Heme Oxygenase-1 Promotes Neuron Survival Through Down-Regulation Of MLK3/MKK7/JNK3 Signaling After Spinal Cord Injury

Objectives1.To isolate,culture and identify primary spinal neurons in vitro;2.To construct adeno-associated virus vector-mediated HO-1 overexpression re-combinant virus?AAV-HO-1?;3.To investigate the effect of AAV-HO-1 on MLK3/MKK7/JNK3 signaling pathway after oxidative stress injury;4.To investigate the effect of AAV-HO-1 on MLK3/MKK7/JNK3 signaling pathway after spinal cord injury?SCI?.Methods1.Trypsin enzymatic digestion and mechanical method were used to isolate pri-mary spinal neurons.Spinal neurons were identified by?-tubulin-III staining and NeuN immunofluorescence staining;2.The overexpresses HO-1 recombinant adeno-associated virus vector was con-structed;3.H₂O₂ was used to establish the oxidative stress injury model.The expressions of HO-1,MLK3,p-MLK3,MKK7,p-MKK7,JNK3,and p-JNK3 proteins and MLK3/MKK7/JNK3 signaling pathways were detected by western blot and Co-immunoprecipitation?Co-IP?after oxidative stress injury model was estab-lished in primary spinal neurons.Then according to different treatment factors,they were randomly divided into the C group,V group,A-C group and A-HO-1 group.Western blot and Co-IP were used to detect the expression of HO-1,MLK3,p-MLK3,MKK7,p-MKK7,JNK3,and p-JNK3 proteins and MLK3/MKK7/JNK3 signaling pathways in different groups after oxidative stress injury.Flow cytometry was used to detect apoptosis;4.Weight compression was used to establish SCI model.The expressions of HO-1,MLK3,p-MLK3,MKK7,p-MKK7,JNK3,and p-JNK3 proteins and MLK3/MKK7/JNK3 signaling pathways after SCI were detected by western blot and Co-IP.Then according to different treatment factors,they were ran-domly divided into the Sham group,V group,A-C group and A-HO-1 group.The V group,A-C group and A-HO-1 group were treated with saline,AAV-EGFP and AAV-HO-1 in the local spinal cord tissue at 7 days before the establishment of SCI model.Fluorescence microscope was used to detect the expression of EGFP in the spinal cord tissue.The motor function of hindlimb was observed by Basso Beattie Bresnahan?BBB?scores before SCI and 1d,3d,7d,14d and 21d after SCI.The expression of HO-1,MLK3,p-MLK3,MKK7,p-MKK7,JNK3,and p-JNK3 proteins and MLK3/MKK7/JNK3 signaling pathways were detected by western blot and Co-IP in different groups;The apoptosis of the spinal cord was detected by TdT-mediated UTP nick end la-beling?TUNEL?.Results1.Primary spinal neurons were successfully isolated and cultured.NeuN staining was positive,and specific marker?tubulin-III was expressed,consistent with the characteristics of spinal cord neurons;2.HO-1 overexpressing recombinant adeno-associated virus vector was con-structed successfully;3.Spinal cord neurons oxidative stress injury model was established successfully.Compared with A-C group,the expression of HO-1 increased significantly,the expression of p-MLK3?p-MKK7?p-JNK3 and the proportion of apoptotic cells decreased significantly in A-HO-1 group?P<0.05?;4.The model of spinal cord injury was established successfully by weight com-pression.Compared with V group and A-C group,the expression of HO-1 in-creased significantly,the expression of p-JNK3?p-MKK7?p-MLK3 and the proportion of apoptotic cells decreased significantly in A-HO-1 group?P<0.05?.Compared with V group and A-C group,the BBB score of A-HO-1group at 3d,7d,14d and 21d after SCI increased significantly.?P<0.05?.Conclusions1.Primary spinal neurons of the high purity and stable biological characteristics were cultured by the trypsin enzymatic digestion combined with the mechani-cal method;2.HO-1 protects primary spinal cord neurons against oxidative stress by down-regulation of MLK3/MKK7/JNK3 pathway;3.HO-1 promotes neuron survival through down-regulation of MLK3/MKK7/JNK3 pathway after SCI.