Mechanism Of DEHP Induced Hepatocyte Apoptosis In Chickens By Regulating PTEN/PI3K/AKT Signaling Pathway Via ROS

Di(2-ethyl)hexyl phthalate(DEHP)is a common environmental endocrine disruptor that induces oxidative stress and causes damage to the endocrine,reproductive,immune,liver,and other organ systems,posing a significant threat to human and animal health.Oxidative stress can activate the PTEN/PI3K/AKT signaling pathway,which is closely related to apoptosis.However,it is unclear whether DEHP induces apoptosis of chicken hepatocyte by regulating the PTEN/PI3K/AKT signaling pathway through oxidative stress.Therefore,in this study,the mechanism of hepatocyte apoptosis induced by DEHP exposure in chickens was studied from the perspective of oxidative stress by combining in vitro and in vivo experiments.One-day-old male HY-LINE VARIETY WHITE laying hens were randomly divided into five groups after pre-feeding for 7 days: blank control group(C group),corn oil control group(Vc group),400 mg/kg DEHP group(L group),800 mg/kg DEHP group(M group)and 1600mg/kg DEHP group(H group).The chickens in different groups were administered intragastrically in succession according the experimental plan,and euthanized at 14 d,28 d and 42 d,respectively.And the liver coefficients were calculated.The activity of serum liver functional enzymes AST and ALT and oxidative stress index were detected by kit;The histopathological changes of chicken liver were observed by paraffin-HE staining and transmission electron microscopy;Real time fluorescence quantitative PCR(q RT-PCR)and Western blot techniques were used to detect changes in m RNA and protein expression of PTEN/PI3K/AKT signaling pathway related genes and apoptosis related genes in liver tissue.The results are as follows:(1)Compared with the C and Vc groups,Liver coefficient in L,M,H DEHP exposure groups increased at 14 d,28 d and 42 d(P<0.05),however,the liver coefficient at 28 d was lower than those at 14 d and 42 d;Meanwhile,the activities of AST and ALT in L,M,H DEHP exposure groups were significantly increased at 28 d and 42 d(P<0.05),and the peak value of group H was reached at 42 d;In the L,M,H DEHP exposed group,the liver enlarged in volume,the capsule appeared rough and dull with the color of dark red.Sometimes the surface showed grayish-yellow spot-like lesions with blunt edges;Histopathological observation showed that in the L,M,H DEHP exposure groups,the arrangement of hepatocyte disordered,more vacuoles of varying sizes appeared in the cytoplasm,and changes such as hepatocyte volume decreased,hepatocytes apoptosis accompanied by changes in cytoplasm,nuclear pyknosis and karyorrhexis.Ultrastructural transmission electron microscopy observation showed that the liver nucleus of group H was pyknotic,the cristae in mitochondria dissolved,the margin of the nuclear membrane was uneven,and the perinuclear space enlarged.(2)Compared with the C and Vc groups,the levels of ROS and MDA contents in liver of chickens in L,M,H DEHP exposure groups were remarkably raised,while the levels of T-AOC and the activities of T-SOD and GSH-PX were lowered significantly(P<0.05).(3)Compared with the C and Vc groups,in L,M,H DEHP exposure groups,the m RNA and protein expression levels of PTEN were up-regulated and the m RNA and m RNA and protein expression levels of PI3K/AKT were decreased at 14 d,28 d and 42 d(P<0.05).(4)Compared with the C and Vc groups,Bax/Caspase-9/Caspase-3 m RNA and protein expression levels were up-regulated and BCL-2 m RNA and protein expression levels were decreased in L,M,H DEHP exposure groups at 14 d,28 d and 42 d(P<0.05).The above results indicated that DEHP exposure affected the growth and development of chickens and led to liver damage,and the degree of damage was positively correlated with the dose of DEHP gavage.At the same time,DEHP exposure caused oxidative stress in liver,activated the PTEN/PI3K/AKT signaling pathway,and induced the apoptosis of chicken liver.To determine whether DEHP could induce apoptosis in chicken hepatocyte by regulating PTEN/PI3K/AKT pathway through oxidative stress,the DEHP exposed chicken hepatocellular carcinoma cell line(LMH)model was established in vitro.Test groups were determined as blank control group(C group),20 μM DEHP(L group),40 μM DEHP(M group),60 μM DEHP(H group),2.5 m M NAC group(NAC group)and 2.5 m M NAC+60 μM DEHP group(NAC+DEHP group).Oxidative stress index was determined by kit.The morphological changes of apoptosis were observed by AO/EB(Acridine orange/ethidium bromide)staining and the proportion of apoptosis was calculated by quantitative analysis.q RT-PCR and Western blot techniques were used to detect changes in m RNA and protein expression of PTEN/PI3K/AKT signaling pathway related genes and apoptosis related genes in LMH cells.The results are as follows:(1)Compared with the C group,ROS levels and MDA contents in L,M,H DEHP exposure groups were remarkably raised,while the levels of T-AOC and the activities of T-SOD and GSH-PX were lowered significantly(P<0.05);While NAC significantly inhibited the overproduction of ROS and MDA,and significantly increased the levels of T-AOC and the activities of T-SOD and GSH-PX(P<0.05).(2)AO/EB staining results showed that compared with the C group,the number of apoptosis were notably increased in L,M,H DEHP exposure groups(P<0.05);And NAC can reduce the number of apoptosis(P<0.05).(3)Compared with the C group,the m RNA and protein expression levels of PTEN were shot up in L,M,H DEHP exposure groups,and the m RNA and protein expression levels of PI3K/AKT were reduced(P<0.05);However,NAC down-regulated the m RNA and protein expression levels of PTEN and up-regulated the m RNA and protein expression levels of PI3K/AKT(P<0.05).(4)Compared with the C group,the m RNA and protein expression levels of Bax/Caspase-9/Caspase-3 in L,M,H DEHP exposure groups were enhanced,yet the m RNA and protein expression levels of BCL-2 were decreased(P<0.05);NAC significantly reduced the m RNA and protein expression levels of related proapoptotic genes,and increased the m RNA and protein expression levels of BCL-2(P<0.05).These results confirm that DHEP exposure can induce apoptosis of chicken hepatocyte through oxidative stress activation of PTEN/PI3K/AKT signaling pathway.In conclusion,DEHP exposure can induce apoptosis of chicken hepatocyte through ROS regulation of PTEN/PI3K/AKT signaling pathway.This study elucidated the new toxic mechanism of DEHP exposure causing hepatocyte apoptosis in chickens,enriched the mechanism of action between oxidative stress and liver injury,provided a new perspective for animal toxicology research,and also provided a reference for comparative medicine.