Effects And Regulatory Mechanisms Of Nox4 During Uterine Decidualization In Mice

During mammalian pregnancy,extensive reconstruction of the uterine tissue and proliferation and differentiation of the endometrial cells,ie decidualization of the uterus,occur.Uterine decidualization is one of the prerequisites for blastocyst implantation,placental formation and pregnancy maintenance.Differentiation of endometrial stromal cells into specialized decidual cells is the key to embryo implantation and survival.Differentiated stromal cells can be selective.Identify and respond to impaired blastocysts,which protect blastocysts from external oxidative stress,control subsequent trophoblast invasion and placental formation,and regulate specific immune cells.It has been reported in the previous period that Nox4?NADPH Oxidase 4?plays an important role in regulating the decidualization process.Nox4 is one of the important members of the NADPH oxidase Nox family.NADPH oxidase is an enzyme system that catalyzes the reduction of molecular oxygen to superoxide and is an important component of the oxidative stress NADPH-ROS pathway.Reactive Oxygen Species?ROS?is a by-product of cell metabolism and is an active molecule derived from molecular oxygen.It is mainly composed of superoxide anion(O₂^?-),hydroxyl radical??OH?,hydrogen peroxide?H₂O₂?,etc..Nox4 mainly produces O₂^?-and H₂O₂,and Nox4 is related to organelles such as endoplasmic reticulum and mitochondria.In a healthy body,ROS and antioxidants are balanced.Oxidative stress occurs when the balance is excessively damaged by ROS.However,excessive ROS can lead to a variety of pathologies,including endothelial dysfunction,atherosclerosis,hypertension,diabetes and acute respiratory distress syndrome.This study investigated the expression,function and regulation mechanism of Nox4 in the uterus of early pregnant mice.Fluorescence quantification was used to study the expression of Nox4 in the early pregnant uterus of mice.The results showed that the expression of Nox4 in the uterus of the 4th day of early pregnancy was significantly higher than that of the mouse uterus on the first day of the early pregnancy,but the expression of Nox4 was significantly lower on the 7th day of the early pregnancy than on the 5th day of pregnancy.Nox4 had no significant effect on uterine stromal cell proliferation and cell cycle.The inhibition of Nox4 expression by specific interference fragment or the increase of Nox4 expression after adding recombinant protein had no significant effect on cell proliferation and cell cycle.Inhibition of Nox4 expression by specific interference fragments significantly increased the expression of decidualization marker genes Prl8a2 and Prl3c1,while the addition of recombinant protein significantly inhibited the expression of decidualization marker genes Prl8a2 and Prl3c1.Further studies have shown that Nox4 can be involved in the regulation of differentiation of uterine stromal cells as a downstream target gene for progesterone.The expression of Prl8a2 and Prl3c1 was significantly increased after the addition of progesterone.The expression of Prl8a2 and Prl3c1 was increased after knocking down Nox4.The expression of Prl8a2 and Prl3c1 was decreased after adding Nox4recombinant protein.It is indicated that Nox4 can mediate the regulation of progesterone on the differentiation of uterine stromal cells.ROS is involved in physiological and pathological processes such as cell proliferation,differentiation,senescence and apoptosis.This experiment interfered with Nox4 and observed the production of ROS in stromal cells.The results showed that the amount of ROS production increased significantly,and O₂^?-was the main component of ROS.The detection showed that the production of O₂^?-also showed an upward trend.Nox4 mainly produces O₂^?-and H₂O₂,which are related to organelles?including endoplasmic reticulum,mitochondria or nucleus?,and endogenous ROS are mainly derived from mitochondria.MitoSox kit can reflect O₂^?-in mitochondria,in interference with Nox4.The content of O₂^?-in the post-mitochondria is on the rise.JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential??m,which can detect changes in cell mitochondrial membrane potential.The decrease in cell membrane potential can be detected by the transition from red fluorescence to green fluorescence,and the change in red-green fluorescence can also be used as an indicator for early detection of apoptosis.In this experiment,it was found that the ratio of red/green fluorescence in uterine stromal cells decreased after interference with Nox4,indicating that the mitochondrial membrane potential in uterine stromal cells decreased after interference with Nox4.Decreased mitochondrial membrane potential is a landmark indicator of early apoptosis,so this experiment examined the apoptosis of the uterine matrix.Flow cytometry revealed that cells that had early apoptosis were significantly increased after interference with Nox4 in uterine stromal cells.In summary,Nox4 regulates the differentiation of mouse stromal cells by progesterone and affects the expression of ROS in early pregnant mice.