Effects And Mechanism Of HMGB1 On Degradation Of Spinal Glial Scar By Regulating The Expression Of MMP-9 In Subacute Phase After Spinal Cord Injury In Rats

Objective:To investigate the expression of matrix metalloproteinase-9(MMP-9)and glial scar related proteins after spinal cord injury(SCI)in rats and the effect of MMP-9 on the degradation of glial scar by inhibiting the expression of high mobility group box-1(HMGB1)or MMP-9.To investigate the molecular mechanism of HMGB1 regulating the expression of MMP-9in subacute phase on the degradation of glial scar in the spinal cord after SCI in rats.Methods:Part Ⅰ:To investigate the expression of HMGB1,MMP-9 and glial scar related proteins in the spinal cord and the injury of spinal axons at different time points after SCI.SD rats at 6-8 weeks were divided into 5 groups:Sham group,SCI Id,3d,7d and 14d group.The SCI model was established by the modified Allen’s method.Western blot(WB)was used to detect the expression of HMGB1,MMP-9 and GFAP in the spinal cord after SCI.The injury of spinal cord and axon at 7 d after SCI were observed by HE staining and modified Bielschowsky staining.Part Ⅱ:To investigate the effects of regulating HMGB1 and MMP-9 on the expression of MMP-9 after SCI in rats,as well as the degradation of glial scar and axonal growth.6-8 weeks SD rats were divided into 4 groups:Sham group,SCI group,SCI+EP(ethyl pyruvate,HMGB1 inhibitor)group and SCI+SB-3CT(MMP-9 inhibitor)group.First,the expression of HMGB1,MMP-9 and GFAP in the spinal cord of rats was detected by WB.Secondly,immunohistochemistry(IHC)was used to detect the expression of GFAP and CSPG in the spinal cord after inhibiting HMGB1 or MMP-9.Modified Bielschowsky staining was used to observe the axonal growth of spinal cord after SCI.Part Ⅲ:To investigate the effect of HMGB1 on the expression of MMP-9 and its molecular mechanism in regulating glial scar degradation and axonal growth after SCI in rats.SD rats(6-8 weeks)were divided into 6 groups:Sham group,SCI group,SCI+EP group,SCI+LPS(lipopolysaccharide,Toll-like receptor 4 agonist)group,SCI+PDTC(nuclear factor-κb inhibitor)group,SCI+SB-3CT group.WB was used to detect the expression of HMGB1,TLR4,NF-κB,MMP-9 and GFAP in the spinal cord tissue of rats in each group.The results were repeated at least five times for all experiments.Experimental results are presented as mean ± standard deviation.If it was consistent with normal distribution and homogeneity of variance,one-way analysis of variance was used to compare the means of multiple samples,and LSD test and Tukey test were used for pairwise comparison between groups.GraphPad Prism 8.0.3 software was used to analyze the data.P<0.05 was considered statistically significant.Results:Part Ⅰ:WB results showed that compared with the the Sham group,the expression of HMGB1,MMP-9 and GFAP in the spinal cord after SCI was significantly higher(P<0.05);the expression of MMP-9 was close to the peak at 3d after SCI,and began to decrease at 7d after SCI,while the expression of GFAP continued to increase.The results of HE staining showed that the structure of spinal cord tissue in the Sham group was complete and uniformly red stained,without abnormality.After SCI,the structural integrity of the spinal cord injury site was destroyed,the cavity appeared,and the morphology of the spinal cord was disordered.Bielschowsky staining showed that the axons in the spinal cord in Sham group were continuous and the structure was normal.After SCI,the axons of the spinal cord were interrupted,the continuity was destroyed,and cavitation was observed.This part showed that the expression of MMP-9 in the spinal cord began to decrease 7 days after SCI,while glial scar continued to form,which should be used as a typical time point for subsequent experiments.Part Ⅱ:The second part of the experiment:To investigate the effects of HMGB1 or MMP-9 on glial scar degradation and axonal outgrowth after SCI in rats.WB results showed that compared with the SCI group,the expression of HMGB1 and GFAP in the SCI+EP group was significantly decreased,and the expression of MMP-9 was increased(P<0.05);In SCI+SB-3CT group,the expression of MMP-9 was significantly decreased,and the expression of GFAP was increased(P<0.05).IHC results showed that the expression of GFAP and CSPG in SCI+EP group was significantly lower than that in SCI group,while the expression of GFAP and CSPG in SCI+SB-3CT group was significantly higher than that in SCI group(P<0.05).Bielschowsky staining showed that there were more axons in the SCI+EP group and less axons in the SCI+SB-3CT group than in the SCI group.These results indicate that inhibiting HMGB1 can up-regulate the expression of MMP-9 and down-regulate the expression of GFAP and CSPG in the spinal cord of rats,and contribute to the degradation of glial scar and axonal growth after SCI in rats.Part Ⅲ:To observe whether HMGB1 plays a role in regulating the expression of MMP-9 and the degradation of glial scar after SCI in rats via TLR4/NF-κB signaling pathway.The results of WB showed that compared with the SCI group,the expression of MMP-9 in the SCI+EP group and SCI+PDTC group increased(P<0.05),but decreased in SCI+LPS group(P<0.05);compared with the SCI group,the expression of GFAP in SCI+EP group and SCI+PDTC group was lower(P<0.05),while the GFAP expression in SCI+SB-3CT group and SCI+LPS group was higher(P<0.05).This suggests that HMGB1 may regulate MMP-9 expression through TLR4/NF-κB signaling pathway to degrade glial scar and promote axonal growth.Conclusions:Inhibition of HMGB1 can promote the expression of MMP-9 in the subacute phase after SCI,degrade glial scar,and promote the growth of spinal axons.TLR4/NF-κB signaling pathway may play a role in this process.