| Pregnancy complications,such as preeclampsia,intrauterine growth restriction,premature delivery,and adverse perinatal outcomes are often accompanied by placental hypoxia,which seriously threatens the health of pregnant mothers and offspring.Nobiletin(NOB),a hexamethoxyflavonoid derived from the citrus pomace,is a nontoxic constituent of dietary phytochemicals approved by the US Food and Drug Administration(FDA)that can be added to daily diets such as orange juice,fruit juice mixes,fruit drinks,sauces,cereal bars,hot cereals,and pastries.Literatures have reported that NOB has an anti-apoptotic effect,but whether it has a protective effect on placental cell apoptosis induced by hypoxia is not well understood.In this study,NOB was used to treat human placental choriocarcinoma cells(BeWo cell line),gestational rats and zebrafish larvae.The effects of NOB on apoptosis of BeWo cells were evaluated by determining the changes of molecules,cell morphology and fetal rats and zebrafish larvae developments with the method of flow cytometry,RT-q PCR,and Western blot.Then,we constructed the hypoxic model for human placental trophoblast cells(human choriocarcinoma JEG-3 cell lines and human placental choriocarcinoma BeWo cell lines)using cobalt chloride,the hypoxic model for the placenta and the brain of fetal rats using uterine perfusion pressure ligation(RUPP)operation on pregnant rats,and the hypoxic model for the brain of zebrafish larvae using a hypoxia incubator containing 5%oxygen.The protective effect of NOB on hypoxia-induced apoptosis of JEG-3 cells,BeWo cells,placental cells,and brain cells of the fetal rats and zebrafish larvae were also explored in this study.Last,the protective mechanism of NOB in reducing hypoxia-induced apoptosis was studied through the molecular docking and dynamics,UV visible spectrum,fluorescence spectroscopy,circular dichroism,and gene knockdown.The main results of the study were as follows:(1)There was no significant difference in late apoptosis rate between the group treated with low-dose NOB(10 or 33μM)and non-NOB-treated group.Compared with non-NOB-treated cells,cell viability was increased,and apoptosis was decreased in response to NOB(100μM)treatment after 48 h of culturing.The treatment of 100μM NOB after 48 h resulted in the up-regulation of BCL2 and BCLX_Lproteins,and the down-regulation of cl-PARP and P53 proteins,suggesting that NOB had a certain protective effect on apoptosis of BeWo cells.(2)Compared with the control group,NOB treatment resulted in the down-regulation of the protein levels of cl-PARP,the m RNA and protein levels of P53,the ratio of BCL2/BAX m RNA and protein in the placenta of pregnant rats and the brain of fetal rats,while TUNEL staining and AO/EB staining test of tissue sections showed no statistical difference.These results suggested that NOB had an anti-apoptotic effect on the placenta of pregnant rats and the brain of fetal rats.(3)There was no developmental toxicity to zebrafish in early life when exposed to NOB at the doses of 10,100 and 1000 n M.There was no significant difference in the behavior of zebrafish larvae among the control group,the 10 n M NOB treatment group and the 100 n M NOB treatment group.Meanwhile,the movement distance and movement speed of the zebrafish larvae were improved after 1000 n M NOB treatment.The m RNA and protein levels of Hif1α,LDH activity and CS activity in the brain of zebrafish larvae were all significantly down-regulated after 1000 n M NOB treatment,indicating that NOB intervention promoted the oxygen metabolism capacity of zebrafish larvae brains.After 1000 n M NOB intervention,the gene expressions of p53,mdm2,and p21 in the zebrafish larvae brain were down-regulated,and the gene ratio of bcl2/bax increased,suggesting that NOB reduced cell apoptosis in the brain of zebrafish larvae.(4)Hypoxia-induced apoptosis models of JEG-3 and BeWo cell lines were successfully constructed by cobalt chloride,respectively.NOB had a protective effect on the apoptosis of JEG-3 and BeWo cell lines induced by hypoxia.The treatment of 100μΜNOB diminished the m RNA and protein levels of HIF1α,and the G1 phase arrest.JEG-3 and BeWo cells treated with 100μΜNOB down-regulated the m RNA and protein expressions of P53 and P21.The BCL2/BAX m RNA and protein ratio of hypoxia-induced JEG-3 cells was up-regulated after 100μM NOB treatment but did not change the BCL2/BAX ratio for BeWo cells.(5)The RUPP operation was used to construct the hypoxia model of the placenta of pregnant rats and the brain of fetal rats.RUPP treatment caused severe hypoxia and damage to the placenta of the pregnant rats and the brain of the fetal rats.These damages were alleviated by NOB pretreatment during pregnancy GD0.5-GD17.5.The m RNA and protein levels of P53,MDM2 and P21 were down-regulated and the m RNA and protein ratio of BCL2/BAX were up-regulated after NOB intervention in the placenta of the pregnant rats and the brain of the fetal rats induced by RUPP.These results indicated that NOB alleviated the cell apoptosis of the placenta of pregnant rats and the brain of the fetal rats induced by RUPP,thereby reducing the RUPP induced-damage in the placenta of the pregnant rats and the brain of the fetal rats.(6)The brain hypoxic model of zebrafish larvae was successfully constructed through an anaerobic incubator containing 5%oxygen.NOB intervention in early life could alleviate brain hypoxia of zebrafish,which was manifested by the down-regulation of Hif 1αm RNA and protein levels,and the decrease of LDH activity.The inhibition of zebrafish larvae behavior caused by hypoxia could be alleviated by NOB intervention,which was revealed in the recovery of zebrafish’s moving distance and speed.Apoptosis-related gene expressions of p53,mdm2 and p21 were increased,and the m RNA ratio of bcl2/bax was decreased in the brain of zebrafish larvae exposed to hypoxia,which could be alleviated after NOB intervention.These results indicated that NOB alleviated the brain hypoxic damage of zebrafish larvae caused by hypoxia and improved zebrafish’s exercise capacity.(7)Molecular docking and dynamics found that NOB spontaneously bond to human p53protein,leading to the change of P53 protein conformation.The intermolecular interaction between NOB and human P53 protein was further confirmed by UV visible spectrum,fluorescence spectroscopy and circular dichroism.Hypoxia caused apoptosis of BeWo cells and cell cycle arrest in G1 phase.Compared with the hypoxic control group,cell apoptosis and cell cycle arrest in the G1 phase were significantly down-regulated after knocking down the P53 gene in BeWo cells.NOB intervention did not change the apoptosis and the distribution of cell cycle in G1 of BeWo cell lines with p53 gene knockdown.These results indicated that NOB could alleviate hypoxia-induced apoptosis by regulating the P53 signaling pathway. |
