| Zearalenone(ZEA)has a very high detection rate in feed grain and feed samples,and pigs are the most sensitive to it.ZEA not only has serious toxic effects on animals,but also harms human health through animal products.Lycopene(LYC)is a kind of fat soluble natural carotenoid,which has powerful antioxidant,anti-inflammatory,anti-cancer,anti cardiovascular disease and detoxification effects.A lot of studies have been carried out on the toxicity of ZEA and the development of protective agents at home and abroad,but the mechanism of toxicity of ZEA on Sertoli cells(SCs)of piglets and the protective effects of LYC have not been reported.Based on the above research status,piglets SCs were used as the research object,to investigate the mechanism of oxidative stress and autophagy in ZEA-induced piglets sertoli cell injury and evaluat the protective effects of LYC based on Nrf2/HO-1 pathway.CCK-8 assay was used to determine the survival rate of piglets SCs after ZEA and/or LYC treatment.The ultrastructural changes of cells were observed by electron microscopy;cell apoptosis rate and ROS level were detected by flow cytometry;the activities of antioxidant enzymes(T-SOD,HO-1,GSH-PX)and the content of MDA were detected by ELISA;the expression and distribution of LC3 and Nrf2 in cells were detected by immunofluorescence;the expression levels of the upstream and downstream genes of Nrf2/HO-1 signaling pathway as well as autophagy and apoptosis related genes were detected by qRT-PCR;WB was used to detect the expression levels of related proteins.Combined with the experimental data,the toxic mechanism of ZEA on piglets SCs and the cytotoxic protective effects of LYC against ZEA were comprehensively analyzed.These results showed that:1.Effects of ZEA and LYC on survival rate and morphology of piglets SCs.The results showed that the cell survival rate was significantly decreased by 25 μM ZEA(P<0.01),and the cell survival rate was significantly affected when LYC concentration was greater than 40 μM(P<0.01).The cells were pretreated with 0-35 μM LYC for 3 h and added with ZEA,when the concentration of LYC was 30 μM,the survival rate of cells was the highest(P<0.01)compared with control group.Therefore,the optimal concentration of ZEA and LYC was determined to be 25 and 30 μM,respectively.The results of laser confocal microscope and electron microscope showed that the morphology and ultrastructure of piglets SCs were seriously damaged by ZEA,cell chromatin aggregates,organelle number decreases,both the endoplasmic reticulum and mitochondria were swollen,the number of mitochondria decreased and the mitochondria were concentrated and the mitochondrial ridges were thickened.LYC pretreament alleviated the cell morphologic changes and organelle damage induced by ZEA.2.Effects of ZEA and LYC on oxidative stress and cell apoptosis in piglets SCs.The results showed that,compared with control group,ZEA significantly increased apoptosis rate,level of ROS and MDA content(P<0.01),significantly decreased the activities of HO-1,GSH-Px and T-SOD(P<0.01),apoptosis rate and ROS level have no significant change in LYC group,the activities of HO-1,GSH-Px and T-SOD in LYC group significantly increased(P<0.05 or P<0.01),MDA content in LYC group was significantly decreased(P<0.01).Compared with ZEA group,LYC+ZEA group HO-1,GSH-Px and T-SOD enzyme activities increased significantly(P<0.01),apoptosis rate,ROS level and MDA content decreased significantly(P<0.01),ML385+ZEA group apoptosis rate,HO-1,GSH-Px and T-SOD activities decreased significantly(P<0.01),ROS level and MDA content were significantly increased(P<0.01),and the apoptosis rate of RAP+ZEA group decreased significantly(P<0.01),the apoptosis rate of CQ+ZEA group increased but not significantly.3.Effects of ZEA and LYC on Nrf2/HO-1 signaling pathway and autophagy in piglets SCs.Immunofluorescence results showed that ZEA significantly increased the expression of LC3 and decreased the nuclear expression of Nrf2,while LYC promoted the nuclear expression of Nrf2.LYC pretreatment downs regulate the expression of LC3 induced by ZEA and improve the expression of Nrf2 inhibited by ZEA.The results of q RT-PCR and WB showed that compared with the control group,the gene and protein expression levels of keap1,LC3,beclin-1 and Bax in ZEA group and ML385 group were significantly increased(P<0.01),Nrf2,HO-1,Gpx1 and bcl-2 expression levels were significantly decreased(P<0.01),and the nuclear protein expression level of Nrf2 was significantly decreased(P<0.01);compared with ZEA group,the gene and protein expression levels of keap1,LC3,beclin-1 and Bax in LYC+ZEA group were significantly decreased(P<0.01),Nrf2,GPX1 and bcl-2 expression levels were significantly increased(P<0.01),the nuclear protein expression level of Nrf2 was significantly increased(P<0.01),and the protein expression level of HO-1 was increased but not significant;the gene and protein expression levels of keap1,beclin-1 and bax in ML385+ZEA group were significantly increased(P<0.01),and the expression levels of Nrf2,HO-1,GPX1 and bcl-2 were significantly decreased(P<0.01),and the mRNA expression level of LC3 was increased but not significantly.In conclusion,ZEA exposure induced morphological changes and ultrastructural damage of piglets SCs,induced abnormal autophagy,increased apoptosis,promoted the production of ROS,inhibited the expression of Nrf2/HO-1 signaling pathway and its downstream genes to cause oxidative stress.LYC pretreatment alleviated the morphological changes and ultrastructural damage of piglets SCs induced by ZEA,by activating Nrf2/HO-1 signaling pathway to eliminate ROS,ZEA-induced autophagy and oxidative stress in piglets SCs were alleviated,and cell apoptosis was reduced. |
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