| Objective:Di-2-ethylhexyl phthalate(DEHP)is a typical environmental endocrine disrupting chemicals(EDCs),always used of plasticizer in various plastic compounds,including baby toys,medical equipment and so on.Epidemiological studies show that exposure to DEHP increases the risk of central nervous system disease.As a large number of widely distributed astrocytes(AS)in the central nervous system,they can secrete a variety of nervous system diseases,If the function of AS damages,the normal development of brain will be impacted.Glial cell derived neurotrophie factor(GDNF)is a neurotrophic factor that plays a major role in neuron survival and growth.There is a link in the secretion function of neurotrophin damage and the occurrence of neurological diseases.But there is no clear report on whether DEHP exposure can effect the secretion of AS neurotrophic factors.This research wants to discuss the influence and mechanism in the secretion of GDNF in AS which are exposure to DEHP by in vitro and in vivo study.Methods:1.Female Wistar rats were randomly assigned into four groups and administered,via oral gavage,0,30mg/kg/d,300mg/kg/dand750mg/kg/d of DEHPin corn oil.Western blot and immunofluorescence staining experiments were performed on the cerebral cortex of pups to detect the expression of neurotrophic factor GDNF and related pathway proteins.2.Primary cultures of AS were prepared from the cerebral cortex of 0-day-old Wistarrats,use CCK8 to detect MEHP tolerance to AS and select the appropriate concentration for experiments.Dual luciferase experiment was used to detect the effect of MEHP on the specific binding of estrogen receptors.Western blot was used to evaluate the expression of GDNF and protein p300 with xenoestrogen added.By western blot and immunofluorescence experiments,the expression of GDNF and relative signal proteins can be detected.The signaling pathwayofERK/c-fos activator Honokiol and inhibitor was used to detect the levels of relative signal proteins.Results: 1.Western blot results showed that the levels of GDNF,p-ERK1/2,c-fos andp300 in the cerebral cortex area of male offspring exposed to 300 and 750mg/kg/d DEHP were significantly decreased compared with the control and the 30mg/kg/daygroup(P<0.05).While the protein expressions of female offspring showed no significant changes in each group(P>0.05).Consistent with theresults of western blot,fluorescence intensity of p-ERK and c-fos in male pups’ medial prefrontal cortex Ⅱ/Ⅲ from the 300and750mg/kg/day groups were also significantly decreased(P<0.05),when compared to the 0 and 30mg/kg/day groups by immunofluorescence and there is also no significant change in groups of femaleoffspring(P>0.05).2.The results showed that the protein expression levels of GDNF,p-ERK1/2 c-fosand p300 proteins in AS from 100μMand 300μM groups were significantly decreased(P<0.05).when compared with the control group by western blot while the expression of ERα protein was not significantly changed(P>0.05).When compared with MEHP alone treatment group,the GDNF and p300 in AS from estrogen and MEHP group and exogenous estrogen administration group were significantly increased(P<0.05).We also found that the result of luciferase experiment from estrogen and MEHP compared with MEHP treatment group was significantly increased(P<0.05).Meanwhile,MEHP combined treatment with Honokiol led to the recovery of levels of GDNF,p-ERK1/2 and c-fosproteins,when compared with MEHP alone treatment group and the difference was statistically significant(P<0.05).Adversely,the levels of GDNF,p-ERK1/2 and c-fosproteins from U0126 and MEHP group were significantly decreased(P<0.05),when compared with U0126 or MEHP treatment groups.Conclusion:1.The expression of neurotrophic factor GDNF secreted by AS was down-regulated when exposure to DEHP.2.DEHP exposure can decrease the expression of GDNF in AS by inhibiting ERK/c-fos signaling pathway.3.The secretory function in AS by DEHP may interfere with the normal estrogen pathway in the brain. |
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