The Mechanism Of Peroxidase 6 Regulating Beef Tenderness Based On Oxidative Stress Pathway

Tenderness is the most important edible quality of beef,and it affects the satisfaction of consumers.The mechanism of beef tenderization has always been a hot and difficult topic in meat science.As a major antioxidant enzyme,Peroxidase 6(Prdx6)regulates the oxidative stress in cells,which affects beef tenderness during postmortem aging.Prdx6 has been shown to serve as a biomarker of beef tenderness.However,the effect and specific pathways of Prdx6 regulating beef tenderness during postmortem aging have not been clearly reported.The left carcass Longissimus Dorsi muscles of six simmental crossbred cattle which have the same premortem management conditions were selected as experimental samples.Meat samples were cut into little pieces and soaked separately in H₂O₂(direct oxidative stress treatment),mercaptosuccinic acid(MA,non-selenium glutathione peroxidase(NSGPx)activity inhibition treatment),N-acetyl-L-cysteine(NAC,inhibiting of oxidative stress and increasing NSGPx activity treatment),and normal saline(control group)to 0,6,12,24,72,168 h.Firstly,the expression and NSGPx activity of Prdx6,shear force and the degradation of cytoskeletal protein were measured to investigate the relationship between Prdx6 and beef tenderness during postmortem aging.Secondly,cell apoptosis rate,protein carbonyl content,and other indicators were measured to investigate the mechanism of Prdx6 regulating beef tenderness based on cell apoptosis and protein oxidation.Finally,proteomics was used to investigate the key proteins and pathways which regulating beef tenderness,and systematically explain the mechanism of beef tenderization.The main results of this study were exhibited as follows:(1)During the postmortem aging,H₂O₂ group significantly increased the reactive oxygen species(ROS)relative content,MA group significantly increased the heat shock protein 70(HSP70)expression.According to these results,there was an increased level of oxidative stress in both groups.In the early postmortem period(about 24 h after slaughter),in order to protect cells against oxidative stress,H₂O₂ group and MA group significantly increased the Prdx6 expression,as well as NSGPx activity in all groups except MA group.On the 3rd day after slaughter,in terms of the shear force,H₂O₂ group was significantly lower than NAC group,and MA group was significantly lower than other groups.The degradation of desmin was similar to shear force in the early postmortem period.These results suggested that,during postmortem aging,direct oxidative stress treatment and NSGPx activity inhibition treatment accelerated the beef tenderization,and the tenderization speed of NSGPx activity inhibition treatment was faster than direct oxidative stress treatment.Therefore,Prdx6's NSGPx activity was negatively correlated with beef tenderness.(2)Prdx6's NSGPx activity could regulate beef tenderness through cell apoptosis and protein oxidation pathways.Ca2+ concentration in sarcoplasm,cell apoptosis rate,Caspase 9and Caspase 3 activity of H₂O₂ group and MA group were significantly higher than NAC group.Cell apoptosis rate and Caspase 3 activity of MA group were significantly higher than H₂O₂ group.These results suggested that direct oxidative stress treatment and NSGPx activity inhibition treatment significantly increased cell apoptosis degree,and cell apoptosis degree of NSGPx activity inhibition treatment was higher than direct oxidative stress treatment.At the meantime,the ?-calpain activity of direct oxidative stress treatment and NSGPx activity inhibition treatment was increased in the early postmortem period.Protein oxidation of H₂O₂ group and MA group were significantly higher than NAC group and control group in 72?168h after slaughter,which suggested that the influence of direct oxidative stress treatment and NSGPx activity inhibition treatment on protein oxidation mainly formed after 72 h postmortem.Therefore,through inhibiting NSGPx activity,the degradation of cytoskeletal protein was increased by promoting Caspases cascade reactions and ?-calpain activity in the early postmortem period.At the same time,inhibiting NSGPx activity also increased protein oxidation and changed protein conformation,which speeding up the tenderization process.(3)Proteomics showed that proteasomal ubiquitin-dependent protein catabolic process related proteins accounted for nearly 1/3 of the total differential proteins at 0 h vs 24 h,which had a remarkable effect on beef tenderization in the early postmortem period.In addition to cellular structural proteins,the differential proteins significantly correlated with tenderness were mainly involved in protein,carbohydrate and lipid catabolism,apoptosis,calcium homeostasis and respiratory electron-transport chain.Catabolism enzymes such as isobutyryl-Co A dehydrogenase(ACAD8),2-phospho-D-glycerate hydro-lyase(ENO2),Acyl-coenzyme A oxidase(ACOXL),carbonic anhydrase(CA);apoptosis related proteins such as acidic leucine-rich nuclear phosphoprotein 32 family member A(ANP32A);and respiratory electron-transport chain related proteins such as NADH-ubiquinone oxidoreductase chain(ND),cytochrome C oxidase subunit 7A1(COX7A1),might be potential factors affecting the beef tenderness,which was worthy of further study.