| Zearalenone(ZEA)is a non-steroidal mycotoxin produced by the genus Fusarium fungi.Research has shown that ZEA is associated with endogenous factors in animals β Estradiol which has a similar structure and can bind to estrogen receptors in mammals.In addition to causing reproductive dysfunction in female animals,it can also cause morphological and biological changes in male reproductive organs,leading to a decrease in male reproductive ability.Orchitis response is one of the important pathogenic factors leading to male infertility.Although many scholars have studied the effects of ZEA on the male reproductive system from aspects such as oxidative damage,cell apoptosis,endocrine interference,etc.,there are little few studies research on whether ZEA can affect the male reproductive system through inflammatory response,especially the lack of relevant molecular mechanism research.Therefore,this experiment investigated the effects and molecular mechanisms of ROSCa2+regulation of NF-κB/NLRP3 and autophagy on the expression of inflammatory factors in testicular sertoli cells by establishing three-dimensional mouse and cellular ZEA poisoning models,so as to provid scientific theoretical basis for the prevention and treatment of ZEA poisoning disease.1.The effect of ZEA induced the secretion of inflammatory factors in sertoli cells and the role of NF-κB/NLRP3 signaling pathwayTo investigate the effect of ZEA on the secretion of inflammatory factors in testicular sertoli cells and the role of NF-κB/NLRP3 signaling pathway,6-week-old BABL/c mice were continuously gavaged with 40 mg/kg-bw ZEA for 5 days to establish a three-dimensional endotoxicity model.Testis were collected and histological changes were observed.The mRNA levels of inflammatory factors IL-6,TNF-α,MCP-1 and IL-1β were detected by fluorescence quantitative PCR.Western blot was used to detect changes in the expression of NF-κB/NLRP3 signaling pathway related proteins.In vitro experiments were conducted using TM4 cells(mouse testis supporting cell line)as the research model.TM4 cells were exposed to different concentrations of ZEA(0,5,10,20μmol/L)or ZEA(20 μmol/L)in combination with NF-κB inhibitors(PDTC)(5 μmol/L)for 24 hours,and the levels of inflammatory cytokines in the supernatant were detected by ELISA kit.We stern blot was used to detect the expression of NF-κB/NLRP3 signaling pathway related proteins.In vivo test results showed that ZEA can make the lumen of the mouse testicular seminiferous tubule smaller.The spermatid at all levels were reduced and arranged in disorder,and the number of mature sperm cells in the lumen was significantly reduced.The qRT-PCR results showed that the-level of gene transcription of IL-1β,MCP-1,IL-6,TNF-α were significantly increased(P<0.05 or P<0.01).The expression of p-IκB and p-NF-κB/NF-κB were significantly increased(P<0.05 or P<0.01).The expression of NLRP3 increased,but the difference was not significant(P>0.05);the expression of IL-1β p17,Caspase1 p20,TNF-α were significantly increased(P<0.05 or P<0.01).The in vitro test results showed that with the increase of ZEA concentration,the contents of IL-1β,TNF-α in TM4 cells supernatant were significantly increased(P<0.05 or P<0.01).The expression of p-IκB and p-NF-κB/NF-κB was significantly increased with 10 μmol/L,20μmol/L of ZEA(P<0.01).The expression level of NF-κB nucleprotein was significantly increased(P<0.01).The expression of NLRP3,Caspase1 p20,IL-1β p17,TNF-α were significantly increased(P<0.05 or P<0.01).After being co-treated by ZEA and PDTC,compared with the cells exposed to ZEA alone group,the contents of IL-1β,TNF-α in TM4 cells supernatant were significantly decreased(P<0.05 or P<0.01).The ratios of p-NFκB/NF-κB,and the expression of NLRP3,Caspase1 p20,IL-1β p17,TNF-α were significantly decreased(P<0.05 or P<0.01).The data suggested that ZEA induced expression of inflammatory factors in sertoli cells through NF-κB/NLRP3 pathway.2.The role of ROS-Ca2+regulates NF-κB signaling pathway in ZEA induced expression of inflammatory factors in sertoli cellsIn order to explore the role of ROS-Ca2+regulates the NF-κB signaling pathway in ZEA induced expression of inflammatory factor in sertoli cells,the TM4 cells were exposed with different concentrations of ZEA(0,5,10,20 μmol/L)or ZEA(20 μmol/L)combined with BAPTA-AM(5 μmol/L),2-APB(5 μmol/L),NAC(10 μmol/L)for 24 h.The flow cytometry was used to detect changes in intracellular Ca2+levels.ELISA kit was used to detect the secretion of inflammatory factors in the TM4 cells supernatant.The NF-κB/NLRP3 pathway related proteins were analyzed by using Western Blot.The data indicated that with the increase of ZEA exposure concentration,the concentration of Ca2+ in the cytoplasm significantly increased(P<0.05 or P<0.01),while the combined treatment with BAPTA-AM or 2-APB were significantly decreased(P<0.05or P<0.01).After the combined treatment of ZEA and 2-APB,compared with the ZEA-treated group alone,the expression of p-IκB,and the ratios of p-NF-κB/NF-κB were significantly decreased(P<0.01).The expression of NLRP3,Caspase1 p20,IL-1β p17,TNF-α were significantly decreased(P<0.05 or P<0.01).Previous studies in our group have shown that the intracellular levels of ROS were increased with ZEA in sertoli cells,while NAC could alleviate the level of ROS.Therefore,further,after being cotreated with NAC,the level of intracellular Ca2+was significantly decreased(P<0.01),and the contents of IL-1β,TNF-α in TM4 cells supernatant were significantly decreased(P<0.01).The ratios of p-NF-κB/NF-κB and the expression of p-IκB,NLRP3,TNF-α,Caspase1 p20 were significantly decreased(P<0.05 or P<0.01),the expression of IL-1β p17 was decreased,but the difference was not significant(P>0.05).The results showed that ROS up-regulateds the expression of NF-κB/NLRP3 pathway related proteins through partial Ca2+ released by endoplasmic reticulum IP3R,thus affecting the inflammatory factors of ZEA induced sertoli cells.3.The role of autophagy in ZEA induced expression of inflammatory factors in sertoli cellsTo investigate the role of autophagy in ZEA induced expression of inflammatory factor in sertoli cells.Western blot was used to detect the expression of autophagy related proteins Beclinl,LC3,and AMPK signaling pathway proteins in testicular tissue.In vitro experiments,TM4 cells were treated with different concentrations of ZEA(0,5,10,20 μmol/L)or ZEA(20μmol/L)combined with AMPK inhibitor Compound C(5 μmol/L)for 24 h.Western blot was used to detect the expression of autophagy related proteins Beclinl,LC3,and AMPK pathway proteins.Immunofluorescence was used to observe the fluorescence aggregation point of LC3;ZEA(20μmol/L)was combined with autophagy inhibitor 3-AM(5 μmol/L)for 24 h.Western blot was used to detec of the NF-κB/NLRP3 pathway related protein expression.In vivo experimental results showed that compared with the control group,the expression levels of LC3,Beclinl,and the ratios of p-AMPK/AMPK were significantly increased with ZEA group(P<0.05 or P<0.01),while the expression levels of p-mTOR/mTOR and p-P70S6K/P70S6K were significantly decreased(P<0.05 or P<0.01).In vitro test results showed that with the increase of ZEA concentration,the expression levels of LC3 and the ratios of p-AMPK/AMPK significantly increased(P<0.05 or P<0.01),the expression levels of p-mTOR/mTOR and pP70S6K/P70S6K significantly decreased(P<0.01).After the combined treatment of ZEA and compound C,compared with the ZEA-treated group alone,the expression of p-AMPK/AMPK and LC3 were significantly inhibited(P<0.01),and the expression of p-mTOR/mTOR,pP70S6K/P70S6K were significantly upregulated(P<0.01),as well as the number and fluorescence intensity of LC3 aggregation points was reduced.Compared with the ZEA alone treatment group,the expression of NLRP3,Caspase1 p20,IL-1β p17,TNF-α ZEA and 3-MA co-treatment groups was significantly decreased(P<0.05 or P<0.01).The results showed that ZEA could promote the expression of inflammatory factors by inducing the autophagy levels of sertoli cells through AMPK signaling pathway.4.The role of ROS-Ca2+ regulates AMPK signaling pathway in ZEA induced autophagy in sertoli cellsIn order to investigate the role of ROS-Ca2+ mediated AMPK signaling pathway in sertoli cells induced by ZEA,the TM4 cells were exposed to ZEA(20 μmol/L)combined with 2APB(5 μol/L),NAC(10 μmol/L)for 24 h.Western blot was used to detect the expression of AMPK signaling pathway and autophagy associated proteins.The number of LC3 aggregation points of TM4 cells were detected by immunofluorescence.The results showed that after the combined treatment of ZEA and compound C,compared with the ZEA-treated group alone,the expression of p-CaMKKβ,p-AMPK/AMPK and LC3 significantly decreased(P<0.05 or P<0.01).The expression of p-mTOR/mTOR and p-P70S6K/P70S6K significantly increased(P<0.05 or P<0.01).After the combined treatment of ZEA and NAC,the expression levels of p-AMPK/AMPK,LC3 and the number of LC3 aggregation points significantly decreased(P<0.05),and the expression levels of p-mTOR/mTOR,pP70S6K/P70S6K significantly increased(P<0.05 or P<0.01).These results indicated that ROS-Ca2+ could activate AMPK signaling pathway and enhance the autophagy level of sertoli cells induced by ZEA.It is concluded that the male reproductive toxicity of ZEA may affect the development of spermatozoa with the inflammation of sertoli cells.ZEA could induce the inflammatory factors of mice sertoli cells by ROS-Ca2+-NFκB and ROS-Ca2+-autophagy pathway. |
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