| Spinal Cord Injury(SCI)is an urgent problem in the medical field.With the development of the world economy and modernization,transportation industry,etc.,the incidence of various accidents leading to spinal cord injury has also increased,and it has imposed a heavy burden on the patients’ families and society.The current research area for the treatment of spinal cord injury focuses on the effective intervention of pathophysiological processes of secondary injury.Recent studies have revealed that Heme Oxygenase-1(HO-1)plays an important role in secondary spinal cord injury.Heme Oxygenase-1 is widely present in body tissues and cells,and its expression is also induced by hypoxia and edema under tissue injury or stress,but the changes of Heme Oxygenase-1expression during secondary injury of spinal cord injury are not verified by a large number of studies.Endoplasmic Reticulum Stress(ERS)is a cellular change in response to a stimulus: when endoplasmic reticulum stress occurs,the cellular organism can promote endoplasmic reticulum recovery by regulating metabolic responses to minimize the stress of blocked protein processing transport in the endoplasmic reticulum;however,if the endoplasmic reticulum stress response is too strong,cytoprotective mechanisms may not be able to counteract the stress,however,if the endoplasmic reticulum stress response is too strong,cytoprotective mechanisms may not be able to counteract the stress injury,and the further development of negative pathophysiological processes such as apoptosis may cause further aggravation of the secondary injury.For spinal cord injury,clarifying the process of endoplasmic reticulum stress changes during secondary injury and finding a balance from it is also one of the entry points to explore the therapeutic measures for spinal cord injury.Apoptosis is a programmed cell death,which is activated by specific genes mediated by the body to maintain the stability of the internal environment under stress.It has been found that apoptosis can be triggered by the activation of the pro-apoptotic transcription factor CHOP and the activation of ER-related caspase-12 when the balance of the body’s internal environment is disrupted,and this may occur in association with excessive endoplasmic reticulum stress.Heme oxygenase 1 is normally expressed on the endoplasmic reticulum in the cytoplasm under physiological conditions and plays a physiological role.Under the action of certain external environmental stimuli and other factors,its carboxy terminus may be dislodged,which leads to its repositioning,regulates related gene expression,and achieves reduced cellular damage from environmental stimulus stress.After nuclear translocation,heme oxygenase 1 can promote its secretion in cells by activating vascular endothelial growth factor transcriptional activity,which can also inhibit endoplasmic reticulum stress-induced endothelial cell apoptosis and promote vascular repair.And these effects may be related to the regulatory role of the blood-spinal cord barrier.In conclusion,the correlation between heme oxygenase 1 and endoplasmic reticulum stress key protein after spinal cord injury,changes in expression with time points,its protective effect of spinal cord injury treatment with the regulation of endoplasmic reticulum stress and induction of apoptosis,and its effect on the regulation of the blood-spinal cord barrier,and whether there is a deeper mechanism of interaction between them,still lack corresponding studies.Therefore,based on the above-mentioned questions,this experiment was conducted in three parts to verify: 1.the changes of HO-1 expression after spinal cord injury in rats and its relationship with the changes of endoplasmic reticulum stress after spinal cord injury;2.the use of SD rats as a model of spinal cord injury and the use of adenovirus-mediated HO-1 intervention therapy to investigate and confirm the therapeutic protective effect of HO-1 in the rat spinal cord injury model,its relationship with endoplasmic reticulum stress and the mechanism of endoplasmic reticulum stress;3.to validate the effect of nucleohemoglobin oxidase-1 on the permeability of the blood-spinal cord barrier in vitro after hypoxia induction by using an in vitro model of blood-spinal cord barrier hypoxia and its mechanism.Objective:Through a series of molecular biology experiments and behavioral and morphological experiments at animal and cellular levels,we investigated and analyzed the changes of molecular mechanism after spinal cord injury,the role and mechanism of action of HO-1 on spinal cord injury,the role of HO-1 and endoplasmic reticulum stress after spinal cord injury and its mechanism of action on the blood-spinal cord barrier;to reveal the role and mechanism of action of HO-1 in spinal cord injury,to provide a theoretical basis and experimental basis for the study of spinal cord injury,and to provide new methods and new ideas for the treatment of spinal cord injury.Methods:Part Ⅰ: Allen’s method rat spinal cord injury percussion model was produced and divided into(SHAM group,4h group,12 h group,1d group,3d group,7d group)by Western Blot(WB)to detect heme oxidase-1 and endoplasmic reticulum stress key protein(GRP78,C-Caspase-12,CHOP)expression at different time points after spinal cord injury,and the correlation between heme oxidase-1 and endoplasmic reticulum stress key proteins expression changes with time points after spinal cord injury was analyzed by Pearson correlation coefficient.Part Ⅱ: Rat spinal cord injury models were made by Allen’s method and divided into SHAM group,SCI group,Ad-GFP group,and HO-1 group,the Ad-GFP group and HO-1group were treated with corresponding therapeutic interventions based on the spinal cord injury model,respectively;motor recovery,structural damage,and neuronal damage after spinal cord injury were assessed by BBB motor function score,Nissl staining,and HE staining;The expression of endoplasmic reticulum stress-related proteins(GRP78,C-Caspase-12,CHOP)in each group was detected by Western Blot;changes in the expression of apoptosis-related proteins(Bax,Bcl-2)were detected by Western Blot and immunofluorescence staining,and apoptosis in each treatment group was further verified by Tunel staining.The changes of HO-1 expression in cytosol and nucleus were detected by Western Blot and immunofluorescence staining,respectively.Part Ⅲ: HCMEC/D3 cell line and HA-sc cell line were used to construct in vitro blood-spinal cord barrier by Transwell,and the injury model was constructed by transfection and chemical hypoxia induction,divided into HO-1CΔ23 group,no-load group and blank control group.Western blot was used to detect the changes of HO-1protein expression in nucleus and cell plasma in HO-1CΔ23 group and blank control group.The changes of HO-1 protein expression in the nucleus and plasma of HO-1CΔ23and blank control groups were detected by Western blot and immunofluorescence staining,and the changes of blood-spinal cord barrier permeability in each group were detected by HRP.Results:Part Ⅰ: The expression of HO-1 stress changed with time after spinal cord injury,and was significantly higher than that of the sham-operated group 4 hours after spinal cord injury,and reached its highest expression at about 3 days,followed by a gradual decrease.The expression of endoplasmic reticulum stress-related proteins(GRP78,C-Caspase-12,CHOP)also changed with time after spinal cord injury,and was significantly higher than that of the sham-operated group 4 hours after spinal cord injury,reaching the highest expression at about 3 days,followed by a gradual decrease.The changes in HO-1 and endoplasmic reticulum stress-related protein expression over time after spinal cord injury showed a consistent trend,and it was speculated that HO-1 expression changes after spinal cord injury were related to endoplasmic reticulum stress changes,and HO-1 might be involved in the regulation of endoplasmic reticulum stress response after spinal cord injury.Part Ⅱ: The results of BBB motor function score of rats showed that the motor function was severely restricted in all model groups after spinal cord injury,but the motor function score was significantly improved in the HO-1 group compared with other groups over time.Nissl staining results suggested that the number of neurons in the anterior horn of the spinal cord was significantly decreased in rats after spinal cord injury compared with the sham-operated group.HE staining results showed that after spinal cord injury,spinal cord tissue was significantly damaged with spinal cord cavity formation,and the ratio of cavity area to spinal cord cross-sectional area was not significantly different between the injury group and the Ad-GFP-empty group,however,the area in the HO-1 group was significantly reduced.Western Blot showed that the expression of endoplasmic reticulum-related proteins(GRP78,C-Caspase-12,CHOP)was significantly increased after spinal cord injury compared to the sham-operated group;the Ad-GFP-empty group showed no significant change compared to the spinal cord injury group,while the HO-1 group Western Blot assay revealed that each intervention group had different regulatory effects on the expression of apoptosis-related proteins,among which the expression of apoptotic protein Bax was significantly increased in the spinal cord injury group and the Ad-GFP no-load group,while the expression of apoptosis-inhibiting protein Bcl-2 was significantly decreased in these two groups,suggesting that after spinal cord injury,the level of apoptosis increased.In contrast,Bax expression was significantly decreased and Bcl-2 expression was significantly increased in the HO-1 group compared with the injury group,and the overall results suggested that HO-1 intervention inhibited apoptosis after spinal cord injury.Tunel apoptosis fluorescence staining results were consistent with the trend of Western Blot results.Western Blot assay results revealed that the expression of HO-1 after spinal cord injury The upregulation of HO-1 expression after spinal cord injury could also lead to an increase in intracytoplasmic HO-1 expression,suggesting that HO-1 intervention could induce HO-1 nuclear translocation,also known as "nuclear HO-1".Accordingly,immunofluorescence staining was performed to examine the changes in HO-1 protein expression in the nucleus and cytoplasm in the null and HO-1 groups,and the results were consistent with the corresponding Western Blot test results.Part Ⅲ: Western Blot detection of nuclear HO-1 and plasma HO-1 changes in the two groups of HCMEC/D3 cells.The HO-1CΔ23 group showed a significant increase in nuclear HO-1 expression compared to the blank control group,while the difference in plasma HO-1 expression between the two groups was not statistically significant.Western Blot detected the changes of CAV-1 and CAV-2 protein in each group compared with the no-load group and blank control,and the expression of CAV-1 and CAV-2protein in the HO-1CΔ23 group was significantly decreased,while there was no significant difference in the expression of CAV-1 and CAV-2 protein in the no-load group compared with the blank control.The results of immunofluorescence detection of CAV-1and CAV-2 proteins were consistent with the Western Blot results.Western detection of tight junction protein ZO-1 and Occludin protein expression in each group,compared with the no-load group and blank control,the expression of ZO-1 and Occludin protein in the HO-1CΔ23 group was significantly increased,and the expression of ZO-1 and Occludin protein in the no-load group compared with the blank control group was significantly increased.Occludin protein expression was not significantly different between the no-load group and the blank control group;the results of immunofluorescence detection of ZO-1,Occludin protein were consistent with the Western Blot results.Conclusions:Part Ⅰ: HO-1 expression changed over time after spinal cord injury,and the trend of endoplasmic reticulum stress-related protein expression showed consistency;HO-1 and endoplasmic reticulum stress-related protein expression changes were correlated after spinal cord injury,and HO-1 may be involved in the regulation of endoplasmic reticulum stress response after spinal cord injury.Part Ⅱ: HO-1 ameliorated spinal cord tissue injury and promoted the recovery of motor function after spinal cord injury;HO-1 could inhibit the expression of endoplasmic reticulum stress-related proteins and inhibit apoptosis after spinal cord injury,confirming that the protective effect on spinal cord after injury might be partly achieved by inhibiting the endoplasmic reticulum stress-induced apoptotic pathway;after spinal cord injury,HO-1 underwent nuclear translocation,HO-1 nuclear translocation occurred,and the expression of HO-1 in the nucleus was significantly higher than that before the injury,and the specific role needs to be further investigated.Part Ⅲ: The therapeutic protective effect of up-regulated heme oxygenase 1expression after spinal cord injury is related to its nuclear translocation,i.e.,"nuclear HO-1";"nuclear HO-1" can reduce the permeability of the blood-spinal cord barrier and maintain the stability of the spinal cord microenvironment.Nucleus HO-1 can reduce the permeability of the blood-spinal cord barrier,maintain the stability of the spinal cord microenvironment,and achieve the protective effect on spinal cord injury. |
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