Protective Effect Of BZP On Oxygen-glucose Deprivation And Reintroduction Induced PC12 Cells Injury

Sodium(±)-5-Bromo-2-(α-hydroxypentyl)benzoate(brandname: Brozopine,BZP)is a new compound with independent intellectual property rights,Which was selected from the Department of chemistry of Zhengzhou University based on the structure modification,structure-activity relationship evaluation,pharmacology and toxicology research,mechanism discussion and so on.In this study,we established the model of oxygen-glucose deprivation and reintroduction(OGD/R)on PC12 cells to explore the protective effect of BZP on OGD/R.Part Ⅰ Protective effects of brozopine on OGD/R induced PC12 cells injuryObjective To explore the protective effect of BZP on OGD/R OGD/R induced PC12 cells injury.1 The establishment of OGD/R model.The PC12 cells were given to the different concentration of Na2S2O4 with low-glucose DMEM at different time and then reoxygenatie for 24 h.Cell morphological changes was observed,theconcentration and incubation time of Na2S2O4 with low-glucose DMEM was recorded when the cell survival rate is about 50 %.2 The dose-effect curve of BZP.The OGD/R model of PC12 cells was pretreated with different concentrations of BZP.Then the dose-effect curve of BZP was drawn to determine the low,medium and high concentration of BZP.3 Testing index.PC12 cells were given to Na2S2O4 with low-glucose DMEM and then reoxygenation for 24 h,and also pretreated with BZP,Br-NBP,and NBP for 24 h respectively.The morphology of PC12 cells was observed by inverting microscope.Cell viability was measured by CCK8 assay,the LDH leakage in the conditioned medium was observed by microplate reader,DNA-binding dye Acridine Orange(AO)was used to detect the morphological characteristic of apoptosis by using a fluorescence microscope.Propidine Iodide(PI)was used to measure apoptosis rate by using Arrary Scan VTI High-Content Screening Reader and Image J Software.In addition,the cell apoptosis detected by Annexin/PI Apoptosis kit.Results 1 The OGD/R model.The PC12 cells were given to 10 mmol/L Na2S2O4 with low-glucose DMEM for 3 h and then reoxygenatie for 24 h according to the cell survival rate.2 The dose concentration of BZP.According to the dose-effect curve of BZP,the dose concentration of BZP were 2,10 and 50 μmol/L respectively.3 Compared with OGD/R group(53.3 ± 2.87 %),pretreatment of the cells with BZP(2,10,and 50 μmol/L)were able to remarkably increase the percentage of inhibition of cells injury by 59.3 ± 4.97 %,68.6 ± 1.75 %,84.6 ± 2.78 % respectively(P < 0.01).There were the difference between BZP 10 μmol/L and Br-NBP 10 μmol/L or NBP 10 μmol/L groups(P < 0.05).The results showed BZP could inhibit cell injury.4 Compared with OGD/R group(917 ± 94.8 U/L),pretreatment of the cells with2,10,and 50 μmol/L BZP were able to increased the percentage of inhibition of LDH leakage remarkably by 26.7 %,29.8 %,and 39.7 % respectively.There was the difference between BZP 10 μmol/L and Br-NBP 10 μmol/L or NBP 10 μmol/L groups(P < 0.05).5 Many axonal arborization were observed around the soma and dendritic trees in Control group.Following exposure of the cells to OGD/R,no dendritic spines could be identified.In contrast,pretreatment of the cells with 2,10,and 50 μmol/L BZP,10 μmol/L Br-NBP,and 10 μmol/L NBP were able to remarkably recover PC12 cells morphology.6 Incubation of cells with 10 mmol/L Na2S2O4 withlow-glucose DMEM for 3 h resulted in shrinkage of the cell and nucleus,and condensation of nuclear chromatin into sharply delineated masses abutting the nuclear membrane.Meanwhile,the nucleus broke up into small fragments(karyorrhexis),and cellular debris is extruded via budding from the plasma membrane,creating cell remnants(apoptotic bodies).In contrast,cells pretreated with different concentrations of Br-NBP(2,10,and 50 μmol/L)inhibited the morphological changes to some extent in a concentration-dependent manner.7 The positive cell rate of PI staining showed that BZP 2,10,and 50 μmol/l groups was 60.3 ± 12.7,49.3 ± 7.98,29.3 ± 11.5 respectively,which was decreased compared with OGD/R group(80.7 ± 14.6;P < 0.05,P < 0.01).Compared with OGD/R group,the apoptotic rates of BZP 2,10,and 50 μmol/L groups(7.34 ± 0.16,5.41 ± 0.17,3.11 ± 0.24,respectively)were obviously reduced according to the Flow cytometry results(9.42 ± 0.347,P < 0.01).In conclusion,BZP could inhibit the apoptosis rate of PC12 cells after OGD/R.Conclusion :BZP exhibited a protective effect on OGD/R induced PC12 cells injury in a concentration dependent manner.Part Ⅱ Protective effect of brozopine on OGD/R induced PC12 cells injuryObjective To explore the protective mechanism of BZP on OGD/R induced PC12 cells injury,so as to provide relevant evidence for clinical application.Materials and Methods Groups were divided in the same way of partⅠ.The GSH-px activities,MDA and NO contents in cells were observed by microplate reader.Mitochondrial membrane potential in PC12 cells were monitored by Rh123-staining.The reactive oxygen species(ROS)were monitored combined with DCFHDA by Arrary Scan VTI High-Content Screening Reader and Image J Software.Intracellular [Ca2+]i was measured using the calcium-sensitive dye Fura 2-AM.To detect the protein expression of Bax,Bcl-2,cleaved Caspase-3,and caspase-3 in PC12 cells by Western blot analysis.Results 1 The PC12 cells were exposed to 10 mmol/L Na2S2O4 with low-glucose DMEM for 3 h and then to reoxygenation for 24 h,and also pretreated with BZP,NBP and Br-NBP respectively.The elevated contents of MDA,NO productions and decreased activities GSH-px in PC12 cells were observed,preincubation with BZP,NBP and Br-NBP at different concentrations significantly attenuated MDA contents and NO productions(P < 0.01)in a concentration-dependent manner.Interestingly,BZP at different concentrations had different effect on GSH-px activity.2 Depolarization of MMP results in the loss of Rh123 from the mitochondria and a decreased in intracellular fluorescence intensity.Compared with OGD/R group,the positive cell rate were 29.2 ± 1.59,36.5 ± 1.76,46.6 ± 2.37 respectively at BZP with different concentrations.The results showed BZP elevated MMP.3 OGD/R group(57.8 ± 1.57)enhanced ROS production in PC12 cells,while 2,10,50 μmol/L BZP attenuated the increase in the positive cell rate by 3.76 ± 0.159,33.7 ± 1.76 and 20.8 ± 2.37 respectively in a concentration-dependent manner(P < 0.01).The results revealed that BZP decreased intracellular ROS level.4 In Krebs’ solution containing calcium,OGD/R group induced an increase in thefluorescence intensity.When the cells were pretreated with BZP at different concentrations,the positive cell rate was significantly reduced.In calcium-free Krebs’ solution,OGD/R group did not induce the increase of fluorescence intensity,and BZP at different concentrations did not further decrease fluorescence intensity.The OGD/R-induced increase in intracellular Ca2+ was dependent on extracellular Ca2+,since removal of extracellular Ca2+ prevented the OGD/R-induced increase in intracellular Ca2+.5 The ratio of Bax/Bcl-2 in BZP 2,10,and 50 μmol/L groups were 1.74 ± 0.31,1.60 ± 0.26,and 1.41 ± 0.10,which were significantly decreased in dose-dependent manner,when the OGD/R group were significantly increased compared with the Control group(P < 0.01).The ratio of BZP cleaved-caspase-3/caspase-3 in BZP 2,10 and 50 μmol/L groups were 1.45 ± 0.32,1.25 ± 0.27,and 1.05 ± 0.17,which were significantly decreased in dose-dependent manner,when the OGD/R group were significantly increased compared with the Control group(P < 0.01).BZP 10 μmol/l group has a better effect than NBP 10 μmol/l group(P < 0.05).The results showed that BZP could inhibit the apoptosis of cells after OGD/R damage in PC12 cells by reducing Bax/Bcl-2 and cleaved caspase-3/caspase-3.Conclusions 1 BZP exhibited the protective effect on OGD/R induced oxidative stress damage in PC12 cells,suggesting that the compound may be a potential therapeutic agent for diseases induced by oxidative damage.The protective effects of Br-NBP were related to the inhibition of lipid peroxidation,ROS formation and restoration of mitochondrial membrane potential and reduction of [Ca2+] i.2 BZP inhibited the protein expression of cleaved-Caspase-3 and Bax,and reduced the ratio of cleaved caspase-3/caspase-3 and Bax/Bcl-2,indicating that BZP has have significant anti-apoptosis effect.