Adipocyte Secreted Exosomal NOX4 Induces Premature Placenta Senescence In Maternal Obesity

Background and purpose:In recent years,there has been an increasing incidence of obesity among women of childbearing age.Some studies suggest that as maternal BMI increases,the telomere length of placental trophoblast cells is continuously shortened,indicating premature senescence in obese placenta.Large amounts of reactive oxygen species（ROS）are accumulated in adipose tissue of obese individuals,and NADPH Oxidase 4（NOX4）is the main source of ROS in obese individuals.Both of them play an important role in mediating DNA oxidative damage and cellular senescence.However,few studies have investigated the effect of adipose tissue on placental premature senescence in obese pregnancy and whether there is a direct communication between adipose tissue and placental tissue.This study aims to investigate whether adipocyte-derived NOX4 impairs placental development via exosomes and its potential mechanism,so as to provide new insights and evidence for reducing adverse pregnancy outcomes in obese pregnancies.Methods:（1）Term placentas were collected from obese and normal pregnancies,and a mouse model of maternal obesity was established through a high-fat diet.SA-β-galactosidase（SA-β-gal）staining,western blot（WB）,and immunohistochemistry（IHC）were used to detect the expression levels of aging-related markers and DNA oxidative damage markers in placental tissues.（2）Subcutaneous adipose tissue of normal and obese pregnant women was collected to extract primary adipocytes,and the cell culture medium was collected to extract exosomes.Exosomes were detected by transmission electron microscopy and Western blotting.The exosomes were labeled with PKH67 and then co-cultured with trophoblasts.（3）NOX4 overexpression(NOX4^OE)and NOX4 knockdown(NOX4^KD)adipocyte models were constructed using lentivirus.Obese adipocytes,normal adipocytes,NOX4^OEadipocytes,NOX4^KDadipocytes,and exosomes extracted from the supernatant of these cells were co-cultured with trophoblast cells,respectively.DCFH-DA probe was used to detect the production of ROS.WB and immunofluorescence（IF）were used to detect the expression of DNA oxidative damage markers and senescence-related markers in the cells.Ed U assay and wound healing assay were used to evaluate trophoblast cell proliferation and migration function,respectively.（4）Exosomes carrying large amounts of NOX4 from obese adipocytes were injected into the tail vein of normal mice during placental development（gestational days 4.5 to 13.5）to investigate the effect of NOX4 on the placenta and pregnancy outcomes in vivo.Results:（1）Histological experiments on normal and obese pregnant placentas revealed that obese placentas exhibited an aging phenotype,accompanied by cell cycle arrest and DNA oxidative damage.Animal experiments further confirmed that obese pregnancies could lead to placental and fetal dysplasia,with lower placental efficiency and fetal weight compared to the normal group.The placenta exhibited an aging phenotype,with increased expression of senescence-related markers and dysplasia of the labyrinth layer.（2）NOX4 was found to be highly expressed in obese adipocytes and was enriched in exosomes.Adipocytes predominantly secreted NOX4 in an exosome-dependent way.Green fluorescence was observed in the cytoplasm and diffused as small spots around the cell nucleus,indicating that adipocyte-derived exosomes could be taken up by trophoblasts in vitro.（3）Trophoblast cells co-cultured with obese adipocyte-derived exosomes showed oxidative DNA damage,cell cycle arrest,significant expression of senescence-related markers,and decreased proliferation and migration ability.Overexpression of NOX4 in exosomes derived from adipocytes further aggravated trophoblast senescence and impaired migration function.In contrast,the knockdown of NOX4 in adipocyte-derived exosomes or the addition of a NOX4 inhibitor alleviated trophoblast senescence and rescued trophoblast migration ability.（4）Injection of NOX4-exosomes derived from obese adipocytes into normal pregnant mice during placental development（gestational days 4.5to 13.5）induced placental aging and adverse pregnancy outcomes.Conclusions:Adipocytes release exosomes containing an amount of NOX4 in obesity.These exosomes enter circulation and are taken up by trophoblasts of the placenta,resulting in robust production of ROS and severe DNA oxidative damage,finally inducing cell cycle arrest and cellular senescence both in vivo and in vitro.In conclusion,adipose tissue can secrete exosomes containing NOX4 which can be delivered to trophoblasts,resulting in severe DNA oxidative damage and premature placental senescence,leading to adverse pregnancy outcomes.Therefore,NOX4 may be a potential therapeutic target to prevent adverse pregnancy outcomes in obesity.