Study On The Mechanism Of Cadmium-Induced Programmed Necrosis Of Chicken Leydig Cells And The Mitigating Effect Of Selenium

Leydig cells are the primary male reproductive cells,responsible for secreting testosterone,which is essential for male reproductive health and fertility,as well as regulating the normal growth,development,and stability of male reproductive organs.Cadmium（Cd）is a widely distributed environmental pollutant that is highly toxic to both animals and humans.The testes are highly sensitive to the toxicity of Cd,which can cause changes in testicular morphology and biochemical characteristics,disrupt the blood-testis barrier,and promote germ cell apoptosis.Selenium（Se）is a trace element in the human body that has a protective effect on cell damage caused by heavy metals.However,there is limited research on the role of Se in alleviating Cd-induced oxidative stress,endoplasmic reticulum stress,and programmed cell death in chicken Leydig cells.Therefore,this study used in vitro cultured chicken Leydig cells as the research material and treated them with Se and/or Cd to explore the toxic effects of Cd on Leydig cells and the alleviating effects of Se,providing new ideas for the prevention and treatment of testicular injury caused by Cd.The 20-week-old chicken Leydig cells were isolated,purified,and cultured.They were treated with different concentrations of Cd and/or Se,and the cell viability was measured using the CCK-8 method.The optimal treatment concentration and duration of Cd and Se were screened based on morphological observations.The experiment was divided into six groups:control group,Cd group,Se group,Se+Cd group,4-PBA+Cd group,and Nec-1+Cd group.The changes in reactive oxygen species（ROS）were detected using the DCFH-DA fluorescent probe method,and the activities of CAT,SOD,GSH,GSH-Px,MDA,and SOD were measured using biochemical methods.The mRNA and protein expression levels of endoplasmic reticulum stress marker genes（GRP78,PERK,ATF6,IRE1,CHOP,JNK）and procedural necrosis marker genes（TNF-α,TNFR1,RIP1,RIP3,MLKL,PARP1,Caspase-8）were detected using fluorescence quantitative PCR and Western-blot.The trends in the changes of mRNA and protein expression levels were analyzed.The experimental results are as follows:1.The optimal concentration was screened by CCK-8 method,and the cell viability tended to be stable at 12 h,24 h,36 h,and 48 h when treated with 20μM CdCl₂,and the difference was significant with other concentrations（P<0.05）.20μM was selected as the concentration of Cd treatment.CCK-8 method was used to select the optimal concentration of Se treatment.When 5μM Na₂SeO₃ treatment was used for 12 h,24 h,36 h,and 48 h,the cell viability tended to be stable at each time,and the difference was significant（P<0.05）.5μM was selected as the concentration of Se treatment.2.Compared with the control group,the activities of CAT,GSH-PX and SOD and the concentration of GSH in Cd group were significantly decreased（P<0.05）,the concentration of MDA and the content of ROS were increased（P<0.05）,and the concentration of MDA and the content of ROS in Se+Cd group were significantly decreased compared with that in Cd group（P<0.05）.The activities of CAT,GSH-PX,SOD and GSH concentration were significantly increased（P<0.05）.3.Compared with the control group,the expressions of ER marker genes GRP78,PERK,ATF6,IRE1,CHOP,and JNK in the Cd group were significantly increased（P<0.05）,and the abnormal expression of mRNA and protein levels of ER stress-related genes could be significantly reduced after 4-PBA pretreatment.The mRNA and protein expressions of GRP78,PERK,ATF6,IRE1,CHOP,and JNK in Se+Cd group were significantly decreased compared with those in Cd group（P<0.05）.4.Compared with the control group,the mRNA and protein expressions of TNF-α,TNFR1,RIP1,RIP3,MLKL,and PARP1 in Cd group were significantly increased（P<0.05）,while the mRNA and protein expressions of Caspase-8 were significantly decreased（P<0.05）.Nec-1 preconditioning could reduce the mRNA and protein expressions of Cd-induced programmed necrosis of chicken Leydig cells but had no effect on the mRNA and protein expressions of endoplasmic reticulum stress marker genes GRP78,ATF6,PERK,IRE1,CHOP,and JNK induced by Cd poisoning.After 4-PBA pretreatment,the expressions of TNF-α,TNFR1,RIP1,RIP3,MLKL,and PARP1 induced by Cd poisoning were increased,and the expression of Caspase-8 was up-regulated.The mRNA and protein expression changes of Cd-induced programmed necrosis of chicken testis Leydig cells in Se+Cd group were significantly reduced（P<0.05）.In conclusion,Cd can induce oxidative stress and endoplasmic reticulum stress in chicken testicular Leydig cells,and lead to programmed necrosis of Leydig cells.Se alleviated Cd-induced programmed necrosis of chicken testicular Leydig cells.