and in vitro antioxidant activity characterization of SAPs.The results showed that the optimal process conditions for the preparation of SAPs by ultrasonic-assisted enzymatic hydrolysis were as follows:enzymatic hydrolysis p H was 8,substrate concentration was 9%,enzyme amount was6665.39 U/g,temperature was 50 ?,enzymatic hydrolysis time was 4 h,ultrasonic time and power were 9 min and 300 W,respectively.After separation and purification,components with molecular weight < 1k Da(SAPs5)had the best activity.The sequence of SAPs5 was identified,and a total of 10 peptide sequences were obtained,including YLFKD,IWNLN,GTYW,CLA,KVW,LRC,SLW,VYV,WLQ and YYK.Through solid-phase technology to synthesis the above peptide sequence,the identified peptide sequences were characterized,and GTYW had the best activity,which was closely related to tyrosine and tryptophan.(2)The experimental mice were divided into control check group(CK),model group(M),positive control group(GSH)and experimental groups(SAPs5 and GTYW),through pre-intervention of SAPs(SAPs5 and GTYW),then construct an acute alcoholic liver injury model to study the preventive effect of SAPs from oxidative stress,lipid metabolism and inflammatory factors.The results showed that compared with the M group,the serum AST/ALT,TC and TG contents,as well as the liver CYP2E1,IL-6,IL-1? and TNF-? contents in the SAPs treatment group were significantly reduced(p <0.05).Although in the SAPs group,the liver MDA content decreased,and the liver GSH content,T-SOD and CAT activity increased to varying degrees,there was no significant difference compared with the M group(p > 0.05).In the pathological section,compared with the model group,the liver cells and hepatic cord structure of the SAPs group were more clear and complete,the kidney cells and structure had not changed.In summary,SAPs can protect the liver by improving the antioxidant capacity,reducing lipid accumulation and the release of inflammatory factors.(3)In order to clarify the mechanism of SAPs(SAPs5 and GTYW)in regulating acute alcoholic liver injury,transcriptomics research was conducted based on the Illumina hiseq sequencing platform.The results showed that the purity and quality of RNA extracted from liver tissues of each experimental mouse were high(OD 260/280? 1.95,OD 260/230 ? 2.14,concentration ? 1000 ng/?L,RIN value ? 7.00).After RNAseq sequencing,the statistics,quality control and comparison results of the sequencing data were good(average error rate of sequenced bases < 0.03%,Q20 > 97%,Q30 >91%,Total mapped > 90%).Through differential analysis,M were set as the control,the up-regulated genes in GSH,SAPs5 and GTYW group were 696,1076,and 1285,while down-regulated genes were 677,661,and 1012,respectively.After functional analysis,it was found that the differential genes were mainly enriched in the reaction process and metabolic pathways related to inflammation,oxidative stress and lipid metabolism after the intervention of SAPs.Under the intervention of SPAs,Acsl1,Aqp8,Aox1,Gclc and Srebf1 were significantly up-regulated,Cxcl1,Il1r1 and Lbp were down-regulated(p <0.05),which can promote lipid metabolism,reduce the release of inflammatory factors,and alleviate oxidative stress damage.