Endoplasmic Reticulum Stress-mediated Coelomocyte Apoptosis During Autolysis Of Sea Cucumber Stichopus Japonicus

Sea cucumbers are one of the most important coastal fisheries seafood.However,a series of environmental and chemical factors lead to autolysis of sea cucumbers.Autolysis of sea cucumbers are induced by ultraviolet radiation,this phenomenon brings a lot of inconvenience to keEPing live and fresh storage industry of sea cucumbers.The early data of research suggested that sea cucumbers are accompanied by apoptosis of coelomocytes during the process of autolysis,changes intracellular Ca²⁺ concentration of coelomocytes and endoplasmic reticulum occurres morphological damage,but the degree of associated with apoptosis is not clear.Based on sea cucumber?Stichopus japonicus?,sea cucumber in the control group was not irradiated by UVA,sea cucumber in the UVA group was irradiated by UVA with the intensity of 15 w/m2 for 30min,sea cucumber in the Ca²⁺-suppression group was irradiated by UVA with the intensity of 15 w/m2 for 30min afrter injecting with calcium chelation agent?BAPTA sodium salt,IC50=1.47mM?into the coelomic cavity.The intracellular free calcium concentration([Ca²⁺]i)of coelomocytes was measured by laser scanning confocal microscope and flow cytometer.Calcium signals-related enzymes(sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase,calcineurin and calpain)were examined by microplate reader.Apoptotic rates of coelomocytes were detected by flow cytometer.The protein expression of endoplasmic reticulum stress-mediated apoptosis factors?CRT,Caspase-12,Caspase-9,Caspase-3,CHOP and Bcl-2?were examined by western blotting and flow cytometry.To investigate Ca²⁺ signals involve in the regulation of coelomocytes apoptosis and the mechanism of endoplasmic reticulum stress mediates coelomocytes apoptosis of sea cucumber.The results of the study are as follows:1.Determination of Ca²⁺ involved in the regulation of apoptosis condition of coelomocytesBased on the theory of Ca²⁺ homeostasis imbalance and overload,sea cucumbers were irradiated by UVA with different times?0,10,20 and 30min?,it was observed that intracellular Ca²⁺ concentration of coelomocytes showed a rEPeatea series of peak(Ca²⁺homeostasis imbalance)after irradiating 20 min and signnificantly increased(Ca²⁺ overload)after irradiating 30 min,which was to determine Ca²⁺ ivolved in the regulation of apoptosis condition of coelomocytes,sea cucumbers were irradiated by UVA with the intensity of 15 w/m2 for 30min.2.The mechanism of Ca²⁺ signals involve in the regulation of coelomocytes apoptosisBase on this experimental condition,sea cucumbers were irradiated by UVA with the intensity of 15 w/m2 for 30min and incubated at room temperature in dark for 0,1,2,3,and 4 h,respectively.Compare'd with the control group,intracellular free calcium concentration of coelomocytes increased gradually with the extended incubation time to reach the highest level at 2h and then decreased.Sarcoplasmic-endoplasmic reticulum Ca2--ATPase,calcineurin and calpain were activated to show a trend of increased and then reduced,and apoptosis rates of coelomocytes reached the highest level at 3h and then decreased.The levels of[Ca²⁺]i,sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase,calcineurin,calpain and apoptosis rates were significantly decreased in the Ca²⁺-suppression group,it was revealed that Ca²⁺ was the initiation signal that involved in the regulation of coelomocytes apoptosis.3.The mechanism of endoplasmic reticulum stress mediates coelomocytes apoptosisDisordered calcium homeostasis leads to Ca²⁺ overload,ER stress is involved to Ca²⁺overload.The protein expression of endoplasmic reticulum stress-mediated apoptosis factors?CRT,Caspase-12,Caspase-9,Caspase-3 and CHOP?significantly increased and the protein expression of anti-apoptotic factor Bcl-2 was significantly reduced.It is supposed that endoplasmic reticulum stress may involve in coelomocytes apoptosis during the process autolysis of sea cucumber.