The MicroRNA Expression Profile Changes Of Pineal In Neonatal Rats After Hypoxic-ischemic Brain Injury And The Effect Of MiR-375 To Pineal Function

Objective: To study the micro RNA expression profile changes of pineal gland in hypoxic-ischemic brain injury in neonatal rats in vivo; To detect the transfection efficiency of mi R-375 in culturing pinealocyte and to investigate the effect of mi R-375 on pineal gland function by building the oxygen glucose deprivation model in culturing pinealocyte in vitro.Method: HIBD model was established according to the modified Rice-Vannucci method from neonatal 7-old day Sprague Dawley(SD) rat. They were randomly divided into sham group, hypoxia- ischemia group. the micro RNA expression profile changes of pineal were detected by microarray and RT-q PCR after 24 h, the changes of AANAT m RNA and mi R-375 expression in culture pinealocyte isolated from neonatal SD rat under oxygen glucose deprivation(OGD) condition were detected by RT-q PCR ELISA was used to detect the changes of MT level were measured by the ELISA kit at 0 h, 12 h, 24 h, 48 h,72 h and 96 h after OGD.Cell viability was measured by CCK-8 and the relation, the effect of mi R-375 on pineal gland function under OGD conditon in pinealocytes transfected by lentivirus LV-mi R- mi R-375 in vitro and under HIBD conditon in neonatal rats transfected by lentivirus LV-mi R- mi R-375 in vivo were detected by RT-q PCR and the ELISA kit.Result:1.Microarray and RT-q PCR showed that the micro RNA expression profile of pineal gland had changed.2.RT-q PCR showed that mi R-375 expression was upregulated increased a peak level at 24 h, afterward gradually restored to the base level(0 h) at 72 h after OGD; AANAT expression was down-regulated, decreased a lowest level at 24 h, afterward gradually restored to the base level(0 h) at 72 h after OGD; Pinealocytes were transfected by LV-mi R-375 for 72 h, mi R-375 expression was obviously increased; AANAT expression were decreased in pinealocytes transfected by LV-mi R-375 for 48 h; mi R-375 expression was increased, reached a peak level at 24 h after OGD in LV-mi R-375--treated with pinealocytes; AANAT expression was decreased, reached a lowest level at 24 h after OGD in LV-mi R-375-pinealocytes.3.ELISA showed that MT level was decreased, reached a lowest level at 24 h,afterward gradually restored to the base level(0 h) at 96 h after OGD in culture pinealocytes; Pinealocytes were transfected by LV-mi R-375 for 72 h, MT level were decreased in pinealocytes transfected by LV-mi R-375 for 72 h; MT level was decreased,reached a lowest level at 24 h and 48 h after OGD in LV-mi R-375-treated with pinealocytes.4.CCK8 showed that the viability of pinealocyte was no changes at 0.5 h and 1 h after OGD, and 2 h of OGD induced pinealocyte injury, while 3 h of OGD significantly aggravated pinealocyte injury.Conclusion:1.The micro RNA expression profile of pineal gland has changed in neonatal rats after HIBD,mi R-375 expression was upregulated obviously.2. mi R-375 expression, AANAT m RNA and MT level have all changed in culture pinealocyte after OGD.3.mi R-375 aggravated OGD- induced pinealocyte damage.4. The activity of pinealocyte was decreased on the time of OGD dependent.