The Molecular Mechanism Of HIPK2-mediated Protective Effects In Spinal Cord Injury

Objective:HIPK2 is a tumor suppressor.It also has been implicated in several functions such as apoptosis and inflammation that are linked to spinal cord injury（SCI）.However,whether HIPK2 ameliorates the neurological pain of SCI remains unclear.Here,we investigated the effects of HIPK2 on neurological function,oxidative stress,levels of inflammatory cytokines and expression of Bcl-2/Bax in an SCI model.Methods:Western blot,H&E staining,Real time PCR,ELISA,TUNEL and immunofluorescence were used to study the molecular mechanism of HIPK2 in the development of spinal cord injury in rats.Results:Firstly,we evaluated the therapeutic effects of HIPK2 on neurological pain in the SCI rat using the Basso,Beattie and Bresnahan scores and H&E staining.Overexpression of HIPK2 significantly elevated the levels of brain-derived neurotrophic factor（BDNF）and glial cell line-derived neurotrophic factor（GDNF）,and it reduced the mRNA expression of Nogo-A and RhoA in SCI rats.Furthermore,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL）assays showed that overexpression of HIPK2 significantly reduced the number of apoptotic cells.Overexpression of HIPK2 also decreased expression of Bax and Caspase-3 and elevated expression of Bcl-2 in the SCI model,indicating that HIPK2 exhibited its protective activity by inhibiting SCI-induced apoptosis.Then,we measured the serum concentrations of malondialdehyde（MDA）,superoxide dismutase（SOD）,catalase（CAT）,and glutathione peroxidase（GSH-PX）.We also determined the mRNA and protein levels of nuclear factor-κB p65 unit,tumor necrosis factor-α（TNF-α）,and interleukin（IL）?1β.HIPK2 overexpression reduced oxidative stress and the levels of inflammatory cytokines compared with SCI control animals.Additionally,acetylation of HIPK2 was reduced in SCI rats.Overexpression of HIPK2 could enhance autophagy by elevating the expression of Beclin-1 and LC3-II while autophagy is regarded as a beneficial regulator to improve spinal cord injury.Together,overexpression of HIPK2 improved contusive SCI induced pain by modulating oxidative stress,Bcl?2 and Bax signaling,and inflammation,and also regulating autophagy.The experimental results showed that reactive oxygen species（ROS）were the most abundant in H₂O₂-induced PC12 cells in the H₂O₂-induced group and H₂O₂+3-MA group.The average fluorescence intensity of ROS in these two groups was significantly different from that in the normal control group（P<0.01）.In the H₂O₂+Ad-HIPk2 and H₂O₂+Ad-HIPk2+SN50 pretreatment group,compared with the H₂O₂ injury group,the ROS content was significantly reduced（P<0.01）.In other words,Ad-HIPK2 can effectively reduce the production of ROS,but 3-MA incubation can increase the production of ROS.In order to study the effect of HIPK2 on inflammatory response in PC12 cells,we used ELISA method to detect the expression of inflammatory factors after oxidative stress injury in cells and supernatant.After H₂O₂ injury,the secretion of IL-1βand TNF-αin the supernatant of PC12 cell culture was far more than uninjured group.the and the secretion of IL-1βand TNF-αin the Ad-HIPK2 group or Ad-HIPK2+SN50 group was significantly decreased in the comparison with the H₂O_2-induced groups.The results of western blot showed that compared with the normal control group,the expression of inflammatory signals NF-κB and NLRP3 in PC12 cells was significantly increased.Overexpression of HIPK2 can down-regulate the expression of NF-κB and NLRP3 in cells,and 3-MA upregulates the inflammatory signaling pathway of NF-κB,indicating that Ad-HIPK2 can promote autophagy and reduce the inflammatory response in spinal cord injured tissues.And immunofluorescence（IF）and western blot results show that compared with normal control group,autophagy-related protein LC3-II and Beclin 1expression increased significantly in H₂O₂-induced group,showing that the oxidative damage of cell autophagy activity.Compared with H₂O₂-induced group,LC3-II and Beclin 1 expression in PC12 cells were increased in Ad-HIPK2 and Ad-HIPK2+SN50group suggesting that Ad-HIPK2 can promote cell autophagy.According to previous reports,the acetylation and hematoxylation status of HIPK2are closely related to its function.Our study showed that in spinal cord injury tissues of rats,the acetylation level of HIPK2 increased while the level of hematoxylation decreased.Furthermore,inhibition of the acetylation level of HIPK2 helped to reduce the inflammatory response in spinal cord injury tissues.On the contrary,increase of the acetylation level of HIPK2 could promote the release of inflammatory factors.Conclusion:HIPK2 might be a promising target to improve the spinal cord injury in clinic.Upregulation of HIPK2 could be helpful to pretect spinal cord from injury.