Sevoflurane Represses The Self-renewal Ability In Mouse Embryonic Stem Cells

Background and ObjectiveWith the advancement of medical technology,the number of pregnant women receiving non-obstetric surgery is gradually increasing.However,the impact of narcotic drugs on fetal development during general anesthesia in pregnant women is always the focus of attention of doctors and patients.Fetal interventional therapy,extensive laparoscopic surgery during pregnancy and part of cesarean section etc.are all required for general anesthesia.Inhalation anesthetics,with the advantages of strong anesthetic efficacy and good controllability,are widely used in clinical practice,including neonatal and pediatric surgery.Sevoflurane is currently one of the most commonly used inhalation anesthetics,whereas,sevoflurane exerts an anesthetic effect by blocking NMDA receptors in the brain and activating GABAA receptors,and whether sevoflurane has developmental neurotoxicity also causes extensive concern of anesthesiologists and research workers.Sevoflurane has a small molecular weight and lipophilicity,so it is easy to pass the placental barrier during anesthesia and may have adverse effects on fetal neurodevelopment.Studies have found that prolonged or multiple sevoflurane exposure during pregnancy can lead to learning and memory damage in offspring mice.The fetus’ s nervous system starts to develop from the fifth to sixth week of pregnancy.The nervous system is the organ with the longest developmental period and the earliest development in all organs.Therefore,the early pregnancy is a crucial period for the development of the fetal central nervous system and the model establishment.Once the developmental damage occurs,it will gradually accumulate in the subsequent development,leading to serious consequences.And the mechanism of sevoflurane influces nervous system development and pattern formation in early pregnancy is not yet clear,demonstrate its mechanism of action will help to provide a theoretical basis on choosing a safer anesthesia drugs for pregnancy during general anesthesia.Materials and methodsMouse embryonic stem cells(E14 mESCs)were purchased from the Shanghai Institute of Biochemistry and Cell Biology and cultured in Dulbecco’s modified Eagle’s medium.All experiments were performed with passage 2-4 generation of mESCs.mESCs were monolayer adherently cultured and were randomly divided into 7 groups: Ctrl group,Sevoflurane group,miR-7a / 7b group,miR inhibitor-Ctrl group and miR-7a / 7b inhibitor group.DAPI was used to observe the apoptotic status of each group of mouse embryonic stem cell nuclei;quantitative real-time PCR was used to detect the expression of stem genes Oct4,Sox2 and Nanog and miR-7a / 7b.Flow cytometry was used to detect cell cycle;Dual-luciferase reporter assay was used to detect whether miR-7a / 7b could bind to mRNA of 3’UTR of Klf4;The changes of Klf4 expression of target gene were detected by Western blot to determine the effect of Klf4 on miR-7a/7b.BrdUrd-positive cells were counted by BrdUrd incorporation assay to detect the number of cells in S phase.Results(1)Sevoflurane inhibits self-renewal ability of mouse embryonic stem cellsCompared with the Ctrl group,Sevoflurane mice ES cells were treated with 4.1% sevoflurane and cultured for 24 h.The expression of Oct4,Sox2 and Nanog in the Sevoflurane group was higher than that in the Ctrl group(P <0.05).The number of BrdUrd positive cells in Sevoflurane group was lower than that in control group,with statistical significance(P <0.05).(2)Upregulation of miR-7a,7b levels in sevoflurane-treated murine embryonic stem cells and up-regulation of miR-7a,7b inhibit the self-renewal ability of ES cellsMiR-7a and miR-7b were up-regulated in Sevoflurane group compared with Ctrl group(P <0.05);mESCs were transfected with miR-7a and miR-7b,Compared with the miR-7a and miR-7b group,the expression levels of Oct4,Sox2 and Nanog and the number of BrdUrd positive cells in miR-7a and miR-7b group were significantly decreased(P <0.05)Overexpression inhibits the self-renewal ability of murine ES cells.(3)Sevoflurane affects self-renewal ability of mouse embryonic stem cells through miR-7a,7bThe mESCs were transfected with miR-7a inhibitor and miR-7b inhibitor respectively to inhibit the expression of miR-7a and 7b,and miR-7a inhibitor and miR-7b inhibitor mESCs were treated with 4.1% sevoflurane.MiR-7a inhibitor group and miR-7b inhibitor group mESCs dry markers of Oct4,Sox2 and Nanog expression levels and BrdUrd positive cells were no significant difference between the number of confirmed that the inhibition of miR-7a,miR-7b Effect of Sevoflurane Secretion on the Ability of Self-renewal of Mouse ES Cells.(4)Klf4 is a downstream target gene of miR-7a,7b,overexpression of miR-7a,7b down-regulated Klf4 expressionCompared with the Ctrl group,the expression levels of Klf in miR-7a and miR-7b groups decreased significantly,with significant difference(P <0.05)ConclusionsSevoflurane Affects Self-renewal Ability of Mouse Embryonic Stem Cells by Regulating miR-7a,7b / Klf4 Signaling Pathways...