Effect And Mechanism Of Zearalenone On The Proliferation And Apoptosis Of Sika Deer Antler Mesenchymal Cells

The top mesenchymal cells are the growth centers of deer antlers,controlling the development of deer antlers.The growth and development of deer antlers are not only controlled by the growth centers but also affected by external factors,with feeding management being one of the most important factors.Sika deer mainly feed on coarse feed,and supplement their diet with concentrate feed made from corn,soybeans,and bran.However,zearalenone(ZEA)is widely present in moldy grains and animal products.ZEA not only directly affects crop growth but also disrupts animal body functions.Flow cytometry and fluorescent quantitative PCR were used to study the effect of ZEA on the proliferation and apoptosis of antler mesenchymal cells and to explore the mechanism of TAZ,Nrf2,GSH,ROS,and mitochondrial function by ZEA.The experimental results showed that the addition of ZEA could inhibit the proliferation activity of antler mesenchymal cells,reduce the number of cells in the S phase.Fluorescent quantitative PCR was used to detect the expression of cell cycle proteins Ccna1,Ccnb1,Ccnd1,Ccnd2,Ccnd3,Ccne1,as well as the expression of cell cycle protein kinases Cdk1,Cdk2,Cdk4,Cdk6,all of which were reduced by ZEA.Further studies found that ZEA could promote apoptosis of antler mesenchymal cells,increase the expression and activity of pro-apoptotic factors Bax and Casp3,and inhibit the expression of anti-apoptotic factor Bcl2.To explore the regulatory mechanism of ZEA on the proliferation and apoptosis of antler mesenchymal cells,we analyzed the effect of ZEA on the cell’s antioxidant capacity and intracellular ROS level.The results showed that adding ZEA could significantly increase the content of oxidative stress marker MDA and intracellular ROS level,while reducing the activities of GPX and GR antioxidant enzymes,as well as the content of GSH antioxidant and the GSH/GSSG ratio.We added GSH or ROS scavenger NAC to antler mesenchymal cells treated with ZEA.The results showed that the intracellular ROS level and oxidative stress marker MDA content were significantly reduced,the cell proliferation activity was enhanced,the number of cells in the S phase increased,and the expression of cell proliferationrelated factors was restored.The apoptosis rate of antler mesenchymal cells was significantly inhibited,the expression of Bax and Casp3 was significantly reduced,and the activity of Casp3 was significantly inhibited,but the expression of Bcl2 was restored.The above results indicate that ZEA can increase the intracellular ROS level by damaging the cell’s antioxidant capacity,thereby affecting the proliferation and apoptosis of antler mesenchymal cells.To further investigate the mechanism by which ZEA regulates proliferation and apoptosis of antler mesenchymal cells,we first examined the regulation of Nrf2 by ZEA.Results showed that the expression of Nrf2 in antler mesenchymal cells was significantly decreased after ZEA treatment.Additionally,the luciferase activity decreased,indicating that ZEA could inhibit the transcriptional activity of Nrf2.Further studies found that antler mesenchymal cells were treated with ZEA and then added with the Nrf2 activator,Oltipraz.Results showed that cell proliferation was significantly enhanced with an increase in the number of S-phase cells,while the apoptosis rate of antler mesenchymal cells was significantly reduced,resulting in the restoration of the expression of proliferation and apoptosis-related factors.Moreover,adding Oltipraz could enhance the activity of GPX and GR antioxidant enzymes,increase the content of GSH and GSH/GSSG ratio,and reduce the level of intracellular ROS and MDA in ZEA-treated antler mesenchymal cells.These results suggest that activating Nrf2 can prevent ZEA from regulating the proliferation and apoptosis of antler mesenchymal cells by improving the cell’s antioxidant capacity.In antler mesenchymal cells,ZEA can inhibit the expression of TAZ,leading to a significant reduction in TAZ/TEAD transcriptional activity.Adding TAZ activator TM-25659 to ZEA-treated antler mesenchymal cells significantly enhanced the cell’s antioxidant capacity,reduced the level of intracellular ROS,restored cell proliferation,and reduced apoptosis.However,adding Nrf2 inhibitor ML385 inhibited the rescue effect of TM-25659 on the above effects.Further studies found that adding TM-25659 could weaken the inhibitory effect of ZEA on Nrf2 expression,significantly increase the transcriptional activity of Nrf2,indicating that Nrf2 is a downstream target gene of TAZ.Mitochondria are important organelles for cellular energy production,and damage to mitochondrial function can lead to cell apoptosis.In order to investigate the effect of ZEA on the mitochondrial function of antler mesenchymal cells,we first analyzed changes in ATP synthesis and mitochondrial membrane potential.After ZEA treatment,the ATP content in antler mesenchymal cells was significantly reduced,and the mitochondrial membrane potential decreased,but this effect could be rescued by the TAZ activator TM-25659.Addition of the Nrf2 inhibitor ML385 blocked the rescue effect of TM-25659 on ZEA,while supplementing with GSH or ROS scavenger alleviated the inhibitory effect of ML385.Meanwhile,after transfection with the mitoDs Red plasmid and adding ZEA,we observed and analyzed changes in mitochondrial morphology in antler mesenchymal cells under a fluorescence microscope.The results showed that the mitochondrial reticular structure in antler mesenchymal cells was damaged after ZEA treatment.The TAZ activator TM-25659 could alleviate the damage to the mitochondrial structure caused by ZEA.However,the Nrf2 inhibitor ML385 blocked the rescue effect of TM-25659 on ZEA.Nevertheless,supplementing with GSH or ROS scavenger could alleviate the inhibitory effect of ML385.These results suggest that ZEA can cause mitochondrial dysfunction in antler mesenchymal cells through the TAZ-Nrf2-GSH-ROS pathway.In summary,ZEA can inhibit the proliferation of antler mesenchymal cells,induce apoptosis,cause mitochondrial dysfunction,and ultimately impair the growth and development of deer antlers,through the TAZ-Nrf2-GSH-ROS pathway.