Protective Effects Of Two Seahorse Peptides On Oxidative Stress Induced By Alcohol In HepG2 Cells

Excessive drinking can cause a wide range of liver diseases such as steatohepatitis,liver fibrosis,cirrhosis,and liver cancer.In particular,alcoholic live disease(ALD)has attracted widespread attention.In this study,two peptides from seahorse were used as experimental materials to investigate their activities.SHP-1(VSIADSKA,Val-Ser-Ile-Ala-Asp-Ser-Lys-Ala)and SHP-2(ENANGP,Glu-Asn-Ala-Asn-Gly-Pro)were studied.The protective effects and mechanism of human hepatoma cell line(HepG2)on alcoholic injury provide a theoretical basis for the prevention and treatment of ALD.The main research contents and results are as follows:1.Cell viability was determined by MTT assay.The experimental results showed that the optimal concentration of alcohol was 0.75 M;in the range of 10-100 ?M,the two peptides had no toxic effect on the cells;compared with the alcohol-injured group(the control group),the relative viability of the cells was obviously enhanced at 10-100 ?M in a concentration-dependent manner.This indicates that the two seahorse peptides have protective effects against alcohol-induced HepG2 cell damage.2.The content of ROS in cells was detected by DCFH-DA method.The results showed that compared with the control group,the two seahorse peptides reduced the intracellular ROS content.That is,seahorse peptides can effectively inhibit the production of ROS.3.The contents of SOD,GSH and GGT in cells were detected by SOD,GSH,and GGT detection kits;the expressions of SOD,GSH and GGT were detected by western blot.The results showed that compared with the control group,the two seahorse peptides could effectively restore the activities of SOD and GSH in the cells and decrease the content of GGT;the expressions of SOD and GSH in the cells increased significantly,while the expression of GGT decreased.4.Western blot was used to detect the expressions of proteins related to apoptosis.The results showed that compared with the control group,the expressions of bax and c-caspase-8/9-3 in the sample group were significantly decreased;while the level of bcl-2 was up-regulated.That is,seahorse peptides could inhibit the apoptosis of HepG2 cells.5.Western blot was used to detect the effects of two sea horse peptides on Akt and NF-?B signaling pathways.The results showed that the expressions of p-Akt,p-IkB-? and p-p65 were significantly down-regulated,compared with the control group.That is,seahorse peptides inhibited Akt and NF-?B signaling pathways.6.Detection of p65 protein into the nucleus by immunofluorescence assay.The experimental results showed that compared with the control group,the two seahorse peptides could significantly inhibit the p65 protein from entering the nucleus from the cytoplasm.7.Western blot was used to detect the effects of two seahorse peptides on MAPK signaling pathway.The results showed that the expressions of p-ERK and p-p38 were significantly decreased compared with the control group.That is,seahorse peptides had inhibitory effects on the MAPK signaling pathway.8.The change of mitochondrial membrane potential was detected by JC-1 staining method.The experimental results showed that two seahorse peptides increased mitochondrial membrane potential,compared with the control group.9.Single-cell gel electrophoresis experiment(comet assay)was used to detect DNA damage.The experimental results showed that two seahorse peptides could effectively reduce DNA damage,compared with the control group.10.ELISA was used to detect the change of TNF-? level.The experimental results showed that two seahorse peptides could reduce the content of TNF-? in cells compared with the control group.In summary,two seahorse peptides protect HepG2 cell from damage caused by alcohol.The mechanism of action may be to inhibit the production of ROS,increase the content of SOD and GSH,inhibit the expression of proapoptotic protein bax,promote the expression of anti-apoptosis protein bcl-2,inhibit the activation of caspase-8/-9/-3,inhibit Akt,NF-?B,and MAPK signaling pathways,increase mitochondrial membrane potential,and reduce intracellular TNF-? levels to protect cells from oxidative stress damage.