| Objective To investigate the relationship between Nrf2 and mitochondrial division in cell line SH-SY5 Y by establishing a cell model of oxygen and glucose deprivation/recovery in vitro and to find out whether this regulatory relationship is via Drp1.Methods A cell model of oxygen glucose deprivation/recovery was created by a three-gas incubator,sugar-free medium and normal medium.According to the references,OGD4 h,6 h,8 h,and 10 h were matched with R0 h,6 h,12 h,18 h,and 24 h to set up various time length combinations.After the treatment of OGD/R,the cell viability was determined by Cell Counting Kit-8(CCK8 kit)and the ideal time length combnation where cells showed obvious apoptosis but still had half of the survival.Tert-butylhydroquinone(t BHQ)、 Brusatol(Bru)and Dimethyl sulfoxide(DMSO)were used as the agonist、inhibitor and vehicle to pretreat cells for 4h followed by OGD/R.Cells were grouped as follows:(1)SH-SY5 Y cells normal group: SH-SY5 Y cells were normally cultured with no other treatment;(2)OGD/R group: 8000 SH-SY5 Y cells were seeded in 96-well plates.After one day of stabilization,the cells were replaced with sugar-free EBSS medium and placed in a three-gas incubator for 4 hours and then the cells were replaced with normal medium and transferred to a normal oxygen-containing incubator for 18 hours(OGD4h/R18h);(3)OGD/R+t BHQ group: SH-SY5 Y cells were pretreated with normal medium containing t BHQ(final concentration 25 μM)for 4 h and then went through OGD/R;(4)OGD/R+Bru group: SH-SY5 Y cells were pretreated with normal medium containing Bru(final concentration 100 n M)for 4h and then went through OGD/R;(5)OGD/R+Veh group: SH-SY5 Y cells were pretreated with normal medium containing DMSO(final concentration 0.1%)for 4 h and then went through OGD/R.Western blot was applid to measure the expression of proteins Nrf2 and Drp1.Meanwhile the morphology of mitochondria in the same batch of cells was observed by the electron microscopy.Results 1.OGD4 h had the highest cell survival rate in all and the cells’ survival rate decreased significantly after R18 h,which was a better time combination representing ischemia-reperfusion injury.2.Western blot showed that the expression of Nrf2 in OGD/R+t BHQ group was significantly higher than that in OGD/R group(P<0.05)indicating that t BHQ could increase the quantity of Nrf2 in cells.Whreas the expression of Nrf2 in OGD/R+Bru group was lower than that in OGD/R group(P<0.05)suggesting that Bru could decrease the quantity of Nrf2 in cells.To conclude,t BHQ could increase the quantity of Nrf2 in cells and Bru could cause the oppsite effect.3.Western blot showed that the expression of Drp1 in OGD/R+ t BHQ group was lower than that in OGD/R group(P<0.05)suggesting that Nrf2 could decrease the quantity of Drp1 in cells.However the expression of Drp1 in OGD/R+Bru group was higher than that in OGD/R group(P<0.05)also suggesting that Nrf2 could decrease the quantity of Drp1 in cells.To conclude,the increase of Nrf2 could decrease the quantity of Drp1 in cells.4.The results of electron microscopy showed that the degree and proportion of mitochondrial division in the OGD/R+t BHQ group were decreased compared with the OGD/R group,but increased in the OGD/R+Bru group(P<0.05).Conclusion The change of Nrf2 in cell line SH-SY5 Y can cause negative correlated changes of Drp1 and the mitochondrial morphology also produced corresponding dynamic changes.Nrf2 may negatively regulated the mitochondrial division via Drp1. |
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