Danshensu Alleviate The Oxidative Stress Response Of Trophoblast Cells Through Wnt/β-catenin Signaling Pathway

BackgroundPreeclampsia(PE)is a special disease that only occurs in pregnant women.It affects about 2% to 8% of pregnant women in the world.It is a significant contributing factor to the rise in maternal and perinatal deaths.It is generally accepted that its pathological basis of its placenta is impaired trophoblast infiltration and insufficient remodeling of placental spiral arteries.The placenta’s antioxidant system,unable to remove enough oxidative stress,weakens the trophoblast cells’ ability to penetrate,resulting in shallow infiltration,inadequate remodeling of the uterine spiral arteries,and shallow placental implantation.This,in turn,leads to hypoxia and ischemia in the placenta.Which further increases the level of oxidative stress,and finally causes a series of oxidative stress damage in pregnant women.The canonical Wnt/β-catenin signaling pathway plays an indispensable role in cell formation,growth,and function through trophoblast invasion and infiltration,as well as the maintenance of cell functions.Danshensu is a known compound extracted from Salvia miltiorrhiza,and its various pharmacological effects have been confirmed,such as antioxidant,antithrombotic,antifibrosis,antibacterial and anti-inflammatory,etc.Studies have demonstrated that PE is caused by the classic Wnt/-catenin signaling system,and Danshensu can effectively alleviate the prognosis of PE mouse models.Therefore,it is considered that Danshensu is theorized to inhibit oxidative stress in order to regulate classic Wnt/β-catenin signaling pathway,thus provided a new theoretical basis for Danshensu to treat PE.PurposesTaking HTR-8/SVneo cells as the research object,it has been studied how the canonical Wnt/-catenin signaling system contributes to trophoblast oxidative stress injury.Our research goal is to investigate the mechanism of Danshensu to alleviate the oxidative stress injury of trophoblast cells,and lay a theoretical foundation for the treatment of Danshensu in patients with preeclampsia.Methods1.When the cells grow to cover 80% of the area of the culture flask or dish,inoculate and subculture.A model of oxidative stress was made: eight hours of hypoxia followed by sixteen hours of reoxygenation.A total of two cycles,48 hours.Set different concentrations of Danshensu(0.001、0.01、0.1、1、10、100μmol/L)to treat the hypoxia/reoxygenation(H/R)HTR-8/SVneo for 24 or 48 hours,use CCK8 to detect the viability of trophoblasts,and select the optimal concentration of Danshensu and the optimal treatment time as the follow-up experimental standards;2.We divided the experiment into four groups: normal control group,normal cultivation + Danshensu group,H/R cultivation group,H/R + Danshensu treatment group.The quantity of reactive oxygen species(ROS)in each treatment group and control group was quantified using fluorescence microscopy;3.RT-PCR was used to measure the transcription levels of DKK1,GSK-3,and CTNNB1(β-catenin protein m RNA)in the Wnt/-catenin signaling pathway in each therapy and control group;4.Western blot was used to detect the protein expression levels of DKK1,Wnt1,GSK-3β,and β-catenin in the classical Wnt/β-catenin signaling pathway in each treatment group and control group.Results1.CCK8 detection indicated that the optimal concentration of Danshensu was 10μmol/L,and the cell viability was obviously restored after 48 hours of treatment.2.Fluorescence microscopy showed that H/R treatment could lead to a significant increase in the ROS accumulation of HTR-8/SVeno,while Danshensu treatment could significantly alleviate the H/R-induced ROS production.In addition,Danshensu treatment had no effect on the intracellular ROS levels of normal cultured HTR-8/SVneo.3.The results of RT-PCR proved that a quite distinctness was discovered between the H/R treatment group and the normal control group with respect to the transcription level of DKK1(P=0.0137);A significant difference was observed in the GSK-3βtranscription levels between the H/R treatment and control groups as compared to the standard control group(P=0.0125);The transcription level of CTNNB1 in the H/R treatment group was highly lower than that in the normal control group,normal culture+ Danshensu treatment group,and H/R+ Danshensu treatment group(P<0.0001、P=0.0001、P=0.0029);There was a decrease in transcription of CTNNB1 in the H/R treatment group compared to the normal control group(P=0.0145).4.The H/R treatment group expressed exceptionally high levels of DKK1 compared to the control group,according to the Western-Blot detection findings(P=0.0480);As compared with the normal control and normal culture + Danshensu treatment groups,the GSK-3β expression level of the H/R treatment group was much lower(P=0.0090,P=0.0407);Compared to the normal control group,normal culture +Danshensu treatment group,and H/R + Danshensu treatment groups,the H/R treatment group expressed significantly less β-catenin.(P=0.0003,P=0.0034,P =0.0062);Compared with the normal control group and the H/R + Danshensu treatment group,the Wnt1 expression level in the H/R treatment group was quite lower(P=0.0436,P=0.0219).Conclusion1.Oxidative stress mainly inhibits the expression of Wnt1 protein and β-catenin protein,blocks the Wnt/β-catenin signaling pathway,damages trophoblasts,accelerates the apoptosis of trophoblasts,and reduces the invasiveness of trophoblasts.2.Danshensu can alleviate trophoblast oxidative stress,rescue Wnt1 protein andβ-catenin protein levels,and rescue Wnt/β-catenin signaling pathway.It has a protective effect on the oxidative stress damage of trophoblasts,and provides a new theoretical basis for the treatment of PE.