Protective Effects Of Shrimp Peptide On Oxidative Damage Of RAW264.7 Cells And Ulcerative Colitis

The pipe whip shrimp(Solenocera crassicornis),also called red shrimp,is an annual crustacean.It is distributed in the southern Yellow Sea,East China Sea and South China Sea.It is an important fishing target in Zhejiang Province and even the whole East China Sea coast.Since the by-products including shrimp heads and shrimp shells produced during processing are mainly used as low-value feed materials,and the deep processing and utilization are still less,and the protein content of by-products is high,therefore it is of great significance to extract polypeptides from the scraps of red shrimp processing.Biologically active peptides are small molecule peptides that are beneficial to the life activities of the body or have specific physiological effects.They can be divided into antimicrobial peptides,immune peptides,antioxidant peptides,neuropeptides,etc.In this paper,the protective effect of peptides from Solenocera crassicornis by-products(SP)on oxidative damage of RAW264.7 cells and the improvement of ulcerative colitis(UC)and intestinal flora in mice were studied through cell model and animal model,providing a theoretical basis for the development of functional food for the prevention of UC.The research methods are as follows:(1)Peptides were extracted from the by-products of red shrimp by enzymatic hydrolysis,and further purified by membrane separation technology to obtain three peptides.The chemical antioxidant activities of the three peptides were compared,and the amino acid composition and molecular weight distribution of the low molecular weight peptides were detected.(2)To study the antioxidant effect of SP in vitro,the oxidative damage model of RAW264.7 cell was constructed by H₂O₂.CCK-8 assay was used to detect the toxicity of different doses of shrimp peptides on RAW264.7 cells and their effects on the survival rate of RAW264.7 cells with oxidative damage.The antioxidant level of RAW264.7 cells was detected by enzyme linked immunosorbent assay(ELISA)and the expression of antioxidant genes in RAW264.7 cells was detected by RT-PCR,and the protein expression of Nrf2-Keap1 pathway was detected by Western blot(WB).(3)Male ICR mice aged 4 weeks were divided into blank group,DSS group,and DSS+SP group(low and high dose groups).The total test period was 21 days.The DSS+SP group was given 300 mg/kg/d and 600 mg/kg/d respectively from the first day to the end of the 21 d of the experiment.Except for the blank group,all the other groups were given water containing 3.5%DSS in the last 7 days of the experiment period.Tissues were collected,body weight,the length of the colon and disease activity index(DAI)were analyzed.The changes of colon in colitis mice were observed by scanning electron microscopy and transmission electron microscopy.Antioxidant indexes GSH-Px and SOD in serum and colon of mice,inflammation-related indexes TNF-?and IL-6,intestinal barrier related indexes LPS and DAO were detected and analyzed.In this study,the venn index and alpha diversity index were used to evaluate the effect of SP on the structure of intestinal flora in mice,and the changes of intestinal microflora in different groups at the phylum and genus level were analyzed.The experimental results are as follows:(1)Shrimp peptides with low molecular weight had better ability to inhibit hydroxyl radicals and superoxide ions.The molecular weight distribution of low molecular weight peptides was mainly in the range of 180-500 Da,the composition of essential amino acid was relatively high,antioxidant amino acids such as Leu and Ala were relatively high.(2)Pre-administration of SP had a preventive and protective effect on H₂O₂-induced oxidative stress in RAW264.7 cells.The survival rate of cells protected by shrimp peptides in advance was significantly increased.Shrimp peptides could increase the activities of SOD,GSH-Px and CAT,and the m RNA expression levels of SOD1,GPX1,Nrf2,NQO1and HO-1 were significantly increased(P<0.05).In addition,the relative protein expression of Keap1 was significantly decreased,while that of Nrf2 was significantly increased(P<0.05),indicating that SP could alleviate oxidative damage by regulating the Keap1-Nrf2 signaling pathway.(3)Compared with the normal group,mice in the DSS group showed symptoms such as weight loss and hematochezia,serious mucosal damage could be observed by HE staining and electron microscopy,while the symptoms of mice in the groups given different concentrations of shrimp peptide were significantly alleviated.Compared with the model group,the levels of GSH-Px and SOD in serum and colon were increased,and the levels of TNF-?,IL-6,LPS and DAO were decreased in the low and high doses of shrimp peptide groups.The sequencing results showed that the abundance of Fimicutes and Proteobacteria in DSS group decreased,while the abundance of Bacteroidetes increased.SP regulated the disorder of intestinal flora by up-regulating the abundance of Firmicutes,down-regulating the abundance of Bacteroidetes and potential pathogenic bacteria Turicibacter,and increasing the abundance of beneficial bacteria Alistipes and Lactobacillus.In summary,shrimp peptides have a certain protective effect on H₂O₂-induced oxidative stress damage in RAW264.7 cells and intestinal inflammation caused by DSS.It may be through up-regulating the expression of antioxidant genes such as HO-1,activating the Keap1-Nrf2 pathway,reducing the pathological damage of colon,reducing the level of inflammatory factors such as TNF-?,increasing the number of beneficial bacteria in intestinal flora,and playing an improvement role in mice with ulcerative colitis.