Inhibitory Effect Of ERK Inhibitor PD98059 On Apoptosis Of SH-SY5Y Cells Induced By Hypoglycemia And Hypoxia

Objective:The ERK inhibitor PD98059 can reduce ROS produced by cerebral ischemia-reperfusion injury and play an anti-apoptotic role,but whether its mechanism is related to reducing ROS and maintaining the balance of mitochondrial morphology and function has not been reported yet.Cerebral ischemia-reperfusion impairs mitochondrial DNA,disrupts electron transport in the respiratory chain complex,and increases ROS production in tissues,while excessive levels of ROS in turn further disrupt mitochondrial homeostasis,which in turn promotes mitochondrial pathway-mediated apoptosis.Our previous study found that the activation of ERK in the brain tissue of rats with cerebral ischemia-reperfusion injury was significantly increased,and the apoptosis rate was also significantly increased,while the ERK inhibitor PD98059 could significantly reduce the ROS in the brain tissue of rats with ischemia-reperfusion injury.And the production of SOD,play a role in brain protection.Therefore,we hypothesized that the mechanism of PD98059 protection against ischemia-reperfusion brain injury is related to the reduction of ROS production and the maintenance of mitochondrial homeostasis.In this experiment,we constructed a hypoglycemic hypoxia-reperfusion reperfusion cell model to simulate ischemia-reperfusion injury in vivo.Different doses of PD98059 were given before glucose deficiency and hypoxia.The purpose of this study was to investigate whether PD98059 is resistant to hypoxia-anoxia-reperfusion injury and maintain mitochondria.Steady state related.Methods:Neuroblastoma cells(SH-SY5Y)with good growth were plated at a certain density.After 24 hours of culture,the cells were randomly divided into five groups:normal control group(Control Group,Control)and DMSO control group.DMSO),Oxygen-Glucose Deprivation/Resuscitation(OGD/R),PD98059 Low-dose(PD-L)and PD98059 High-dose(PD-H),DMSO,PD-L And PD-H group were given 0.04%DMSO,10?M and 20?M PD98059,Control group and OGD/R group were added fresh complete medium.After 24 hours,OGD/R group,PD-L group and PD-H group The OGD/R treatment was carried out,followed by reoxygenation for 24 hours,and the Control group and the DMSO group were routinely cultured.The experimental indicators were as follows:Western blotting was used to detect the expression of p-ERK and ERK proteins,and the activation of ERK pathway was observed.The survival rate of cells was determined by CCK-8 method to determine the optimal hypoxia time The nucleus was stained with Hoechest 33258.The apoptosis was observed.The double staining of SH-SY5Y cells was determined by Annexin V-FITC/PI staining and flow cytometry.The apoptosis rate of the cells was determined.The apoptosis proteins Claved-caspase3/caspase3 and Bcl-2 were detected.The expression of Bax was further determined by apoptosis.The ultrastructure of mitochondria was observed by transmission electron microscopy.The changes of ROS and mitochondrion membrane potential(MMP)were observed by fluorescence microscope.The T-SOD and MnSOD were detected by UV spectrophotometer.Activity,Western blot was used to detect the expression of mitochondrial proteins OPA1,MFN1,p-DRP1/DRP1 and Cyto c,and the changes of mitochondrial fusion and division function were observed.Results:1.At 1h,2h and 3h after hypoxia,the expression of p-ERK was gradually increased by Western blot,and the expression of ERK was unchanged.2.Under different hypoxia time,the expression of claved-caspase3 was gradually increased with the prolongation of hypoxia,and the expression of caspase3 was unchanged.At the same time,the survival rate of cells under different hypoxia time was detected by CCK-8 method:Control group:(100±0.33)%,hypoglycemia and hypoxia 1h(OGD 1 h/R 24h)group:(81±0.17)%,hypoglycemia and hypoxia 2h(OGD 2h/R 24h)group:(566±0.25)%,glucose-deficient hypoxia 3h(OGD 3h/R 24h)group:(35±0.14)%,hypoglycemia and hypoxia 4h(OGD)4h/R 24h)group:(26 ± 0.10)%.It can be seen that as the time of hypoxia increases,the survival rate of cells gradually decreases.3.After the addition of the ERK inhibitor PD98059,the expression of p-ERK protein was significantly inhibited,and the expression of ERK was unchanged.At the same time,the expression of Claved-caspase 3 was significantly decreased,the expression of Caspase 3 was basically the same,and the ratio of Bcl-2/Bax was gradually increased.Hochst staining showed that the nuclear morphology of PD-L group and PD-H group was lighter than that of ODG/R group,the nuclear morphology gradually became normal,nuclear pyknosis decreased,and nuclear membrane damage decreased.The apoptotic rate of cells detected by flow cytometry was(8.5±5.2)%in the Control group,(13.4±5.8)%in the DMSO group,(50.4±10.7)%in the OGD/R group,and the PD-L group was(37.6±1.1)%,and the PD-H group was(27.9±4.6)%.DMSO vs Control:p>0.05;OGD/R vs Control:p<0.01;OGD/R vs PD-L:p<0.05;OGD/R vs PD-H:p<0.01.4.Fluorescence microscopy showed that PD98059 can significantly reduce the production of intracellular ROS and increase the mitochondrial membrane potential.Ultraviolet spectrophotometer showed that the activity of T-SOD in OGD/R group was significantly lower than that in Control group(P<0.01).In PD-H group,the activity of T-SOD was significantly higher than that in OGD/R group.p<0.01);MnSOD also gradually increased with the increase of PD98059 dose.5.Transmission electron microscopy showed that the mitochondria were swollen in the OGD/R group,and vacuolization occurred.The ridges were blurred.In the PD-L group and the PD-H group,mitochondrial vacuolization gradually decreased and the outer membrane became intact.Gradually clear.6.Western blot analysis of mitochondrial proteins revealed that the expression levels of MFN1,OPA1,p-DRP1,DRP1 and Cyto c increased in the OGD/R group.In the PD98059 group,the expression levels of these proteins gradually increased with increasing drug concentration.reduce.Conclusions:The ERK inhibitor PD98059 blocks the mitochondrial homeostasis caused by OGD/R and protects mitochondrial morphological and functional integrity,thereby inhibiting mitochondrial pathway-mediated apoptosis.