| Objective: Intrauterine placental hypoperfusion(IUPH)is a common pathological feature of a variety of fetal abnormalities,which can affect the development of the fetal nervous system in the second trimester and bring a poor prognosis for the fetus.With the development of assisted reproductive technology,complex twins complications have increased,and feto-scopic surgery has been carried out extensively.The degree of intrautero-placental hypoperfusion increases the frequency of occurrence.Progesterone not only acts as a hormone during pregnancy,but itself is also a neurosteroid,which can exert neuroprotective effects to a certain extent.The neuroprotective effect of progesterone in the second trimester neurodevelopmental stage is still unclear.The purpose of this study was to establish a uteroplacental hypoperfusion pregnant rat model to simulate the pathological effects of intrauteroplacental hypoperfusion on fetal brain development in the second trimester,and supplement progesterone to treat the offspring of uteroplacental hypoperfusion pregnant mice to explore the role of progesterone in uteroplacental hypoperfusion.Mechanisms that exert neuroprotective effects under the model.Methods: 1.The intrautero-placental hypoperfusion pregnant mouse model was established at 18.5 days of gestation by surgical ligation of bilateral uterine arteries,and divided into SHAM operation group,uteroplacental hypoperfusion group,SHAM operation plus progesterone treatment group and intrautero-placental hypoperfusion plus progesterone group,the fetal mice were collected at 24 h,48h,and 72 h after the model was established at multiple time points.RT-q PCR was used to detect the relative expression levels of apoptosis-related genes Bcl-2,Bax,Caspase-3,etc.,and Western blotting was used.The expression levels of apoptosis-related proteins Bcl-2,Bax and Caspase-3 were detected by TUNEL method,and the apoptosis of neurons in fetal rat brain tissue sections was detected by TUNEL method.2.After the establishment of the uteroplacental hypoperfusion model of pregnant mice,the offspring were kept and reared until 28 days after birth.The neurobehavioral Morris water maze test was performed to detect and evaluate the effect of uteroplacental hypoperfusion and progesterone treatment on the learning and memory of the offspring after birth.For the influence of ability,the corresponding brain tissue sections were taken after the end of neurobehavioral study,and the pathological changes of brain tissue brought by uteroplacental hypoperfusion and progesterone treatment were observed by HE staining and Nissl body staining.3.Using western blot to detect the BDNF/PI3K/Akt signaling pathway related proteins BDNF,PI3 K,p-Akt/Akt expression levels.4.The neuronal cell line HT22 cells were cultured in vitro under hypoxia to simulate the pathological process of uteroplacental hypoperfusion on fetal rat brain neurons,and progesterone was added to the culture medium for treatment,and RT-q PCR was used to detect apoptosis.The relative expression levels of apoptosis-related genes Bcl-2,Bax,Caspase-3,etc.,and the expression levels of apoptosis-related proteins Bcl-2,Bax,Caspase-3,etc.were detected by western blotting,and cell viability was detected by CCK8 method and flow cytometry.The level of apoptosis was detected by surgery,and the apoptosis was detected by TUNEL method.5.At the cellular level,the PI3K/Akt signaling pathway inhibitor LY294002 was used to explore the mechanism that progesterone might activate the BDNF/PI3K/Akt signaling pathway to provide anti-apoptotic effects under hypoxia.Results: 1.The establishment of a model by bilateral uterine artery ligation can reduce the blood flow of the uterine artery to the fetal mouse,and can establish a uteroplacental hypoperfusion pregnant mouse model.2.The results of RT-q PCR showed that the anti-apoptotic gene Bcl-2 decreased,the pro-apoptotic genes Bax and Caspase-3 increased in the brain tissue of E19.5,E20.5,and E21.5 fetal mice after uteroplacental hypoperfusion.Maternal supplementation of progesterone can increase Bcl-2 and decrease Bax and Caspase-3.3.Western Blot results showed that the expression of anti-apoptotic protein Bcl-2 in E19.5,E20.5,and E21.5 fetal rat brain tissue decreased after uteroplacental hypoperfusion,and the pro-apoptotic proteins Bax and Caspase-3 increased,while After progesterone supplementation to the mother,Bcl-2 in uteroplacental hypoperfused fetal mice can be increased,and Bax and Caspase-3 can be decreased.4.The TUNEL method detected E19.5,E20.5 and E21.5fetal rat brain tissue sections.It can be seen that the apoptosis level of brain tissue increased under uteroplacental hypoperfusion.After progesterone supplementation to the mother,fetal rat brain tissue The level of apoptosis decreased.5.For the neurobehavioral test of the offspring after birth,the Morris water maze test was used to detect,in the positioning cruise experiment,there was no significant difference in the average swimming speed between the groups,and in the escape latency,compared with the control group,In the uteroplacental hypoperfusion group,with the progress of the experimental days,the escape latency time was prolonged,and the difference between the two groups was statistically significant.Compared with the uteroplacental hypoperfusion group,the escape latency was reduced,and the difference between the two was statistically significant.6.In the neurobehavioral Morris water maze space exploration experiment,compared with the postnatal offspring of SHAM group,the number of crossing the platform and the length of stay in the fourth quadrant of the platform decreased,and the difference was statistically significant.After progesterone supplementation to the mother,compared with the offspring of the IUPH group,the number of crossing the platform and the length of stay in the fourth quadrant of the offspring increased,and the difference was statistically significant.7.In the experiment of the effect of progesterone on HT22 cells under hypoxia,the results of RT-q PCR indicated that the expression of proapoptotic genes Bax and Caspase-3 increased under hypoxia,but decreased after administration of progesterone;the anti-apoptotic gene Bcl-2 expression decreased,but increased after progesterone supplementation.8.The effect of progesterone on the apoptosis of HT22 cells under hypoxia was detected by flow cytometry and TUNEL.The results showed that the apoptosis level of HT22 cells increased under hypoxia,while the apoptosis level of HT22 cells treated with progesterone decreased.9.Western Blot detected the PI3K/Akt signaling pathway protein in fetal rat brain tissue,and found that the expression levels of PI3 K and p-Akt/Akt decreased in the IUPH group at the time points of E19.5,E20.5,and E21.5,and progesterone was administered.After treatment,the expression levels of PI3 K and p-Akt/Akt increased at each sampling time node in the IUPH+PROG group.10.In HT22 cells,the results of Western Blot detection indicated that the expression levels of BDNF,PI3 K and pAkt/Akt decreased after hypoxia,and the expression levels of BDNF,PI3 K and pAkt/Akt after treatment with cellular progesterone increased,and the activation of PI3K/Akt signaling pathway by progesterone could be reduced after the use of PI3K/Akt pathway-specific inhibitors.Conclusion: 1.Hypoperfusion of uteroplacenta can induce neuronal apoptosis in fetal mice,and progesterone supplementation can reduce the occurrence of apoptosis.2.The learning and memory of the offspring of the uteroplacental hypoperfusion model after birth were lower than those of the SHAM operation group,and the learning and memory ability of the offspring was improved to a certain extent after supplementing progesterone during pregnancy.3.Under uteroplacental hypoperfusion,progesterone may reduce neuronal apoptosis by activating PI3K/Akt signaling pathway in brain tissue cells.4.Under hypoxia,progesterone may reduce hypoxia-induced apoptosis by activating BDNF/PI3K/Akt signaling pathway in HT22 cells.5.Progesterone may provide neuroprotective effects under uteroplacental hypoperfusion in the second trimester. |
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