Endoplasmic Reticulum Stress And Mitochondria Damage Of Coelomocytes During The Autolysis Of Stichopus Japonicus

Sea cucumber has a high nutritional value and economic value,but because of its autolysis characteristics,resulting in huge economic losses,seriously restricting the healthy and sustainable development of the sea cucumber industry.The preliminary experimental data of this group showed that the process of autolysis was accompanied by apoptosis,but the specific mechanism has not been elucidated.Flow cytometry and confocal laser microscopy were used in this paper.Firstly,a method for the detection of the phagocytic rate was established.It is concluded that the coelomcyte is an ideal target for the immune stress experiment.And the model of seacucumber coelomcyte based on this research,the application of LSCM,FCM,Western blotting and fluorescence microplate technique,detection of sea cucumber induced by UV autolysis process,endoplasmic reticulum and mitochondria stress injury and its relationship with apoptosis.This study will provide a theoretical basis for the quality control of the preservation of sea cucumber,and provide a scientific basis for further study on the cellular immune function of the sea cucumber from the molecular level.The results are as follows:1.Changes of phagocytosis in the process of sea cucumber autolysisThe use of FCM and LSCM to detect the phagocytosis rate of coelomocyte of fluorescent microspheres in vivo and in vitro,optimize the seacucumber phagocytosis rate detection method(in vitro incubation method)and concentration(1:1500).This method was used to determine the changes of phagocytosis rate,mortality rate and apoptosis rate after UVA-induced(15w/m2.30min)incubation for different duration,found with the extension of standing time,phagocytic rate is inversely proportional to the rate of apoptosis and death,and determined the sea cucumber coelomocyte can be used as experimental model of immune cells study on stress reaction.2.Endoplasmic reticulum stress and mitochondrial damage in the process of sea cucumber autolysisIn this study,the structural damage of the endoplasmic reticulum and mitochondria in the coelomocyte of the sea cucumber were detected by FCM and LSCM.It was found that the content of in the endoplasmic reticulum increased significantly at 2h,and the content of mitochondrial fluorescence increased significantly at 1h.According to the structural characteristics of endoplasmic reticulum and mitochondria,the results indicated that the structural damage of mitochondria in the coelomocyte of sea cucumber was prior to the structural dam^age of endoplasmic reticulum.The study on Caspase-12,GRP78 and mitochondrial membrane potential,mitochondrial complex I activity,found that both Caspase-12 and GRP78 had obvious transition at 2h,and the mitochondrial membrane potential and mitochondrial complex I in 1 h and initial began to drop significantly.It is speculated that the function of mitochondria may precede the damage of endoplasmic reticulum.3.The distribution of endoplasmic reticulum and mitochondria in the coelomocyte of sea cucumberBy measuring the content of calcium ion in endoplasmic reticulum and mitochondria,found that calcium concentration in mitochondria has changed significantly in the beginning,obvious change of calcium concentration in the endoplasmic reticulum occurred after 2 h.It is inferred that mitochondrial calcium uptake in the cytoplasm first to maintain their balance,and then,in order to maintain the homeostasis of cells,endoplasmic reticulum release calcium ions,resulting in the damage of its structure and functionIt is conferred that the apoptosis of Stichopus japonicus coelomocytes induced by UVA might undergo the pathway of endoplasmic reticulum and mitochondria,which led to the damage of endoplasmic reticulum and mitochondria in coelomocytes;the degree of damage was developed with prolongation time.And the damage of mitochondria may precede the injury of endoplasmic reticulum.