The Protective Role And Mechanism Of Placental Lipophagy In Gestational Exposure Cadmium-induced Fetal Growth Restriction

Background:Cadmium（Cd）,is a known developmental poison.Previous studies of Cd have found that non-selective autophagy play a protective role in Cd damage to the placenta and fetal development.However,the role of placental lipophagy（a selective autophagy）in Cd-induced fetal growth restriction（FGR）remains unclear.Objective:This study investigated the protective role and mechanism of placental lipophagy in Cd-caused FGR.Methods:The methods of case control study,cell experiment and whole animal experiment were used in this paper.Firstly,a population case-control study was conducted to investigate the association between placental lipophagy and all-cause fetal growth restriction.A total of 144 human placentas were collected,including 17placentas small for gestational age（SGA）and 127 placentas suitable for gestational age（AGA）.After controlling for confounding factors such as maternal age,pre-pregnancy body mass index and gestational age,a total of 6 pairs of SGA and AGA placentas were matched for lipophagy related protein levels（ATGL and LC3B）and lipid drops（LDs）oil red O staining.Subsequently,the effect of Cd treatment on lipophagy of human placental trophoblast cells was studied by cell culture.The cell experiment consisted of two parts.In experiment 1,the placentas of full-term pregnant women were collected for human primary placental trophoblast isolation in order to investigate whether Cd treatment activated the lipophagy of human primary placental trophoblast cells,and Cd chloride（Cd Cl₂,20μM）was given for 0 h,6 h,12 h and 24 h.In experiment 2,to investigate the role of lipid autophagy receptor ATGL in Cd-induced placental lipophagy,human JEG-3 cells were pretreated with Atgl si RNA before exposure to Cd,and lipophagy related protein levels were detected and lipid drops oil red O staining was performed.Finally,the effects of Cd exposure during pregnancy on placental lipophagy and fetal development were studied in animal experiments.Animal experiments consist of 5 experiments.In experiment 1,in order to explore the effects of Cd exposure during pregnancy on fetal growth and development and placental lipophagy,healthy C57BL/6J pregnant rats were randomly divided into different treatment groups and given normal saline（control）and Cd chloride（Cd Cl₂,1.0 and 2.0 mg/kg,i.p）respectively on GD8.Experiment 2:To investigate the role of placental lipophagy inhibition in Cd-induced FGR,healthy pregnant mice were pretreated with 3-methyladenine before being treated with Cd.Experiment 3:To further verify the role of placental lipophagy loss in Cd-induced FGR,Dpp3-Cre/Atg5^flox/-mice were treated with cadmium chloride（Cd Cl₂,2.0mg/kg）on GD8.Experiment 4:To explore the role of placental lipophagy activation in Cd-induced FGR,healthy pregnant mice were given rapamycin before Cd treatment.Experiment 5:To investigate the protective effect of progesterone（P4）supplementation on Cd-induced FGR,healthy pregnant mice were treated with P4.On GD16,maternal serum,placenta and amniotic fluid were collected for further experimental analysis,including detection of the contents of P4 and free cholesterol（FC）in maternal serum,autophagosomes and LD-containing autophagosomes in Cd-treated mouse placentae were observed by transmission electron microscopy,the levels of placental lipophagy related proteins were detected by protein western blotting,and LDs were observed by oil red O staining.The co-localization of ATGL and LC3B was detected by immunofluorescence.Results:Firstly,case-control study results showed that all-cause SGA placenta-specific lipid autophagy receptor ATGL and lipophagy associated proteins（Atg5 and LC3B-II）were expressed at higher levels than AGA placentas,suggesting a positive association between placental lipophagy and FGR.Cd exposure activated lipophagy in human placental trophoblast cells and mouse placental tissue,both in vivo and in vitro.In addition,Atgl si RNA pretreatment of human JEG-3 cells significantly increased the down-regulation effect of Cd on lipophagy related protein expression,and increased Cd-induced placental lipid accumulation.These results suggest that environmental Cd treatment can induce lipophagy activation in human placental trophoblast cells and mouse placental tissue.In animal studies,cadmium exposure during pregnancy has also been found to induce FGR and decreased placental progesterone levels.In order to elucidate the protective effect of placental lipophagy on Cd-induced FGR,the intervention experiment of related lipophagy was conducted.Lipophagy inhibitors significantly exacerbate FGR caused by Cd exposure.To further confirm the above results,a mouse model of Atg5 knockout was established in this study,in which absence of autophagy aggravated FGR caused by Cd exposure.Pharmacologically activated placental lipophagy results showed that pretreatment with activator significantly alleviated FGR caused by Cd exposure.These results suggest that activation of placental lipophagy alleviates Cd-induced FGR.Lipophagy can degrade LDs to produce FC,which is the main synthetic material of P4.Pretreatment with lipophagy inhibitor increased Cd and decreased the contents of FC and P4 in maternal serum,placenta and amniotic fluid.Cd exposure in pregnant mouse also significantly decreased the level of Atg5^-/-placental FC and P4e content.In contrast,pretreatment with lipophagy activator significantly reversed Cd and reduced the contents of FC and P4 in maternal serum,placenta and amniotic fluid.The results of placental oil red O staining showed that pretreatment with lipophagy inhibitor significantly increased the accumulation of placental LDs caused by Cd.Cd treatment also caused the accumulation of large LDs in Atg5^-/-placentae.On the contrary,lipophagy activator pretreatment significantly alleviated Cd-induced placental LDs accumulation.These results suggest that activation of placental lipophagy alleviates Cd-induced reduction of FC and P4 in placenta and amniotic fluid.P4 is essential for fetal development.In pregnant mouse,P4 supplementation significantly reversed Cd reduction in fetal weight and top hip length,and reversed Cd increase in the incidence of FGR.At the same time,P4 supplementation during pregnancy also alleviated Cd exposure,reduced Atg5^-/-fetal weight and top hip length,and significantly alleviated Cd increased the incidence of Atg5^-/-FGR.In addition,we found that P4 supplementation during pregnancy significantly alleviated Cd and decreased serum,placenta and amniotic fluid P4 content in wild-type mice and Atg5^-/-pregnant mice.These results suggest that P4 supplementation during pregnancy alleviates Cd-induced FGR.Conclusion:Placental lipophagy degrades LDs to produce FC,promotes P4 synthesis,and thereby protects against Cd-caused FGR.