Preparation And Mechanism Of Active Peptide From Quinoa Bran Against Hepatocellular Cancer

Hepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system.Due to high recurrence and metastasis rates,the survival rate of HCC is very low.Sorafenib,a multi kinase inhibitor,is currently the first line treatment for patients with advanced hepatocellular carcinoma.However,this drug is highly susceptible to drug resistance and adverse reactions,and only prolongs the survival period of 3 months,making the current clinical treatment of HCC far from satisfactory.Therefore,it is urgent to find novel against hepatocellular cancer active molecules with high efficiency and low toxicity.Chenopodium quinoa Wild,as a characteristic coarse grain crop in Shanxi,is rich in a variety of nutrients,and is the only perfect nutritional food recognized by the Food and Agriculture Organization of the United Nations.Quinoa has a higher content of protein than other common grains,and has a reasonable proportion of various amino acids.It is a raw material for high-quality protein.Quinoa bran,the outer seed coat of quinoa,has high nutritional value and rich protein content,just like quinoa.Due to the fact that high molecular weight proteins can increase the burden on the digestive tract of patients with gastrointestinal dysfunction or postoperative weakness,based on the advantages of active peptides that do not require digestion compared to proteins,are easier to absorb into the bloodstream,and have strong activity,it indicates that the development of active peptides with against hepatocellular cancer effects is particularly important.At present,there have been research reports on the health effects of quinoa bran active peptides both at home and abroad,mainly focusing on hypotension,hypoglycemic,antioxidant,and other aspects.It is suggested that quinoa bran active peptides have the potential to alleviate chronic diseases,but there are few reports on anti-tumor activities.Therefore,in this study,quinoa bran active peptides with against hepatocellular cancer activity were obtained from quinoa bran through biomimetic digestion,named QBPP.Through tricine gel electrophoresis and 12% SDS-PAGE,it was preliminarily determined that QBPP was some small peptides with low molecular weight.The against hepatocellular cancer effect of QBPP was systematically evaluated from three aspects of cell proliferation,apoptosis and migration,and further explored its molecular mechanism of anti hepatoma.The research results are as follows:(1)Preparation of QBPP and evaluation of its against hepatocellular cancer effect.The crude protein of quinoa bran was preliminarily obtained by acetone ammonium sulfate precipitation,and then the quinoa bran active peptide with against hepatocellular cancer activity was obtained by in vitro bionic digestion,named QBPP.The QBPP was preliminarily identified by tricine gel electrophoresis and 12% SDS-PAGE.MTT and cell cloning experiments showed that QBPP could inhibit the proliferation of hepatoma cells Bel-7402 and Hep G2 in a concentration dependent manner,but had no effect on normal human hepatocytes.Flow cytometry and Western blot experiments showed that QBPP treatment could significantly induce the decrease of mitochondrial membrane potential in hepatoma cells Bel-7402 and Hep G2,thereby inducing apoptosis.(2)The p38 MAPK signaling pathway mediates QBPP to exert against hepatocellular cancer effects.The p38 MAPK pathway plays an important role in the occurrence and development of tumors.Transcriptomic sequencing results showed that after QBPP treatment,a total of 635 differential genes were identified in Hep G2 cells,including 380up-regulated genes and 255 down-regulated genes.At the same time,KEGG enrichment showed that the MAPK signaling pathway was significantly enriched.The thermograms of differentially expressed genes in the MAPK pathway showed that MAPK8(JNK)and MAPK14(p38)were up-regulated,while MAPK1(ERK)was down-regulated,but MAPK14(p38)was most significantly up-regulated.Further,RT-PCR and Western blot techniques were used to verify the key role of p38 MAPK signaling pathway in QBPP’s against hepatocellular cancer response.In addition,SB203580,an inhibitor of the p38 MAPK pathway combined with QBPP,has a weaker antiproliferative and apoptotic effect on against hepatocellular cancer compared to QBPP alone.These results suggest that QBPP can inhibit the proliferation and induce apoptosis of liver cancer cells by activating the p38 MAPK pathway.(3)QBPP inhibits the migration of liver cancer cells by activating the p38 MAPK signaling pathway.Through cell scratch test,transwell test,and intercellular adhesion test,it was found that QBPP can significantly inhibit the migration of liver cancer cells Bel-7402 and Hep G2,and promote the adhesion between liver cancer cells.Further,Western blot analysis confirmed that QBPP inhibits the EMT effect of liver cancer cells by activating the p38 MAPK pathway,thereby blocking the migration of liver cancer cells.In summary,this study took Chenopodium album,a characteristic resource in Shanxi,as the research object.Through acetone ammonium sulfate precipitation and in vitro simulated digestion,the Chenopodium album active peptide QBPP with against hepatocellular cancer effect was obtained from Chenopodium album,and its effective amino acid components were identified.Through transcriptomics,it was revealed that QBPP can play a molecular mechanism of against hepatocellular cancer by activating the p38 MAPK pathway,To provide scientific basis and material basis for the development of quinoa derived active peptides as candidates for anti liver cancer drugs and the research and development of nutritional intervention products.