The Mechanism Of Puerarin Alleviating Pre-eclampsia Symptoms By Inhibiting Ferroptosis In Trophoblast Cell

BackgroundPre-eclampsia(PE)is a specific disease during pregnancy,the main manifestations are new onset hypertension and proteinuria after 20 weeks of gestation and multiple organ dysfunction may occur in severe cases.Many studies have shown that PE is caused by multiple factors,mechanisms and pathways among which oxidative stress and ferroptosis are important links in the pathogenesis of PE.At present,the incidence of PE in China is about 5.6-9.4%,With the improvement of economic level and the adjustment of fertility policy,the number of woman who choose to marry late and have a second child gradually increases,and the risk of PE also increases.It is urgent to explore new ways to treat PE.Puerarin is the main active ingredient of pueraria lobata,which belongs to isoflavones and has an effect of antioxidant.It is used to treat angina pectoris and myocardial infarction clinically.Previous studies have reported that the systolic and diastolic blood pressure of the hypertensive rat decreased significantly after intragastric administration of puerarin.Our previous research confirmed that intragastric administration of pueraria lobata decoction can effectively prevent the occurrence and development of PE in mice.At present,a few studies have reported that puerarin can alleviate PE-like symptoms,but there is still a lack of systematic discussion and sufficient research basis.Therefore,in combination with previous reports,this study explored whether puerarin monomer could inhibit the pathogenesis of PE at both animal and cell levels,and explored the relevant molecular mechanisms.ObjectiveThis study aims to explore the molecular mechanism of puerarin affecting the pathogenesis of PE by regulating the oxidative stress of trophoblasts and mediating the change of placental function.Methods1.The animals were divided into three groups:control group,L-NAME group and L-NAME+purarin group.PE mouse model was induced by subcutaneous injection of L-NAME.After puerarin treatment,the systolic blood pressure,urinary protein level,body weight and placental pathological changes of mice were observed The expression of HO-1,GPX4 and SLC7A11 protein in placenta and the change of serum GSH content were detected2.Oxidative stress model of trophoblasts was induced by hypoxia/reoxygenation(H/R)and CoC12 respectively.After treatment with puerarin,ROS content was detected by flow cytometry and fluorescence staining.At the same time,Erastin induced the ferroptosis model of trophoblasts,and explored the effect of puerarin on oxidative stress and ferroptosis of trophoblasts by detecting the changes of HO-1,GPX4,SLC7A11,GSH and MDA.The expression level of HO-1 GPX4 and SLC7A11 was detected by Western blot.3.Based on the JASPAR database,the potential transcription regulators of HO-1 were screened.Through the construction of candidate transcription factor knockdown and over-expression trophoblast cell lines,RT-qPCR,western blot and dual luciferase reporter gene experiment,we explored the molecular mechanism of puerarin acting on transcription regulatory factors and participating in PE pathogenesis.Result1.Compared with the control group,the L-NAME group increased the expression of HO-1 in placenta,decreased the expression of GPX4 and SLC7A11,and decreased the content of GSH in serum.Compared with L-NAME group mice,puerarin supplementation can significantly reduce the systolic blood pressure and urine protein of pregnant mice,reduce the abortion rate,increase the weight of offspring,restore the normal tissue structure of placenta,and down-regulate the expression of HO-1,up-regulate the expression of GPX4,SLC7A11,and significantly increase the content of serum GSH.2.Compared with the trophoblast oxidative stress model,after puerarin treatment,the content of ROS in cells decreased significantly,the expression of HO-1 decreased significantly,GPX4,SLC7A11 and GSH increased significantly,and the content of MDA decreased significantly;Compared with the control group,the expression of GPX4 and SLC7A11 in trophoblast cells increased significantly after HO-1 knockdown.3.Compared with the knockdown control group,the expression of HO-1 decreased significantly and the expression of GPX4 increased significantly after the knockdown of CREB;Compared with the overexpression control group,the expression of HO-1 was significantly increased and the expression of GPX4 was significantly decreased after overexpression of CREB.The dual luciferase reporter gene experiment showed that there was a direct binding site between CREB and HO-1.Compared with L-NAME group mice,the expression of CREB in placenta of L-NAME+purarin group mice was significantly decreased.ConclusionPuerarin reduces oxidative stress of trophoblasts and inhibits ferroptosis by down-regulating CREB/HO-1 signaling pathway,which affects the occurrence and development of PE.