Study On Mechanism Of Bnip3-mediated Mitochondrial Autophagy In Mechanically Injured PC12 Neurons

Spinal cord injury(SCI)is the most serious complication of spinal injury and often leads to severe dysfunction below the injury segment.SCI has a high disability rate and mortality rate,which puts a heavy burden on individuals and society.The prevention,treatment and rehabilitation of spinal cord injury has become a major topic in the medical field.Spinal nerve cells are difficult to regenerate and grow slowly,which makes it difficult to recover after nerve tissue damage.Therefore,the pathological and repair mechanisms of spinal cord injury are mainly focused on the prevention and reversible regulation of secondary spinal cord injury.Neuronal apoptosis is an important mechanism of secondary injury to SCI.Therefore,the main strategy is to reduce neuronal damage,inhibit apoptosis,and control the inflammatory response to promote spinal cord repair.In recent years,hot mitochondrial autophagy is also worth exploring.In recent years,BNIP3 has been found not only to participate in the process of apoptosis,but also to participate in the development of autophagy through a variety of mechanisms.Therefore,BNIP3 as a key protein of both,its mechanism in SCI still needs further discussion,aiming at the BNIP3 entry point,exploring the regulation of the balance between autophagy and apoptosis,and reducing the apoptosis of spinal cord neurons.Part I:PC12 induced differentiation to establish mechanical damage in vitro model to observe the expression of autophagy Objective:To explore the expression of autophagy,BNIP3 and membrane potential after mechanical injury of PC12 neurons.Method:(1)Culture and passage of PC12 cells,and differentiation of PC12 was induced by NGF.On the 74 th day,the in vitro neuronal mechanical injury model was established according to the method of Haiping Que;(2)The cytoplasmic proteins were extracted at different time points(0h,1h,4h,8h,24h)after mechanical injury,and the expression of autophagy(LC3,P62)and BNIP3 was analyzed by western blot.(3)Mitochondrial membrane potential levels were measured at different time points after injury using the JC-1 kit.Result:(1)LC3II/I increased at 4h after injury(P<0.05),and autophagy substrate P62 decreased(P<0.05);(2)Using autophagy lysosomal inhibitor BAF(100 nM),LC3II/I increased significantly(P<0.05),but autophagy substrate P62 accumulated(P<0.05);(3)8 hours after injury,the JC-1 green/red fluorescence ratio increased significantly(P<0.05);(4)BNIP3 showed a significant increase after 8 hours of injury,and then continued to increase,reaching a peak at 24 hours(P<0.05).Conclusion: BNIP3 may regulate the flow of autophagy through MPT,competition with BCL-2 binding,and inhibition of Rheb.Overexpression of BNIP3 within 24 hours of mechanical injury of PC12 neurons can enhance autophagic flow,inhibit the occurrence of apoptosis,and increase cell viability.However,there is no significant difference between silent BNIP3 and normal cell lines,and there may be other compensatory mechanisms.Part II: Effect of BNIP3 overexpression and silencing on PC12 neurons in mechanical injuryObjective: To investigate the regulation of BNIP3 overexpression and silencing on autophagy and apoptosis after PC12 neuronal mechanical injury,and the effect on PC12 neurons after injury.Method:(1)Detecting the expression characteristics of BNIP3,a stable strain of lentivirus infection,and whether it meets the requirements;(2)Establish 4 groups(sham group,SCI group,SCI?BNIP3 overexpression group,SCI?BNIP3 sh RNA group).A mechanical injury model was established with the first part;the protein was extracted 24 h after injury to detect the expression of autophagyrelated proteins;(3)mitochondrial membrane potential levels between groups were detected 24 h after injury;(4)Apoptosis of cells was detected by tunel 24 h after injury;(5)Apoptosis and cell viability were measured by flow cytidine-FITC/PI double staining 24 h after injury.Result:(1)The detection of LC3II/I,SCI group,SCI?BNIP3 overexpression group,SCI?BNIP3 sh RNA group were significantly increased(p<0.05,compared with sham group);there was no significant difference between SCI group and SCI?BNIP3 sh RNA group.The statistical significance(p>0.05);SCI?BNIP3 overexpression group was significantly higher than SCI group,SCI?BNIP3 sh RNA group.(p<0.05).These results confirmed that overexpression of BNIP3 enhanced autophagic flow,but BNIP3 silenced had no significant effect on autophagic flow.Considering this may be the existence of other compensation mechanisms.(2)The results of flow detection of apoptosis and TUNEL detection of apoptosis were similar.The apoptosis ratios of SCI group,SCI?BNIP3 overexpression group and SCI?BNIP3 sh RNA group were significantly increased(p<0.05,compared with sham group);there was no statistically significant difference between SCI group and SCI?BNIP3 sh RNA group.(p>0.05);the SCI?BNIP3 overexpression group showed a significant decrease in the apoptotic ratio of the SCI group and the SCI?BNIP3 sh RNA group.(p<0.05).The cell viability is the opposite.(3)The above results showed that within 24 hours of mechanical injury of PC12 neurons,the autophagic flow of BNIP3 overexpressing cells was significantly enhanced(p<0.05),apoptosis was significantly decreased(p<0.05),and cell viability was relative to The SCI group and the BNIP3 silent group were significantly increased(p<0.05).Conclusion: BNIP3 may regulate the flow of autophagy through MPT,competition with BCL-2 binding,and inhibition of Rheb.Overexpression of BNIP3 within 24 hours of mechanical injury of PC12 neurons can enhance autophagic flow,inhibit the occurrence of apoptosis,and increase cell viability.However,there is no significant difference between silent BNIP3 and normal cell lines,and there may be other compensatory mechanisms.