Study On The Role And Regulatory Mechanism Of Gal-3 In Inflammation Of Acute Spinal Cord Injury In Rats

Objective:To elucidate the role and regulation mechanism of Galectin-3?Gal-3?in the acute inflammatory response of spinal cord injury?SCI?in rats.Methods:Part I:Expression and role of Gal-3 in rat acute spinal cord injury model?in vivo?;?1?12 male SD rats were randomly divided into control group and SCI group?n=6 per group?.The Allen's method was used to combat spinal cord modeling,and the rats were subjected to blood sampling from the orbital vein 24 hours after surgery.BBB scores were obtained at24h,48h and 72h,respectively,and then the spinal cord tissues were sacrificed for water content determination and hematoxylin-eosin?HE?staining.Enzyme-linked immunosorbent assay?ELISA?was used for detection.Expression levels of serum p65,TNF-?,IL-1?,IL-6,MDA,SOD,CAT and GSH-PX.The expression of Gal-3 mRNA was determined using real-time fluorescent quantitative PCR?RT-qPCR?.?2?Eighteen male Sprague-Dawley rats were randomly divided into control group,SCI group and SCI+GB1107 group?n=6 per group?,and the above experimental procedure was repeated.Part II:Possible mechanism of Gal-3 promoting inflammatory response in acute spinal cord injury in rats?in vivo?;?1?12 male SD rats were randomly divided into control group and SCI group?n=6 per group?.Rats and spinal cord tissues were sacrificed at 24 h for gene chip technology,and the regulatory targets of Gal-3 in the SCI model were screened.?2?Twelve male Sprague-Dawley rats were randomly divided into a negative control group and a Gal-3 intervention group?n=6 per group?.Western blot was used to detect Gal-3,TXNIP and NLRP3.Protein expression.?3?Eighteen male Sprague-Dawley rats were randomly divided into control group,SCI group and SCI+GB1107 group.Western blot was used to detect the expressions of Gal-3,TXNIP and NLRP3 protein.Part III:Gal-3 promotes neuroinflammation after spinal cord injury via ROS/TXNIP/NLRP3signaling pathway?in vitro?;?1?LPS induced PC12 cells to construct an in vitro neuroinflammation model.PC12 cells were collected 48 h later and randomly divided into controls.Western blot was used to detect the expression of Gal-3,thioredoxin interacting protein?TXNIP?and NOD-like receptor family protein 3?NLRP3?in group,LPS group and LPS+si-Gal-3 group.The levels of ROS,MDA,SOD,CAT,GSH-PX and IL-1?in the cells were detected by immunofluorescence staining.?2?To determine the effect of reactive oxygen species?ROS?inhibitor on the regulation of Gal-3 on oxidative stress,which were randomly divided into control group,LPS group,LPS+si-Gal-3 transfection group and LPS+si-Gal-3+H₂O₂ group,LPS+ROS inhibitor and LPS+ROS inhibitor+Gal-3group were used to detect the expression levels of ROS,MDA,SOD,CAT,GSH-PX and IL-1?in each group by ELISA.TXNIP was detected by Western blot.And expression of NLRP3 protein.?3?To clarify the role of TXNIP/NLRP3 in Gal-3-induced neuroinflammation:PC12 cells were randomly divided into 6 groups:control group,LPS group,LPS+si-TXNIP group,LPS+si-TXNIP+Gal-Three groups,LPS+si-NLRP3 group and LPS+si-NLRP3+Gal-3 group were used to detect the expression of IL-?in each group by ELISA.The expressions of TXNIP and NLRP3 protein were detected by Western blot.Results:The BBB scores of the first part of the 1SCI group were significantly lower than those of the control group?P<0.05?.The BBB scores of the SCI+GB1107 group were significantly higher than those of the SCI group?P<0.05?.The tissue water content of the SCI group was obvious.Compared with the control group,the SCI+GB1107 group was significantly higher than the SCI group?P<0.05?.The control group had intact spinal cord tissue,normal cell morphology,clear nucleus and cytoplasm staining,and tissue necrosis and cell degeneration in the SCI group,edema,structural disorder,with varying degrees of bleeding and neutrophil infiltration as the main performance,SCI+GB1107group of spinal cord injury significantly relieved,tissue necrosis,cell degeneration,edema,hemorrhage and other conditions have improved;4 and Compared with the control group,the levels of serum p65,TNF-?,IL-1?,IL-6 and MDA in the SCI group were significantly increased,and the activities of SOD,CAT and GSH-PX were significantly decreased.Compared with the SCI group,the SCI+GB1107 group was compared.The levels of serum p65,TNF-?,IL-1?,IL-6 and MDA in rats were significantly decreased,and the activities of SOD,CAT and GSH-PX were significantly increased?P<0.05?.Compared with the control group,the spinal cord of SCI group Gal-3 mRNA expression levels in tissues increased significantly;and SCI group Ratio,SCI+in rat spinal cord tissue GB1107Gal-3 mRNA expression was significantly decreased?P<0.05?.The second part:1Compared with the control group,the 8 most important genes were up-regulated in the SCI group,7 genes were down-regulated;8 kinds of GO analysis biological processes involved.PPI analysis of differentially expressed genes totaled 23 nodes,a total of seven pairing relationships,the signaling pathway is mainly concentrated in NLRP3 and TXNIP protein.Compared with the control group,the Gal-3,TXNIP and NLPR3 proteins in the Gal-3 intervention group were significantly higher than the negative control group;compared with the SCI group,the expression of Gal-3,TXNIP and NLPR3 protein was significantly decreased in the SCI+GB1107 group?P<0.05?.Part III:1 Compared with LPS group,the expression levels of IL-1?,MDA and ROS in LPS+si-Gal-3 transfection group were significantly decreased,and SOD,CAT and GSH-PX were significantly increased?P<0.05?.The expression level of LPS+ROS inhibitor+Gal-3 group was significantly higher than that of LPS+ROS inhibitor group?P<0.05?;overexpression of Gal-3 induced TXNIP/NLRP3 protein expression and increased IL-1?level.Conclusion:In vivo SCI model,Gal-3 expression is up-regulated,and inhibition of Gal-3 expression can attenuate neuroinflammatory response after spinal cord injury.In vitro induction of PC12 model,inhibition of Gal-3 expression can attenuate neuroinflammation and ROS production after injury;ROS can regulate the effect of Gal-3 on oxidative stress.In conclusion,Gal-3 promotes neuroinflammatory responses following SCI by activating the ROS/TXNIP/NLRP3 signaling pathway;Gal-3 may be a potential therapeutic strategy for SCI.