Zearalenone Induces Intestinal Oxidative Stress And Its Mechanism In Post-weaning Piglets

Zearalenone(ZEA)is a common pollutant in cereal crops and has varying degrees of toxicity to animals and humans.Exposure to ZEA will affect the reproduction of all kinds of livestock,and the most affected species is pigs.The annual loss caused by ZEA to pig farms in the world is as high as millions of dollars,so the research on ZEA has become a hot spot.However,the research on ZEA at home and abroad mainly focuses on the reproductive organs of pigs and mice,and there are few studies on the effect of ZEA on the intestines of postweaning piglets.This study aims to study the intestinal oxidative stress induced by ZEA in post-weaning piglets and its mechanism through a combination of in vivo and in vitro experiments,and to explore the Keap1-Nrf2 signaling pathway that may be a key target for the prevention and treatment of ZEA-induced intestinal injury.This provides theoretical basis for the study of the molecular mechanism of ZEA changing nutrient absorption and the revision of feed hygiene standards in China.1.ZEA-induced intestinal oxidative stress and its mechanism in post-weaning pigletsIn this experiment,forty healthy post-weaning piglets(Duroc × Landrace × Yorkshire)with body weight of 14.01±0.86 kg were randomly allocated into 4 treatments(10 replicates per treatments,1 pig per replicate).Post-weaning piglets in the Control group were fed basal diet,and those in the Experiment group were fed supplemented with diets 0.5 mg/kg ZEA,1.0mg/kg ZEA,1.5 mg/kg ZEA(ZEA0.5,ZEA1.0,ZEA1.5),respectively.The experiment include 10 d pre-trial period and then 35 d test period,and aimed to study the effects of ZEA(0.5～1.5 mg/kg)on the duodenum,jejunum and ileum in post-weaning piglets.The results showed that compared with the control group,the activities of aspartate aminotransferase,alkaline phosphatase and lactate dehydrogenase in serum of ZEA group were significantly increased(P < 0.05).Serum urea nitrogen and high-density lipoprotein in the ZEA1.5treatment group increased significantly(P < 0.05),while the total protein decreased significantly(P < 0.05).The morphological results of the small intestine showed that ZEA induced a significant increase in the villus height and crypt depth of duodenum and jejunum(P < 0.05),the arrangement was loose and disordered,the number of small intestinal glands decreased,and the thickness of the mucosa became thinner.In the ZEA0.5 and ZEA1.0treatment groups,the ratio of villus height to crypt depth in the duodenum and ileum were increased significantly(P < 0.05),and the ratio of villus height to crypt depth in the jejunum was decreased(P < 0.05).With increasing concentrations of dietary ZEA,the activities of antioxidant enzymes T-SOD and GSH-PX,the relative expression of Keap1 at m RNA and protein level in the duodenum,jejunum and ileum decreased linearly and quadratically(P<0.05),and the MDA content,the relative expression of Nrf2,Gpx1 and Ho1 at m RNA and protein level,the expression of Nrf2 and Gpx1 immunoreactive substances increased quadratically(P < 0.05).The order of Nrf2 immunoreactivity in different intestinal tissues is jejunum > ileum > duodenum,and the order of Gpx1 is ileum > jejunum > duodenum.2.ZEA affects nutrient absorption and induces oxidative stress in porcine intestinal epithelial cells and its mechanismIn order to explore the regulation mechanism of ZEA-induced intestinal oxidative stress,the test first constructed an in vitro culture model of porcine jejunal epithelial cells(IPEC-J2)to determine whether ZEA induced oxidative stress in IPEC-J2 cells.Construct a IPEC-J2 cell model,add different concentrations of ZEA(10,20,40,80,160 μmol/L)when the cells are in the logarithmic growth phase and continue to culture,and set the blank control group,after 24 and 36 h collect cells for follow-up experiments.The results showed that the cell morphology and cell viability changed significantly when different concentrations of ZEA treated IPEC-J2 cells for 24 and 36 hours.When the cells were treated with 40 μmol/L ZEA for 36 h,the cell viability was about 50%,and the cells were seriously damaged and began to die in large numbers.So the selected ZEA concentration was 10,20,40 μmol/L(ZEA10,ZEA20,ZEA40),and the time gradient was 24 and 36 hours for the following study.The results showed that with the increase of ZEA(10,20,40 μmol/L)concentration and the prolongation of the action time,the apoptosis rate of IPEC-J2 cells increased significantly(P < 0.05),the TEER value of IPEC-J2 cells reduced significantly(P < 0.05),indicating that the toxic effect of ZEA on IPEC-J2 cells has a time and dose effect.The relative expression of SGLT1 and GLUT2(36 h)at m RNA and protein level in IPEC-J2 cells increased linearly and quadraticly(P < 0.05).SGLT1 immunofluorescence results showed that with the increase of ZEA concentration,the fluorescence intensity around the cytoplasm and the cell membrane decreased,indicating that the expression of SGLT1 in the cell membrane was significantly reduced.The results of ROS flow cytometer histogram and immunofluorescence showed that the mean fluorescence intensity(MFI)of ROS increased significantly(P < 0.05)with the increase of ZEA concentration at 24 h and 36 h compared with the control group,and the immunofluorescence intensity around the nucleus increased.When the cells were treated with40 μmol/L ZEA for 36 h,the expression of ROS reached the highest level.The T-SOD and GSH-PX activities and relative expression of Keap1 at m RNA and protein level in IPEC-J2 cells decrease quadratically(P < 0.05)with the increase of ZEA concentration,while MDA level and relative expression of Nrf2,Nqo1,Ho1,and ROS at m RNA and protein level showed linear and quadratic(P < 0.05)increase.The immunofluorescence results of Nrf2 showed that with the increase of ZEA concentration,the fluorescence intensity around and in the nucleus increased significantly,indicating that Nrf2 began to move to the nucleus.When the ZEA concentration was 40 μmol/L at 24 h,the expression of Nrf2 increased significantly,and the invasion of Nrf2 into the nucleus was very obvious.3.Study on the effect of Sh RNA interference Nrf2 expression on ZEA-induced oxidative stress in porcine jejunal epithelial cellsThe experiment were constructed a model of IPEC-J2 cells and set blank(Control),negative control group(Sh-control),positive control group(Sh-Nrf2),to determine the effect of Nrf2-mediated signal pathway on ZEA-induced oxidative stress in IPEC-J2 cells.When the successfully transfected IPEC-J2 cells by Sh-Nrf2 were in the logarithmic growth phase,10,20,and 40 μmol/L ZEA(Sh-Nrf2+ZEA10,Sh-Nrf2+ZEA20,Sh-Nrf2+ZEA40)were added,and the cells were collected after 24 and 36 h culture for subsequent experiments.The results of the study showed that the apoptosis rate of IPEC-J2 cells in the Sh-Nrf2 group was significantly higher than that of the Control and Sh-control groups at 24 h and 36 h(P < 0.05).Compared with the Sh-Nrf2 group,in IPEC-J2 cells that interfered with Nrf2 expression,ZEA significantly increased in a time and dose-dependent manner of the apoptotic rate(P <0.05),indicating that both the interference cell Nrf2 expression and high concentration ZEA can induce apoptosis.Compared with the Control and Sh-control groups,the T-SOD and GSH-PX activities,the Nrf2 immunofluorescence intensity and the relative expression of Keap1,Nrf2,Nqo1,and Ho1 at m RNA and protein level of IPEC-J2 cells in the Sh-Nrf2 group were significantly reduced(P < 0.05),and the MDA level,the relative expression level of ROS immunofluorescence,m RNA and protein were significantly increased(P < 0.05)at24 and 36 h.Compared with the Sh-Nrf2 group,the activities of T-SOD and GSH-PX and relative expressions of Keap1 at m RNA and protein level in the Sh-Nrf2+ZEA20 and ShNrf2+ZEA40 treatment groups were significantly reduced(P < 0.05),the MDA level,and the fluorescence intensity around and within the nucleus of ROS and Nrf2,and the relative expressions of Nrf2,Nqo1,Ho1,and ROS at m RNA and protein level significantly increased(P < 0.05).When the ZEA concentration was 40 μmol/L,the expression of Nrf2 reached the highest at 24 h,the ROS expression reached the highest at 36 h.In summary,this study draws the following conclusions:(1)The addition of ZEA(0.5-1.5 mg/kg)in the diet changed the metabolism and intestinal morphology of post-weaning piglets,induced intestinal oxidative stress,which in turn affected the healthy growth of postweaning piglets.(2)When ZEA induced intestinal oxidative stress by activating the Keap1-Nrf2 signaling pathway,and therefore affected the intestinal development.(3)ZEA induced oxidative stress,promoted the occurrence of apoptosis,and changed the glucose absorption capacity and antioxidant capacity of IPEC-J2 cells.(4)The Keap1-Nrf2 signaling pathway was activated in oxidative stress induced by ZEA in IPEC-J2 cells.With the prolongation of ZEA treatment time,the expression of Nrf2 gradually decreased,and the effect of Keap1-Nrf2 signaling pathway gradually weakened.(5)Interfering with the expression of Nrf2 in IPEC-J2 cells affected the activation of the Keap1-Nrf2 signaling pathway and reduced the ability of cells to resist ZEA-induced oxidative stress.Therefore,the Keap1-Nrf2 signaling pathway had an important protective effect in ZEA-induced intestinal oxidative stress.