| Freshwater fish contains high-quality protein,making a great contribution to people’s livelihood.Recently,the demand for refrigerated freshwater fish has been increasing.However,the deterioration of flesh quality occurred in postmortem fish muscle,causing the decrease of texture,decrease of water holding capacity and the changes of color,resulting in wasted raw materials and causing economic losses.The changes in flesh quality in the pre-storage period were attributed to the activation of endogenous enzymes.There were many reports on the mechanism of flesh quality deterioration in freshwater fish.However,the existing theories could not fully explain the deterioration of flesh quality during cold storage condition.In recent years,the theory of apoptosis was widely used to explain the tenderization process of postmortem beef meat,which could provide a new perspective to explain the mechanism of beef tenderization.However,freshwater fish were more susceptible to tenderization,which was bad for the consumption of fish products.Also,whether the apoptosis process was involved in the deterioration of flesh quality in postmortem fish muscle was not yet clear.Up to now,there were limited studies on the activation of apoptosis in freshwater fish during postmortem storage,and the relationship between apoptosis initiation and the changes of flesh quality needs to be clarified.In this study,grass carp（Ctenopharyngodon idella）was selected as subject and the dorsal muscle above the lateral line was used as the test sample.This study investigated the relationship between the flesh quality parameters and apoptosis activation under postmortem storage.The present research also investigated the effects of apoptosis inhibitors on the activation of apoptosis,the changes of flesh quality as well as the possible mechanism in postmortem fish muscle.This study also investigated the apoptotic pathways in postmortem fish muscle,which could provide reference to apoptosis activation in postmortem fish muscle under cold storage condition.The present research also investigated the possible apoptotic inducing factors in postmortem fish muscle,including energy metabolism,the profound changes of mitochondria,oxidative damage and heat-shock proteins during postmortem storage,which revealed the roles of these inducing factors involved in apoptosis activation.These works could help to further improve the theory of apoptotic activation,reveal the mechanism of the deterioration of flesh quality and provide references for freshwater fish preservation aiming at enhancing flesh quality during cold storage condition.The main findings were as follows:1.The changes of muscle structural,deterioration of flesh quality,cell death patterns and the relationship between apoptosis and flesh quality were investigated in postmortem fish muscle.The deterioration of flesh quality was increased with the increase of storage time,showing the decrease of shear force and water holding capacity,while showing the increase of drop loss.In the meanwhile,the disrupted structural integrity,including the disruption of the myofibril structure,the degradation of skeletal proteins,the release of AG and NAG enzymes in centrifugal juice and the overexpression of tight junction proteins were occurred in postmortem fish muscle.The above results showed that the deterioration of flesh quality was tightly connected with muscle damage.HE staining showed typical apoptotic features（cell shrinkage,chromatin condensation and nucleus breakage）without inflammatory response,indicating that apoptosis rather than necrosis was involved in postmortem fish muscle.The apoptosis was further confirmed with the increase of caspases activities,the formation of fragmented DNA,and the increase of positive apoptotic nuclei in postmortem time.Pearson correlation analysis showed that apoptotic cells were positively correlated with drop loss（P<0.01）and negatively correlated with shear force and water holding capacity（P<0.01）.2.This study also investigated the effects of apoptosis inhibitors on the changes of flesh quality and the related action behaviors.With the increase of postmortem time,apoptotic enzyme inhibitors of Z-VAD（OMe）-FMK（FMK）and Ac-DEVD-CHO（DEVD）could delay the increase of caspase-3,caspase-8,caspase-9 activities.FMK and DEVD could delay the reduction of sensory scores（color,odor,morphology and elasticity）,the loss of moisture and increase of tenderness,suggesting that apoptosis behaviors could affect the deterioration of fish quality as well as the damage of muscle structure.Apoptotic inhibitors could inhibit the degradation of cytoskeletal proteins of desmin,troponin T,the degradation of the intramuscular connective tissue of Decorin and Elastin,and the production of the protein degradation products of TVB-N and TCA soluble peptides,thereby delaying the deterioration of texture and reducing water loss in fish muscle.The inhibition of apoptosis could affect the activation of cathepsin（B,L and D）,serine protease,calpain as well as the release of endogenous enzymes of AG and NAG.Apoptotic inhibitors could reduce the increase of ROS,decrease oxidative damage caused by the oxidation of lipid and protein,thereby delaying the deterioration of fish quality.3.The apoptosis triggering mechanisms were determined in postmortem fish muscle,including the activation of apoptotic pathways,the expression of apoptosis-related proteins and the transcriptional regulation of apoptotic signaling molecules.With the increase of storage time,the increase of Fas L m RNA levels and the activation of caspase-8 and caspase-3 indicated that the death receptor apoptotic pathway（Fas L/caspase-8/caspase-3）was involved in activation of apoptosis in postmortem fish muscle.The changes of cytochrome c redox status indicated that mitochondrial cytochrome c released into cytoplasm and oxidized.The changes of cytochrome c redox status were associated with the increased levels of the pro-apoptotic protein Bax,decreased levels of the anti-apoptotic protein Bcl-2 and increased Bax/Bcl-2 ratios.The changes of transcriptional levels of Mcl-1,Apaf-1 and IAPs,as well as increased gene expressions of JNK and p38 MAPK suggested that mitochondrial apoptotic pathway[（JNK and p38 MAPK）/（Bax,Bcl-2 and Mcl-1）/cytochrome c/Apaf-1/caspase-9/caspase-3]was involved in apoptosis process in postmortem fish muscle.Meanwhile,mitochondria-nuclear translocation of AIF indicated that caspase-independent pathway was also involved in postmortem fish muscle.The triggering apoptotic mechanisms associated with multiple cytokines transcriptional levels showed that the up-regulated pro-apoptotic mediators[IFN-γ2,TNF-α,IL-6,IL-1β,IL-17D,IL-12p35 and IL-10（except IL-15）]and the down-regulated anti-apoptotic mediators of[IL-8 and IL-11（except TGF-βand IL-4）]were benefit to apoptosis at early stage,which were regulated by NF-κB and TOR,respectively.Results suggested that transcriptional regulation of multiple cytokines exerted a positive outcome on triggering apoptosis.4.Changes of apoptosis-inducing factors were studied and the correlations between apoptosis parameters and freshness indicators were also analyzed in postmortem fish muscle.With the increase of postmortem time,ATP levels gradually decreased.The decrease of ATP was closely related to the profound changes of mitochondria.Profound changes occurred in mitochondria during storage,including increased swelling（both long and short diameter）,decreased mitochondrial membrane potential,increased MPTP opening,decreased membrane fluidity,increase of mitochondrial Ca2+.With the increase of postmortem time,protein expression of HSP27 and HSP90 was significantly higher than at 0 h（P<0.05）,and the increased expression of HSPs might have an important role in inducing apoptosis.Increased ROS,lipid oxidation and protein oxidations were involved in postmortem fish muscle,indicating that oxidative damage contributed to the apoptosis process.The reduced antioxidant capacity was closely related to the decrease of non-enzymatic antioxidant substances（GSH）and the decrease of antioxidant enzymes Cu Zn SOD,Mn SOD,CAT,GPx and GR.In addition,the decreased gene expressions of antioxidant enzymes（Cu Zn SOD,Mn SOD,CAT,GPx1a,GSTR,GR）and signaling molecule of Nrf2 were consistent with the reduced activity of antioxidant enzymes.Apoptosis-related parameters（apoptosis rate,mitochondrial membrane fluidity,mitochondrial AIF,mitochondrial swelling degree,mitochondrial membrane potential and mitochondrial MPTP）were highly correlated with K value.In summary,this study scientifically investigated the changes of muscle integrity,deterioration of flesh quality,the relationship between apoptosis and flesh quality.Apoptosis inhibitors could affect the changes of apoptotic enzymes activities and apoptosis-mediated cascades.The inhibitors of FMK and DEVD could decrease the deterioration of fish quality by inhibiting the reactions of the apoptotic cascades,reducing the activity of apoptotic and endogenous enzymes,inhibiting the degradation of cytoskeletal proteins and intramuscular connective tissue and reducing the production of protein degradation products,decreasing the damage to the muscle structure and structural integrity and decreasing oxidative damage in postmortem fish muscle.The death receptor pathway,mitochondrial apoptotic pathway and the AIF mediated caspase-independent pathway were involved in the activation of apoptosis in postmortem fish muscle.The changes of apoptosis-inducing factors such as ATP,mitochondria,oxidative damage and heat shock proteins exerted a positive outcome on triggering apoptosis in postmortem fish muscle.Six apoptosis-related indicators could be used to reflect fish freshness,including apoptosis rate,mitochondrial membrane fluidity,mitochondrial AIF,mitochondrial swelling,mitochondrial membrane potential and mitochondrial MPTP.This study could enhance the theory of apoptosis and quality deterioration of freshwater fish during cold storage,and provide theoretical reference for cold storage and transportation,changes of flesh quality and evaluation of freshness in freshwater fish. |
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