Effect Of RNAi-Silencing CAPN1 Gene On Neurotoxicity And Apoptosis Induced By A? 25-35 In Primary Neurons

Purpose and Background:Alzheimer's disease(AD)is the most common chronic neurodegenerative disease.Calpains are over-activated in AD and aggravate disease progression.Therefore,inhibition of calpain is of great importance for the treatment of AD,but due to a large variety of calpains which participated or involved in various physiological activities.There is currently no specific calpain inhibitors.Therefore,in this study,RNAi was used to silence the CAPN1 gene.The effect of silence the CAPN1 gene in primary cortical neuronal neurotoxicity and apoptosis induced by A?25-35 AD-like cell model was observed.Method:Experimental Groups: 1)Primary fetal rat cortical neurons + PBS;2)Primary fetal rat cortical neurons + PBS + 10 ?M A?25-35;3)primary fetal rat cortical neurons + CAPN1 shRNA lentivirus + 10 ?M A?25-35;4)primary fetal rat cortical neurons + CAPN1 shRNA lentivirus + 10 ?M A?25-35+ 2ug/ml p25;after lentiv -irus added and cultured 48 hours,a variety of drugs was added and then cultured 72 h.Cell viability was detected by CCK-8 assay.Annexin V-PI was used to detect apoptosis.Real-time PCR was used to detect the expression of CAPN1 mRNA after CAPN1 RNAi transfection.?-calpain,CDK5,GSK-3?,p-tau/tau protein expression were detected by western blot.Result:(1)The results of CCK8 assay showed that compared with NC control group,the viability of NC + A? group was significantly decreased,and the viability of cells transfected with CAPN1 shRNA was significantly improved(p <0.05).Compared with CAPN1 shRNA + A? group,CAPN1 shRNA + A? + p25 group had no significant difference in cell viability(p> 0.05);(2)The results of Annexin V-PI apoptosis test showed that compared with NC group,the rate of apoptosis of NC + A? was significantly increased(p <0.01);After transfection of CAPN1 shRNA,compared with the model group,the apoptosis rate of CAPN1 shRNA+A? group was significantly decreased(p<0.05).In the CAPN1 shRNA+A?+p25 group,apoptosis was increased versus that in the CAPN1 shRNA+A? group,but there was no statistically insignificant(p>0.05).(3)The results of RT-PCR showed that CAPN1 mRNA expression was significantly inhibited in CAPN1 shRNA + A? group compared with NC + A? group;compared to CAPN1 shRNA+A? group,mRNA expression level in shCAPN1 + A? + P25 group was increased but there was no statistically insignificant(P> 0.05);(4)The protein expression was detected by western blot.The expressions of ?-calpain,CDK5,p-tau/tau protein,and GSK-3? protein in NC + A? group were significantly increased(p <0.01 or <0.05)compared with NC group.Compared with the model group,the expression of ?-calpain,GSK-3?,p-tau/tau protein and CDK5 protein were significantly decreased(p <0.01 or p <0.05).After p25(CAPN1 shRNA+A?+P25 group)added,CDK5,GSK-3?,p-tau / tau protein levels were increased(P<0.05),?-calpain protein expression was not significantly changed(p> 0.05).Conclusion:RNAi silencing CAPN1 gene can down-regulate ?-calpain and significantly improve the neurotoxicity induced by A?25-35,which mechanisms may be through down-regulating kinase protein of CDK5,GSK-3?,p-tau protein expression level.