Experimental Study Of High Mobility Group Box 1 On Regulating Neuroinflammation And Ferroptosis After Spinal Cord Injury

Objective Spinal cord injury(SCI)has a high disability rate and poor prognosis,and its pathogenesis is very complex.The primary injury can cause multiple continuous cascades,among which neuroinflammation and neuronal ferroptosis are the main secondary damage factors.High-mobility group protein box 1(HMGB1)is a kind of damage-associated molecular pattern molecule(DAMP).Studies have shown that its expression upregulated after SCI.HMGB1-p38/JNK signaling pathway can not only participate in regulating inflammatory response,but also regulate ferroptosis in other diseases.However,it has not been reported in SCI.This study aims to verify the role of the HMGB1-p38/JNK signaling pathway in the inflammatory response after microglia activation and neuronal ferroptosis through in vitro and in vivo experiments.The SCI model rats treated with HMGB1 inhibitor glycyrrhizic acid(GA).Then the changes in neuroinflammation and ferroptosis at the injured site of SCI rats were observed after HMGB1 inhibitor treatment.This study aims to reveal the mechanism of the dual role of HMGB1 in regulating neuroinflammation and ferroptosis,and provides experimental evidence for HMGB1 to be a therapeutic target for SCI.Methods 1.The compression SCI model was established by clipping the T10-level spinal cord of SD rats with aneurysm clips.Twenty-four rats randomly divided into the sham group(n=12)and the SCI group(n=12).The rats in the sham group were only treated with laminectomy without spinal cord clamping,while those in the SCI group were treated with spinal cord clamping.The Basso,Beattie,and Bresnahan Scale(BBB)scores of hindlimb motor function were performed once a day for 3 days after awakening.The spinal cord tissues were collected for HE staining and reverse transcription-quantitative polymerase chain reaction(RT-q PCR)to detect the expression of HMGB1.2.Lipopolysaccharide(LPS)was used to induce the inflammatory response in HAPI microglia(LPS-HAPI).CCK-8 assay used to find the optimal concentration and time of LPS intervention.HAPI microglia treated with LPS(100 ng/m L)for 12 hours,and then the cells were collected for RT-q PCR.The mRNA expression level of HMGB1,ionized calcium binding adapter molecule 1(IBA1)and inflammatory factors,such as tumor necrosis factor-α(TNF-α),interleukin(IL)-1β and-6 were detected.The cell model of the inflammatory response of HAPI microglia was successfully constructed in vitro.Subsequently,HMGB1 inhibitor GA was used to treat LPS-induced HAPI cells,which were divided into the LPS treatment group and the GA+LPS treatment group.The mRNA and protein levels of HMGB1 and inflammatory factors(TNF-a,IL-1β,and IL-6)were detected by RT-q PCR and Western Blot(WB).HMGB1 overexpression plasmid was constructed and transfected into LPS-HAPI cells treated with GA.The cells were divided into the GA+LPS treatment group,GA+LPS+NC(negative control)group and the GA+LPS+HMGB1 group to observe whether the anti-inflammatory effect of GA could be reversed after overexpression of HMGB1.The expression levels of critical proteins in the p38/JNK signaling pathway(p38,p-p38,JNK,and p-JNK)were detected by WB assay.3.In order to investigate the effect of HMGB1 inhibitor GA on neuronal ferroptosis,PC12 cells were induced with ferroptosis inducer erastin(erastin-PC12).CCK-8 experiment used to explore the optimal intervention concentration and time of erastin drugs,and then the ferroptosis inhibitor Fer-1 was used to observe whether the ferroptosis effect of erastin could be partially reversed.WB assay was used to detect the expression of ferroptosis-related protein acyl-Co A synthetase long-chain family member 4(ACSL4)and glutathione peroxidase 4(GPX4).Malonaldehyde(MDA)assay and cell iron content assay were used to detect the changes of cell lipid peroxidation level and cell iron content,respectively.The erastin-induced PC12 cells were treated with HMGB1 inhibitor GA and divided into the erastin treatment group and the GA+erastin treatment group.RT-q PCR and WB were used to detect the changes in the expression of HMGB1,ACSL4 and GPX4.MDA assay and cell iron content assay were also used to detect the changes of cell lipid peroxidation level and cell iron content,respectively.The overexpression plasmid of HMGB1 was constructed and transfected into the GA-treated erastin-PC12 cells.The cells were divided into the GA+erastin group,GA+erastin+NC(negative control)group,and the GA+erastin+HMGB1 group to observe whether overexpression of HMGB1 could reverse the anti-ferroptosis effect of GA.The expression levels of critical proteins in the p38/JNK signaling pathway(p38,p-p38,JNK,and p-JNK)were detected by WB assay.4.The compression SCI model was established by clipping the T10 segment of the spinal cord with aneurysm clips in vivo.The spinal cord was clipped with a 70 g FT220 T aneurysm clip for 10 seconds,and GA(100 mg/kg)was injected intraperitoneally once a day immediately after the operation.After 3 days,the SD rats were sacrificed after deep anesthesia,and the injured spinal cord tissues were collected for subsequent experimental studies.A total of 90 SD rats were randomly divided into three groups: sham group,SCI group,and SCI+GA treatment group,with 30 rats in each group.They were used for the RT-q PCR experiment,WB experiment,MDA experiment,tissue iron content determination experiment,and immunohis-tochemical staining experiment,respectively,with 6 rats in each experiment.HE staining was used to observe the tissue injury and the degree of inflammatory cell infiltration after SCI.RT-q PCR and WB experiments were used to detect the mRNA and protein expression changes of HMGB1 and inflammatory factors(TNF-a,IL-1β,and IL-6).Immunohistochemistry was used to detect the number of HMGB1-positive cells,and immunofluorescence staining was used to detect the expression of microglia marker ionized calcium-binding adapter molecule 1(IBA1).The mRNA and protein expression levels of ACSL4 and GPX4,two important molecules closely related to ferroptosis,were detected by RT-q PCR and WB assay.MDA test and tissue iron content determination were used to detect the changes of MDA content and tissue iron content after GA treatment.Prussian blue staining was used to observe the deposition of iron in the spinal cord tissues.Immunofluorescence staining was used to observe the expression of the neuronal marker protein Neu N in the spinal cord tissues.Results 1.Morphology of SCI and expression level of HMGB1 mRNA in SCI tissues.Under the naked eye,the spinal cord tissue of the SCI group showed noticeable pinch marks and tissue damage seriously,which appeared dark red.The hind limbs of the SCI group rats completely paralyzed,and the BBB score was 0.HE staining showed that the spinal cord tissue in the sham group had complete morphology,uniform distribution of cells,normal cell morphology,and no hemorrhage or edema.The spinal cord tissue in the SCI group showed structural disorder,severe damage,unclear boundary between gray matter and white matter,a large number of inflammatory cells infiltration,and extensive hemorrhage and swelling at the injury site.The results of RT-q PCR showed that the expression level of HMGB1 mRNA in SCI tissues was significantly increased 3 days after SCI,and the difference was statistically significant.2.HMGB1-p38/JNK signaling pathway regulates the inflammatory response of HAPI microglia.The results of the CCK-8 assay showed that 100 ng/m L LPS did not affect on HAPI cell viability,while 500 ng/m L LPS significantly reduced HAPI cell viability after 12 hours.RT-q PCR showed that LPS(100 ng/m L)treatment for 12 hours increased the expression of HMGB1,microglia marker IBA1,and inflammatory factors(TNF-a,IL-1β,and IL-6)in HAPI cells.The results showed that 100 ng/m L LPS could effectively activate HAPI cells and induce an inflammatory response.RT-q PCR and WB analysis showed that the expression of HMGB1 mRNA and protein significantly decreased after HAPI cells treated with 100 m M GA for 24 hours(P<0.01).Compared with the LPS group,GA pretreatment for 12 hours significantly down-regulated the mRNA expression of HMGB1,IBA1,and inflammatory factors(TNF-α,IL-1β,and IL-6)in LPS-HAPI cells.WB results showed that the protein levels of HMGB1 and inflammatory factors(TNF-a,IL-1β,and IL-6)decreased after GA treatment.Therefore,GA can inhibit the inflammatory response of HAPI microglia by reducing the expression of HMGB1.After transfection of HMGB1 overexpression plasmid into HAPI cells co-treated with GA and LPS,RT-q PCR results showed that the expression of HMGB1 increased after transfection of HMGB1 overexpression plasmid(P<0.001).Compared with the control group,HMGB1 transfection significantly up-regulated the mRNA expression of IBA1 and inflammatory factors(TNF-α,IL-1β,IL-6)(P<0.001).WB showed similar changes in protein expression.GA significantly inhibited the phosphorylation of p38 and JNK,but had no significant effect on the total protein expression of p38 and JNK.These results suggest that the anti-inflammatory effect of GA maybe mediate through HMGB1-p38 /JNK signaling pathway.3.HMGB1-p38/JNK signaling pathway regulates ferroptosis in PC12 cells.CCK-8 assay showed that the viability of PC12 cells was decreased by about 50% after 5 μM erastin induction for 24 hours.Ferroptosis inhibitor Fer-1(5 μM)partially reversed the effect of erastin and significantly increased the cell viability of PC12 cells.After erastin treatment for 24 hours,the protein expressions of HMGB1 and ACSL4 were increased,the expression of GPX4 was decreased,and the MDA content and iron content were increased.These effects of erastin could be partially reversed by using ferroptosis inhibitor Fer-1.These results indicated that erastin could induce ferroptosis in PC12 cells.After PC12 cells were treated with 100 m M GA,RT-q PCR and WB results showed that the expression of HMGB1 mRNA and protein levels in the GA intervention group were decreased compared with the control group.RT-q PCR showed that compared with the erastin group,the mRNA expression of HMGB1 and ACSL4 in GA+ erastin group decreased,and the mRNA expression of GPX4 increased.WB showed that the expression of HMGB1 and ACSL4 in the GA+ erastin group was down-regulated,while the expression of GPX4 was significantly increased.In addition,MDA and iron contents in the GA+erastin group were significantly lower than those in the erastin group.These results suggest that GA may inhibit erastin-induced ferroptosis in PC12 cells by down-regulating HMGB1.Subsequently,the recombinant HMGB1 overexpression plasmid was transfected into PC12 cells in the GA+erastin group.After transfection,HMGB1 mRNA expression was significantly increased.RT-q PCR and WB results showed that the expression levels of ACSL4 mRNA and protein in the GA+erastin+HMGB1 group were increased,and those of GPX4 mRNA and protein were decreased.Compared with the GA+erastin group,MDA content and cell iron content were significantly increased,which was reflected in the PC12 cells of the HMGB1 overexpression plasmid group.These results suggest that HMGB1 can reverse the anti-ferroptosis effect of GA,and high expression of HMGB1 promotes the ferroptosis of cells.WB showed that GA inhibited the phosphorylation of p38 and JNK,but had no significant effect on the total expression of p38 and JNK.These results suggest that the anti-ferroptosis effect of GA is mediated through HMGB1-p38 /JNK signaling pathway.4.HMGB1-p38/JNK signaling pathway regulates neuroinflammation and ferroptosis in the injured spinal cord of SCI rats.HE staining showed that the spinal cord tissues were severely damaged on the third day after SCI,with a large number of inflammatory cell infiltration and hemorrhage.In contrast,the inflammatory cell infiltration was reduced in the GA treatment group.RT-q PCR and WB showed that HMGB1 and inflammatory factors(TNF-α,IL-1β,and IL-6)were significantly increased on the third day after SCI,while the expression levels of HMGB1,TNF-a,IL-1β,and IL-6 were decreased considerably after GA treatment.Immunohistochemical analysis confirmed that the expression of HMGB1 decreased after GA treatment.Immunofluorescence staining showed that IBA1 expression increased on the third day after SCI and decreased after GA treatment,which inhibited microglial activation.RT-q PCR and WB results showed that the mRNA and protein levels of ACSL4 were increased,and the mRNA and protein levels of GPX4 were decreased in SCI tissues,which were reversed by GA treatment.The results showed that the contents of MDA and tissue iron in the spinal cord were significantly increased 3 days after SCI,but decreased significantly after GA treatment.In addition,Prussian blue and DAB staining showed increased iron deposition in the injured spinal cord tissue 3 days after SCI.Iron deposition decreased in the SCI tissue after GA treatment.Immunof-luorescence staining showed that the positive expression of the neuronal marker Neu N decreased on the third day after SCI.The positive expression of Neu N increased in the spinal cord of the GA treatment group compared with the SCI group.GA significantly inhibited the phosphorylation of p38 and JNK,but had no significant effect on the total expression of p38 and JNK.In vivo experiments showed that GA could alleviate neuroinflammation and ferroptosis after SCI in rats by inhibiting the activation of the HMGB1-p38/JNK signaling pathway.Conclusions(1)On the third day after SCI,the tissue injury was severe,with local infiltration of a large number of inflammatory cells and increased expression of HMGB1.(2)HMGB1-p38/JNK signaling pathway regulates the inflammatory response of HAPI microglia.(3)HMGB1-p38/JNK signaling pathway regulates ferroptosis in PC12 cells.(4)HMGB1-p38/JNK signaling pathway regulates neuroinflammation and ferroptosis in the injured spinal cord of SCI rats.