Effects Of Cd²⁺ On Toxicity Pathway Of Prophenoloxidase Activating System Activity In Freshwater Crayfish Procambarus Clarkii

Objective: In recent years,the pollution situation of water environment has become increasingly serious.Cadmium is one of the typical pollutants,which can cause toxic effects such as oxidative stress and immunosuppression on aquatic animals,jeopardizing their health.The phenoloxidase activating system?pro PO-AS?is the main immune defense system in crustaceans,the results showed that it is closely related to the health status of crustaceans.Generally,activating phenoloxidase would be controlled rigorously and precisely,however,the toxicity pathway of cadmium on pro PO-AS is not clear.Key transcription factors?NF-?B p65,Nrf2?and signal transduction pathway?p38 MAPK?have attracted much attention in the response of aquatic animals to heavy metal stress and their regulation of oxidative damage and immune?or inflammatory?system.Environmental stress induces the synthesis and expression signaling pathway molecules in the hepatopancreas,such as p38 MAPK,NF-?B,Nrf2.The endogenous H₂ S of signaling pathway molecules has vital immunoregulatory functions,which is a frontier and hotspot factor for studying stress response,signal transduction and immune regulation in our country and abroad.In addition,it has a wide range of physiological functions in human,mammalian and plant cells,such as oxidative stress response,disease development,stress resistance.The signaling pathways and H₂ S may also play an important role in the natural immune defense,which can be disturbed by cadmium.In this study,we investigated the key protein of the signaling pathway and the gas signal molecule H₂ S,and analyzed the activity of the low,medium and high sub-lethal concentrations Cd²⁺ induced pro PO-AS,preliminary discussion on the toxicity mechanism of Cd²⁺ perturbation of natural immune defense,aiming to provide a theoretical basis for the prevention and treatment of crustacean diseases.Methods: The current study assessed the sublethal toxicities induced byCd²⁺ in the fresh water crayfish Procambarus clarkii.The static test method of actute toxicity test was used.The nominal concentrations of Cd²⁺(1.0,5.0,10.0mg·L^-1)corresponding to 1/42,1/8,1/4 of the 96 h LC50(42mg·L^-1).The study included samples from fresh water crayfish,haemolymph,and hepatopancreas.The oxidative stress indicators ROS,major immune factors?THC,pro-PO,PO and SP?and endogenous hydrogen sulfide content were determined by Biochemical method.The expression levels of NF-?B p65,Nrf2,CBS,p38 and p-p38 in hepatopancreas was determined by immunohistochemistry.The correlation was analyzed by Pearson's method.Results: 1.The ROS level in hepatopancreas and hemolymph was increased because of Cd²⁺,thus producing oxidative stress.After 96 hours exposing to Cd²⁺,the ROS level raised sharply with the growing concentration?P<0.05?.Such exposure caused typical pathological damage on hepatopancreas by 1 mg·L^-1,5 mg·L^-1 and 10 mg·L^-1 Cd²⁺.During the whole period,the three Cd²⁺ treatment groups induced p38 expression in hepatopancreas and induced phosphorylation in different degrees,what's more the effect of 5 mg·L^-1 Cd²⁺ treatment group was extremely obvious?P<0.05?.The expression levels of p38 and p-p38 were inhibited by 10mg·L^-1 Cd²⁺.Compared with the control group,the expression levels of key transcription factors NF-?Bp65 and Nrf2 were drastically raised?P<0.05?,meanwhile the level of 5mg·L^-1Cd²⁺ group was obviously higher than other exposure groups,indicating that it was activated to different extent.Correlation analysis showed that p-p38 MAPK was positively correlated with NF-?Bp65 and Nrf2,and negatively correlated with NF-?Bp65 and Nrf2.Cd²⁺ higher the expression levels of p38 MAPK,p-p38 MAPK,NF-?B p65 and Nrf2,which may be the toxic effects of Cd²⁺.2.During Cd²⁺exposure,the expression levels of p38 MAPK,p-p38 MAPK,NF-?B p65 and Nrf2 was enhanced,the PO,SP and pro PO activities were inhibited,which may be related to p38 MAPK,NF.-?B p65 and Nrf2 are mediated by the toxic effects of Cd²⁺.3.After 48 hours of exposure to Cd²⁺,immunohistochemical analysisshowed that the expression levels of CBS in the low concentration group(1mg·L^-1)was overwhelmingly higher than other groups?P<0.01?.The content of endogenous H₂ S also changed to different extent compared to the control group,showing that the change of endogenous H₂ S is closely related to Cd²⁺ exposure.Correlation analysis showed that the expression levels of CBS and NF-?B p65?PO had a significant correlation.The results showed that the endogenous H₂ S of the gas signal molecule may also be involved in the change in the activity of the Cd²⁺ stimulated phenoloxidase activation system.Conclusion: This study showed that under heavy metal Cd²⁺ stress,the body produced the large amount of ROS,which led to oxidative stress.The signal transduction pathway?p38 MAPK?was activated in hepatopancreatic tissues,and the expression of downstream transcription factors?NF-?B p65,Nrf2?was enhanced.PO,SP and pro PO activities were inhibited to varying degrees,which may be related to the toxic effects of Cd²⁺ mediated by p38 MAPK,NF-?B p65 and Nrf2.The endogenous H₂ S of the gas signal molecule may also be involved in the change in the activity of the Cd²⁺stimulated phenoloxidase activating system.Through the study of key proteins and gaseous media in signaling pathway,the effects of Cd²⁺ on the activity of pro PO-AS of Procambarus clarkii were analyzed,the toxicity mechanism of environmental Cd²⁺ exposure to the natural immune defense of invertebrates such as Procambarus clarkii,and provided a theoretical basis for disease control of aquaculture animals.It is of great significance for the healthy breeding of aquatic animals.