Study On The Molecular Mechanism Of ZEA-induced Apoptosis In Porcine ESCs Based On JNK Pathway Activated By Endoplasmic Reticulum/mitochondria Pathway

Zearalenone（ZEA）is an estrogen-like mycotoxin with high detection rate in agricultural products and feeds,which seriously endangers the healthy development of animal husbandry.Studies have shown that ZEA has reproductive toxicity and can affect the reproductive performance of animals.At present,the studies on reproductive toxicity of ZEA in China and abroad mainly focus on male animals,and there are few reports on the toxicity damage of reproductive cells in sows.Therefore,in this experiment,porcine endometrial stromal cells（ESCs）were used as the research object to study the molecular mechanism of ZEA-induced apoptosis of porcine ESCs based on the activation of JNK pathway through endoplasmic reticulum/mitochondria pathway.In this study,the cell viability was detected by CCK-8 method,and the optimal concentration of ZEA and inhibitor was selected by this method.The ultrastructure changes were observed by transmission electron microscope.The cell growth status and the expression and distribution of Cyt C were detected by laser confocal microscope;the apoptosis rate,mitochondrial membrane potential（MMP）,reactive oxygen species（ROS）and Ca²⁺level were detected by flow cytometry;the relative expression levels of ERS genes,apoptosis-related genes and JNK pathway genes were detected by q RT-PCR.The relative expression of ERS protein,apoptosis-related protein and JNK pathway protein were detected by Western-blot,and the toxic effect of ZEA on porcine ESCs and its molecular mechanism of action were comprehensively evaluated.The experimental results showed that:1.Toxic effects of ZEA on porcine ESCs.The results showed that when the concentration of ZEA was 15μM,the cell viability was significantly decreased（P<0.01）,and reaching half inhibitory concentration.When the concentration of ERS inhibitors（4-Phenylbutyric acid,4-PBA）was 1.5μM,the concentration of Mitochondrial fission inhibitor 1（Mdivi）was 1.0μM,there was no damage to the ESCs,and the damage to the cells caused by ZEA was significantly alleviated（P<0.01）.The results of laser confocal microscopy showed that with the increase of ZEA concentration,the green fluorescence decreased and the red fluorescence increased gradually with Calcein-AM staining,indicating that the proportion of dead cells was increased than that of living cells.The results of transmission electron microscopy showed cell shrinkage,organelle rupture,chromatin aggregation,and cell membrane damage were observed,indicating that ZEA could induce the ultrastructure damage of porcine ESCs in a dose-dependent manner.2.ZEA induced apoptosis by activating JNK pathway through ER pathway.The results showed that compared with the control group,the levels of ROS,Ca²⁺and the apoptosis rate in ZEA group were highly significantly increased（P<0.01）,while there was no significant change in 4-PBA group（P>0.05）.The levels of ROS,Ca²⁺and the apoptosis rate in P+Z group were highly significantly decreased compared with ZEA group（P<0.01）.The results by q RT-PCR and WB showed that the expression of CHOP,GRP 78,HSP 70,PERK and ASK 1 genes and protein in ZEA group were extremely significantly increased compared with the CON group（P<0.01）.The expression of Caspase 3,Caspase 9 and Bax/Bcl-2 genes and protein in ZEA group were significantly increased or extremely significantly increased compared with the control group（P<0.05 or P<0.01）.Compared with the control group,the relative expression of JNK m RNA in ZEA group was extremely significantly increased（P<0.01）,the expression level of nuclear protein p-JNK was extremely significantly increased（P<0.01）.The group of 4-PBA compared with the control group,each gene had no significant change,compared with the ZEA group,the genes and protein expression of CHOP,GRP 78,HSP 70,PERK,ASK 1,Caspase 3 and Caspase 9 in the P+Z group were significantly or extremely significantly decreased（P<0.05 or P<0.01）,JNK m RNA in the P+Z group was extremely significantly decreased（P<0.01）,and the expression level of nuclear protein p-JNK in the P+Z group was extremely significantly decreased（P<0.01）.3.ZEA induced apoptosis by activating JNK pathway through mitochondrial fission pathway to induce apoptosis.The results showed that compared with the control group,the levels of ROS,Ca²⁺and the apoptosis rate in the ZEA group were extremely significantly increased（P<0.01）,MMP in the ZEA group was extremely significantly decreased（P<0.01）,and there was no significant change in the Mdivi group（P>0.05）.After adding inhibitors,the levels of ROS,Ca²⁺and the apoptosis rate in M+Z group were extremely significantly decreased than those in ZEA group（P<0.01）,MMP in M+Z group extremely significantly increased compared with the ZEA group（P<0.01）.The results of immunofluorescence showed that the released of Cyt C was obvious in ZEA group（P<0.01）.Cyt C detected by WB showed that ZEA could extremely significantly increase the intracellular Cyt C expression（P<0.01）.The expression of Cyt C protein in M+Z group was significantly lower than that in ZEA group（P<0.01）.The results of q RT-PCR and WB showed that compared with the control group,the expression of Mfn 1 and Mfn 2 genes and protein in ZEA group were extremely significantly decreased（P<0.01）,and the expression of Fis 1 and Drp 1,Bax/Bcl-2,Caspase 3 and Caspase 9 genes and protein were extremely significantly increased（P<0.01）.Compared with the CON group the expression of JNK m RNA in ZEA group was significantly increased（P<0.01）,and the expression of nuclear protein p-JNK was significantly increased（P<0.01）.After addition of Mdivi,the genes and protein expression levels of Mfn 1 and Mfn 2 in the M+Z group were significantly higher than those in the ZEA group（P<0.05）.The genes and protein expression of Fis 1,Drp 1,Bax/Bcl-2,Caspase 3 and Caspase 9 in the M+Z group were significantly or extremely significantly decreased（P<0.05 or P<0.01）,the JNK m RNA in the M+Z group was extremely significantly decreased（P<0.01）,and the relative expression of nuclear protein p-JNK in the M+Z group was extremely significantly decreased compared with the ZEA group（P<0.01）.In conclusion,exposure of porcine ESCs to ZEA can produce excessive ROS and Ca²⁺caused cell MMP decreased,increased the apoptosis rate,caused cell ERS and mitochondria fission,and induce apoptosis after activation of JNK signaling pathway.When inhibiting the ER and mitochondrial pathway,it can effectively alleviate ZEA damage on pig ESCs and reduce apoptosis.