ICS Ⅱ Mitigates Amyloid Beta 25-35-induced PC12 Cells Apoptosis Through Activation Of The BDNF/TrkB Signaling Pathway

Objective:The present study was designed to investigate the effects of icariside Ⅱ（ICS Ⅱ）on amyloid beta 25-35(Aβ_25-35)-induced PC12 cells apoptosis,and explore its potential mechanisms in depth.Methods:PC12 cells were divided into control,25μM ICS Ⅱ alone,20μM Aβ_25-35alone,20μM Aβ_25-35+6.25μM ICS Ⅱ,20μM Aβ_25-35+12.5μM ICS Ⅱ,20μM Aβ_25-35+25μM ICS Ⅱ,20μM Aβ_25-35+20μM sildenafil,10μM ANA-12 alone,25μM ICS Ⅱ+10μM ANA-12,20μM Aβ_25-35+10μM ANA-12,20μM Aβ_25-35+10μM ANA-12+25μM ICS Ⅱ,20μM Aβ_25-35+10μM ANA-12+20μM sildenafil,ANA-12 was added to the particular cell cultures 1 h before ICS Ⅱ treatment.After 48 h,MTT assay was used to evaluate the effect of ICS Ⅱ or ANA-12 on cell viability induced by Aβ_25-35.The leakage rate of lactate dehydrogenase（LDH）and the content of phosphodiesterase5（PDE5）were detected by enzyme linked immunosorbent assay（ELISA）.The levels of intracellular and mitochondrial reactive oxygen species（ROS）and apoptosis of cells were detected by flow cytometry,Mito-SOX dye and TUENL cell apoptosis detection kit.The protein expression of Bax,Bcl-2,BDNF,TrkB and the levels of pro-caspase-3,active-caspase-3 and phosphorylation of CREB were detected by Western blot,respectively.Results:Compared with control group,Aβ_25-35decreased the PC12 cell viability（P<0.01）and increased leakage rate of LDH（P<0.05）as well as the apoptosis rate（P<0.01）;Moreover,Aβ_25-355-35 not only reduced the level of ROS both in intracellular and mitochondria（P<0.01）,but also lowered the content of PDE5（P<0.01）.Additionally,the expression of Bax（P<0.05）and the level of active-caspase-3（P<0.01）were significantly increased,whereas,the protein expressions of Bcl-2（P<0.05）,BDNF（P<0.01）,TrkB（P<0.05）and the levels of pro-caspase-3（P<0.05）and CREB phosphorylation（P<0.05）were decresaed following Aβ_25-355-35 exposure.However,ICS Ⅱ significantly increased cell viability（P<0.01）,decreased LDH leakage rate（P<0.05）and apoptosis（P<0.05）,Notably,ICS Ⅱ reduced intracellular and mitochondrial ROS generation（P<0.01）as well as intracellular PDE5 content（P<0.05）;In addition,ICS Ⅱ significantly down-regulated the expression of apoptosis-related protein Bax（P<0.05）and the level of active-caspase-3（P<0.05）,and significantly repressed the decline of pro-caspase-3 level（P<0.05）,and significantly up-regulated the expression of Bcl-2（P<0.01）,BDNF（P<0.05）,TrkB（P<0.05）protein and the CREB phosphorylation（P<0.05）.Interestingly,ANA-12,a TrkB inhibitor,almost abolished the beneficial effects of ICS Ⅱ on Aβ_25-35-induced neuronal cell injury（P<0.05）.Conclusion:Under the experimental conditions,ICS Ⅱ mitigates Aβ_25-35-induced neuronal cell injury by upregulating the BDNF/TrkB/CREB signaling pathway.