Study On The Mechanism Of α-klotho In Placenta Senescence Of Advanced Age Pregnancy

Background: In recent years,the number of Advanced maternal age(AMA)pregnancy is increasing.AMA pregnancy is a serious threat to the health of pregnancy and is also one of the main causes of perinatal and long-term complications in offspring.AMA pregnancy is associated with higher risks of adverse perinatal outcomes,which may result from premature senescence of the placenta.Premature placental senescence may be the main cause of pregnancy complications in AMA pregnancy.α-Klotho is a well-known anti-aging protein;however,its expression and effect on the placenta of AMA pregnancies have yet to be fully elucidated.Purpose: The purpose of this study is to explore whether α-Klotho is a determinant of senescence in placental trophoblasts.This study aims to clarify the effect of trophoblast α-Klotho on placental senescence and explore the feasibility of using trophoblast α-Klotho as the target to regulate placental senescence in AMA pregnancy.Furthermore,we aim to explore whether AMA is an independent risk factor for the loss of placental α-Klotho expression beginning in the early phase of pregnancy.Methods: The huma placental tissues(term placenta and early pregnancy villi)were collected.AMA mouse model was established.The expression patterns of α-Klotho in mouse and human placentas of AMA pregnancies were determined by Western blotting and IHC staining.α-Klotho-knockdown(Sh-KL)and overexpression(OE-KL)of JAR cells were generated using lentiviruses.The expression of α-Klotho in JAR cells was manipulated to investigate its role in trophoblastic senescence,and Transwell assays were performed to assess trophoblast invasion.Apoptosis rates of JAR cells were assessed using flow cytometry.The proliferative ability of JAR cells was detected by EDU assay.The downstream genes regulated by α-Klotho in JAR cells were first screened by m RNA sequencing in α-Klotho-knockdown and control JAR cells and then confirmed in by q RT-PCR.α-Klotho-deficient mice were generated by injecting klotho interfering adenovirus(Ad-Klotho)via tail vein on GD8.5.Results: Human term placentas and first trimester villi from AMA pregnancies showed significantly upregulated expression of multiple well-known biomarkers of senescence,including p53,p21 and p16,compared with that in control placentas.α-Klotho is ubiquitously expressed in various types of placental trophoblast cells,including cytotrophoblasts(CTBs),cell column trophoblasts(CCT),syncytiotrophoblasts(STB),and interstitial extravillous trophoblast(i EVT)cells.α-Klotho was expressed in both the labyrinth zone(Lz)and junctional zone(Jz)areas in the mouse placenta.α-Klotho expression was dysregulated in the placentas of AMA pregnancies.Ablation of α-Klotho resulted in not only a senescent phenotype and loss of invasiveness in JAR cells but also a reduction in the transcription of cell adhesion molecules(CAMs)genes.Overexpression ofα-Klotho significantly improved invasion but did not alter the expression of senescence biomarkers.α-Klotho-deficient mice exhibited the phenotype of placental senescence,manifested as increased expression of senescence marker including p53,p21 and p16.The area of blood sinuses in theα-Klotho-deficient placenta at GD14.5 and GD18.5 was significantly compromised.α-Klotho-deficient mice exhibited an abnormal junctional zone(Jz)/ labyrinth zone(Lz)area ratio.α-Klotho-deficient mice exhibited a remarkable reduction in both placental and fetal weight throughout the later gestational stages.Compared with negative control cells,α-Klotho-deficient cells showed significant alterations in the expression levels of 1876 m RNAs(1019 upregulated and 857 downregulated.α-Klotho knockdown leads to the down-regulation of the placental cell adhesion molecule pathway.Conclusion: In conclusion,AMA results in reduced α-Klotho expression in placental trophoblasts,therefore leading to premature senescence and loss of invasion(possibly through the downregulation of CAMs),both of which ultimately result in placental malformation and adverse perinatal outcomes.The decreased expression of α-Klotho induced by AMA may be an independent risk factor for placental senescence.α-Klotho may regulate the senescence of trophoblast cells by regulating the expression of senescence related biomarkers,and α-Klotho mediates the invasion ability of trophoblast cells by regulating the CAMS pathway.