The Effect Of Isorhynchophylline On β Amyloid Peptides (25-35) Induced PC12 Cells Apoptosis

Objective:To research the effect of Isorhynchophylline（IRN）onβamyloid peptides 25-35(Aβ_25-35)damaged in rat adrenal pheochromocytoma cells（PC12 cells,PC12）apoptosis,and to investigate the possible pharmacological effect and mechanism.Methods:（1）PC12 cells were set up and subcultured.（2）The PC12 cells of logarithmic growth phase were divided into normal control group,model group and IRN groups with 5 concentration gradients.The IRN groups were respectively treated with IRN of different concentration gradient in PC12 cells after 2 hours,then added20mol/L Aβ_25-355-35 into culture to co-incubate for additional 24 hours.Cell viability was detected by CCK-8 kit.The cell proliferation rate was determined by crystal violet colorimetry.The concentrations of three IRN interventions were screened.（3）Cell apoptosis rate was detected by flow cytometry.（4）The content of malondialdehyde（MDA）,Superoxide dismutase（SOD）and glutathione peroxidase（GSH-PX）activity was detected by colorimetric method.Intracellular reactive oxygen species（ROS）was determined by fluorescence spectrophotometry（DCFH-DA）.（5）Mitochondrial membrane potential was detected by Rhodamine 123 fluorescence staining.（6）ELISA was used to detecting tumor necrosis factor alpha（TNF-α）.（7）The expression of nuclear factor kappa B（NF-κB）,Interleukin 1β（IL-1β）,cysteine-containing aspartate specific proteases-3（caspase-3）,B-cell lymphoma-2（Bcl-2）,Bcl-2 Associated X Protein（Bax）and Cytochrome C（Cyt C）were detected by Western blot.Results:（1）Compared with the cell survival rate[（99.32±0.21）%]and proliferation rate[（99.56±0.42）%]in control group,the survival rate[（51.01±2.24）%]and proliferation rate[（50.86±2.36）%]in model group were reduced（P<0.05）;Compared with model group,the cell survival rate[（55.62±2.70）%,（59.05±2.06）%,（76.03±1.97）%,（66.26±1.41）%,（57.22±2.86）%,（52.32±2.65）%]and proliferation rate[（58.74±2.44）%,（76.22±1.86）%,（67.56±1.82）%]of IRN groups were increased（P<0.05）,which the concentration of IRN 20μmol/L group increased significantly.Then determined the low,medium and high concentrations of IRN were 5μmol/L,20μmol/L,80μmol/L.（2）The results of Flow Cytometry showed that compared with control[（2.50±1.02）%]group,the apoptosis rate[（25.31±2.62）%]of model group was increased（P<0.05）;Compared with model group,the apoptosis rate[（23.10±1.52）%,（14.07±2.02）%,（22.67±1.12）%]of IRN group were decreased（P<0.05）.（3）ComparedwiththeactivityofSOD[（366.89±7.85）U/mgprot],GSH-Px[（372.16±6.58）mU/mg]and the level of MDA[（586.14±24.81）nmol/mg prot]incontrolgroup,theSOD[（162.26±5.15）U/mgprot]and GSH-Px[（92.65±11.35）mU/mg]activities were decreased while the MDA[（1343.42.49±26.42）nmol/mg prot]level were increased in model group（P<0.05）;Comparedwithmodelgroup,theSOD[（195.11±18.26）U/mgprot,（296.64±4.12）U/mgprot,（272.34±4.26）U/mgprot]andGSH-Px[（181.72±10.24）mU/mg,（278.82±3.68）mU/mg,（248.73±9.42）mU/mg]activities were increased while the MDA[（958.76±21.96）nmol/mg prot,（628.16±14.46）nmol/mg prot,（716.32±17.88）nmol/mg prot]level were decreased in IRN group（P<0.05）.（4）Compared with control group,the fluorescence of the PC12 cells in the model group,the mitochondrial membrane potential dissipation and ROS all increased（P<0.05）;Compared with model group,the fluorescence of the PC12 group in the IRN group,the membrane potential and ROS decreased（P<0.05）.（5）Compared with control[（546.34±11.78）pg/m L]group,the concentration of TNF-αin the model[（894.18±11.96）pg/m L]group was increased（P<0.05）;Compared with model group,the concentration of TNF-αin the IRN[（838.24±10.16）pg/m L,（718.26±10.82）pg/m L,（786.44±8.26）pg/m L]group were decreased（P<0.05）.（6）Compared with control group,theexpressionlevelofNF-κB（2.43±0.01）,Bax（2.32±0.01）,Caspase-3（2.35±0.01）,IL-1β（2.72±0.01）and Cyt C（1.21±0.01）were increased while Bcl-2（0.74±0.01）was decreased（P<0.05）;Compared with model group,the expression level of NF-κB[（1.58±0.02）,（2.19±0.02）],Bax[（1.36±0.02）,（1.44±0.02）],Caspase-3[（0.82±0.02）,（0.75±0.02）],IL-1β[（1.65±0.02）,（1.30±0.02）]and Cyt C[（0.78±0.02）,（0.93±0.02）]were decreased while Bcl-2[（1.55±0.02）,（1.45±0.02）]was increased in IRN-20 and IRN-80 group（P<0.05）.Conclusion:IRN can increase the survival rate and proliferation rate induced by Aβ_25-35,decrease the cells apoptosis via stabilize the mitochondrial membrane potential induced by Aβ_25-35.The mechanism may through reduce the level of ROS and MDA,increase the activity of SOD and GSH-PX and increase the antioxidant capacity of cells;IRN can reduce the expression of TNF-αand IL-1βand inhibit the expression of NF-κB and inflammatory signal pathway;Stabilize the mitochondrial membrane potential,enhance the expression of Bcl-2 and inhibit the expression of Bax,and then regulate the expression of Cyt-C and Caspase-3.