The Mechanism Of DEHP-induced Autophagy And Apoptosis Of Mouse HT22 Hippocampal Neuronal Cells

Background:Di-2-ethylhexyl phthalate(DEHP)is the most commonly used plasticizer that makes plastic more flexible and elastic.DEHP binds to plastic materials with very weak covalent bonds,which can easily leach out from these plastic materials into the environment under high temperatures.Therefore,DEHP can be found in air,ground water and soil because of the constant release into the environment,which has raised concerns pertaining to continuous exposure of human beings.DEHP can cross the blood-brain barrier(BBB)and accumulates in the brain.DEHP has been shown to inhibit cholesterol synthesis in the brain and affect the myelination process through disturbing lipid metabolism and distribution.DEHP can cause damage of dopaminergic neurons in the mesencephalon and finally inhibit the synthesis of dopamine,an important neuromodulator involved in learning,cognition and memory,which affect reference memory,self-righting ability and spatial learning.The hippocampus is located under the cerebral cortex,which plays an important role in the formation and consolidation of memory.The dendrites of hippocampal neurons could be affected by many kinds of chemicals,such as inorganic arsenic and persistent organic pollutants.The hippocampus can be affected by DEHP either maternal exposure or acute postnatal exposure.However,it is still unknown about the underlying mechanism on DEHP-caused the damage of the hippocampus.In the current study,we investigate the potential mechanism on DEHP-induced autophagy and apoptosis of mouse hippocampus via mouse model and the HT22 hippocampal neuronal cell,an immortalized hippocampal neuronal cell line,which may provide a novel insight into the mechanism for DEHP-induced nerve injury in human.Part I DEHP induced apoptosis and autophagy of hippocampus in mice Objective:To investigate whether DEHP induce apoptosis and autophagy of hippocampus in mice and whether oxidative stress is involved in DEHP-induced apoptosis and autophagy of hippocampus in mice.Methods:Adult mice were intragastrically(i.g.)administered with 0,100,200 or 400 mg DEHP/kg/day for 3 weeks,then the hippocampus tissues were stained with hematoxylin and eosin(HE).Western blot was used to determine the protein levels of cleaved Caspase-8,cleaved Caspase-3,Bax and Bcl-2,as well as the protein levels of autophagy related protein Atg5、Beclin1 and LC3.The level of GSH and the activities of GSH-PX and SOD were determined by oxidation-antioxidation assay kits.Results:Adult mice were intragastrically(i.g.)administered with 0,100,200 or 400 mg DEHP/kg/day for 3 weeks,hematoxylin and eosin(HE)staining showed that DEHP had no significant effect on the morphology of the hippocampus tissues.However,the protein levels of cleaved Caspase-8,cleaved Caspase-3 and Bax markedly increased in the DEHP-treated group,whereas there was a significant decrease in the Bcl-2protein level.These results suggested that DEHP could induce apoptosis of hippocampus in mice.To investigate whether DEHP can induce autophagy of hippocampus in mice,the protein levels of Atg5、Beclin1 and LC3 were detected by Western blot after mice were administered with 0,100,200 or 400 mg DEHP/kg/day for 3 weeks.We found that DEHP could significantly increase the protein levels of Atg 5 and Beclin 1,as well as the ratio of LC3-II/LC3-I in the mouse hippocampus tissue,indicating that DEHP could induce autophagy of mouse hippocampus tissue.To investigate the potential mechanism,the contents of GSH and MDA and the activities of antioxidant enzyme GSH-PX and SOD were determined after mice were administered with 0,100,200 or 400 mg DEHP/kg/day for 3 weeks.DEHP was shown to significantly increase the content of MDA,whereas markedly decreased the level of GSH and the activities of GSH-PX and SOD.These results suggested that DEHP induced oxidative stress of mouse hippocampus tissue.Conclusion:1.DEHP had no significant effect on the morphology of the hippocampus tissues.However,DEHP could markedly increase the protein levels of cleaved Caspase-8,cleaved Caspase-3 and Bax,while decrease Bcl-2 protein level,indicating that DEHP could induce apoptosis of hippocampus in mice.2.DEHP could increase the protein levels of Atg 5 and Beclin 1,as well as the ratio of LC3-II/LC3-I in the mouse hippocampus tissue,suggesting that DEHP could induce autophagy of mouse hippocampus tissue.3.DEHP could increase the content of MDA,whereas markedly decrease the level of GSH and the activities of GSH-PX and SOD in the mouse hippocampus tissue,implying that DEHP induced oxidative stress of mouse hippocampus tissue.Part II DEHP inhibited cell viability and induced apoptosis of mouse HT22 hippocampal neuronal cells via oxidative stress Objective:To investigate whether DEHP induce apoptosis of mouse HT22 hippocampal neuronal cells and its potential mechanism by in vivo experiment.Methods:Mouse HT22 hippocampal neuronal cells was treated with the indicated concentration of DEHP in the absence or presence of N-Acetyl-L-cysteine,an inhibitor of oxidative stress.Cell viability was observed by CCK8 kit.LDH release was detected by LDH release kit.Western blot was used to determine the protein levels of cleaved Caspase-8,cleaved Caspase-3,Bax and Bcl-2.Hoechst33342 staining was utilized to observe nuclei and apoptotic body in the cells.Annexin V-FITC/PI double staining was used to count the Annexin V-positive staining cells.The level of GSH and the activities of GSH-PX and SOD were determined by oxidation-antioxidation assay kits.Results:To investigate whether DEHP caused cytotoxicity of mouse HT22 hippocampal neuronal cells,the cells were treated with 0,2.5,5,10,20,40 or 80 μM DEHP for 24 h.Compared to the control group,there was no significant change in 2.5 and 5.0 μM DEHP-treated cells(P> 0.05).While 10,20,40 or 80 μM DEHP dramatically inhibited cell viability of mouse HT22 cells(P<0.05).LDH release was detected after the cells were treated with 0,10,20 or 40 μM DEHP for 24 h,we found that DEHP could cause LDH release from the cells in a dose-dependent manner,suggesting that DEHP could cause cytotoxicity of mouse HT22 cells.Meanwhile,the protein levels of cleaved Caspase-8,cleaved Caspase-3 and Bax markedly increased in the DEHP-treated cells,whereas there was a significant decrease in the Bcl-2 protein level.Hoechst33342 staining showed that nuclei were round and homogenously stained in the control cells;while there was an increase in the DNA condensation and apoptotic body in the DEHP-treated cells.Apoptosis was further confirmed by Annexin V-FITC/PI double staining,we found that DEHP dramatically increased the total numbers of Annexin V-positive/PI-negative(early apoptosis)and Annexin Vpositive/PI-positive staining cells(late apoptosis).These results implied that DEHP could induce apoptosis of mouse HT22 cells.To investigate the potential mechanism,the contents of GSH and MDA and the activities of antioxidant enzyme GSH-PX and SOD were determined after the mouse HT22 cells were treated with 0,10,20 or 40 μM DEHP.DEHP was shown to significantly increase the content of MDA,whereas markedly decreased the level of GSH and the activities of GSH-PX and SOD.These results suggested that DEHP induced oxidative stress of mouse HT22 cells.In order to confirm whether oxidative stress was involved in DEHP-induced apoptosis of mouse HT22 cells,we further investigated cell viability and apoptosis after the cells were treated with 0 or 40 μM DEHP for 24 h in the presence or absence of 5 m M N-Acetyl-L-cysteine(NAC),an inhibitor of oxidative stress.40 μM DEHP significantly inhibited cell viability and increased the release of LDH from the cells;meanwhile,compared with the DEHPtreated group,the inhibition of cell viability and the release of LDH were rescued in the NAC plus DEHP group to some extent.Furthermore,inhibition of oxidative stress could rescue the induction of apoptosis by DEHP.These results indicated that DEHP could induce apoptosis of mouse HT22 cells via oxidative stress.Conclusion:1.DEHP significantly inhibited cell viability and induced apoptosis of mouse HT22 hippocampal neuronal cells.2.DEHP could induce oxidative stress of mouse HT22 hippocampal neuronal cells.3.DEHP inhibited cell viability and induced apoptosis of mouse HT22 hippocampal neuronal cells via oxidative stress.Part III: Role of autophagy in DEHP-induced apoptosis of mouse HT22 hippocampal neuronal cells Objective:To investigate whether DEHP induce autophgy of mouse HT22 hippocampal neuronal cells and the role of TRAF4/RPS6KB1 in DEHP-induced autophgy of the cells.To determine the role of autophagy in DEHP-induced apoptosis of mouse HT22 hippocampal neuronal cells via autophgy inhibitor 3-MA.Methods:Mouse HT22 hippocampal neuronal cells was treated with the indicated concentration of DEHP,then Western blot was used to determine the protein levels of Atg 5,Beclin 1 and LC3,transmission electron microscopy(TEM)was utilized to observe autophagic vacuoles in the cells,and the protein levels of TRAF4 and RPS6KB1 was detected by Western blot.To investigate whether TRAF4 can activate RPS6KB1 in the mouse HT22 hippocampal neuronal cells,Western blot was used to determine the protein levels of RPS6KB1 and p-RPS6KB1 and autophagy through over-expression and knockdown of TRAF4 in the cells.Results:Mouse HT22 hippocampal neuronal cells was treated with 0,10,20 and 40 μM DEHP for 24 h,then Western blot was used to determine the protein levels of Atg 5,Beclin 1 and LC3.We found that DEHP could significantly increase the protein levels of Atg 5 and Beclin 1,as well as the ratio of LC3-II/LC3-I.Furthermore,TEM showed that there was a significant increase in autophagic vacuoles in the DEHPtreated cells.These results indicated that DEHP could induce autophagy of mouse HT22 hippocampal neuronal cells.In order to confirm whether oxidative stress was involved in DEHP-induced autophagy of mouse HT22 hippocampal neuronal cells,we further investigated autophagy after the cells were treated with 0 or 40 μM DEHP for 24 h in the presence or absence of 5 m M N-Acetyl-L-cysteine(NAC),an inhibitor of oxidative stress.We found that NAC could decrease the protein levels of Atg 5 and Beclin 1 and the ratio of LC3-II/LC3-I in the DEHP-treated cells.Compared with the DEHP-treated group,there was a decrease in autophagic vacuoles in the NAC plus DEHP-treated cells.These results suggested that DEHP induced autophagy of mouse HT22 hippocampal neuronal cells via oxidative stress.To further investigate the potential mechanism of DEHP-induced autophagy of mouse HT22 hippocampal neuronal cells,the cells were treated with 0,10,20 and 40μM DEHP for 24 h.DEHP was showed to decrease the protein levels of TRAF4 and p-RPS6KB1,indicating that DEHP could inhibit TRAF4/RPS6KB1 signaling.Overexpression of TRAF4 was shown to increase the contents of p-RPS6KB1,while knockdown of TRAF4 decreased the protein levels of p-RPS6KB1.Furthermore,overexpression of TRAF4 could decrease the contents of Atg 5 and Beclin 1 and the ratio of LC3-II/LC3-I induced by DEHP.These results showed that TRAAF4 could inhibit DEHP-induced autophagy of mouse HT22 hippocampal neuronal cells.To determine the role of DEHP-induced autophagy in the viability of mouse HT22 hippocampal neuronal cells,the cells were treated with 0 or 40 μM DEHP for24 h in the presence or absence of 1 m M 3-MA,an inhibitor of autophagy.Compared with DEHP-treated cells,3-MA plus DEHP further inhibited cell viability and increased the release of LDH from the cells.There was a significant increase in the protein levels of cleaved Caspase-8,cleaved Caspase-3 and Bax,as well as a dramatical decrease in the protein level of Bcl-2;furthermore,3-MA could increase the positive cells of Annexin V-FITC in the DEHP-treated cells,indicating that inhibition of autophagy could increase the induction of apoptosis of mouse HT22 hippocampal neuronal cells.These results suggested that autophagy played a protective role in DEHP-induced apoptosis of mouse HT22 hippocampal neuronal cells.Conclusion:1.DEHP could induced autophagy of mouse HT22 hippocampal neuronal cells,and oxidative stress was involved in DEHP-induced autophagy of the cells.2.DEHP could inhibit TRAF4/RPS6KB1 signaling;TRAF4 could increase the protein level of p-RPS6KB1 and inhibited DEHP-induced autophagy of mouse HT22 hippocampal neuronal cells.3.Inhibition of autophagy could increase the induction of apoptosis of mouse HT22 hippocampal neuronal cells,and autophagy played a protective role in DEHP-induced apoptosis of the cells.