Neuroprotective Effect Of Bergenin On Spinal Cord Injury In Rats

Objective:The therapeutic effect of bergenin in rats with SCI was observed to confirm the neuroprotective effect of bergenin on SCI.To explore the protective mechanism of bergenin on spinal cord injury and provide theoretical and experimental basis for its future application in the treatment of neurodegenerative diseases such as spinal cord injury.Methods:（1）Modeling and grouping:220g female SD rats were selected,and the spinal cord injury（SCI）model was prepared by PSI-IH method,divided into sham surgery group,control group and treatment group.Bergenin treatment,the dose of100mg/kg,twice a day.The control group used the same solution（1%DMSO in normal saline）as in the treated group.（2）Behavioral testing:BBB score（Basso Beattie Bresnahan Score）was used to score rats with 1 to 6w injuries.Grid walk and Footprint analysis were used to assess motor recovery at 6w after the SCI.（3）Histopathological analysis:the damaged void area was detected by HE staining.The axonal myelination area of the spinal cord section was detected by LBF staining.（4）The number of motor neurons in the anterior horn of the spinal cord was determined by Nissl staining and the apoptosis of the local neuronal cells damaged 7d after SCI was determined by immunofluorescence double staining with SCI.SH-SY5Y and PC12 cells were used to observe the protective effect of bergenin on the damaged cells.（5）Transcriptomic sequencing（RNA-Seq）:extract total RNA of rat spinal cord7d after injury for transcriptome sequencing,bioinformatics analysis of differentially expressed genes and differentially enriched pathways.（6）Oxidative stress detection:to detect hydrogen peroxide（H₂O₂）,malondialdehyde（MDA）,glutathione peroxidase（GSH-PX）,and superoxide dismutase（SOD）content in the spinal cord tissue at different time points.（7）Inflammatory factor detection:RT-q PCR and Western Blot detected the pro-inflammatory factor IL-1β,IL-6,and TNF-αexpression in rat spinal cord tissue at 7d after SCI.（8）Detection of macrophage subsets:the number of damaged local macrophage subsets at 7d after SCI was detected by immunofluorescence double-dye method.（9）Signaling pathway detection:PPARγspecific antagonist GW9662 was injected intraperitoneal before bergenin administration,and PPARγ,RXRαprotein expression and NF-κB p65phosphorylation were measured in spinal cord tissues 7d after SCI.Results:（1）Rat SCI model was successfully prepared.（2）BBB score showed that treatment group score was higher at 3,4,5,6w than control group（P<0.05）.Grid walking showed lower walking error rate at 6w（P<0.05）.Footprint analysis showed that treatment group scored higher at 6w than control group（P<0.05）.（3）HE staining found that the damage cavity area was less than the control group 1mm and 2mm from the treatment group（P<0.05）.LBF staining showed that the myelination retention was 1mm higher than the control group（P<0.05）.（4）Nissl staining indicated that the number of neurons in the treated group was higher 3mm away and4mm away from the injured center（P<0.05）.Immunofluorescence double staining showed that the number of neuronal cells were locally apoptotic 7d after SCI.In vitro experiments showed that parcabolin could increase the viability of H₂O₂ after damaging SH-SY5Y and PC12 cells（P<0.05）.（5）The RNA seq is shown,the treatment group,when compared to the control group,there were 644 genes upregulated,there were 461 differential genes downregulated.upregulation of GO enrichment includes glutamate receptor activity,NAD,neurotransmitter receptor activity,GTP enzyme activity,ion transport,downregulation functions include immune response,cytokine activity,cytokine receptor binding,chemokine activity,and chemokine receptor binding.upregulation of KEGG enrichment includes neuroactive ligand-receptor interactions,glutamatergic synapses,synaptic vesicle cycles,calcium signaling pathways,downregulated pathways include TNF signaling,IL-17 signaling,Type-C lectin receptor signaling pathway,cytokine-cytokine receptor interaction,ECM-receptor interaction,etc.（6）Oxidative stress testing showed that H₂O₂ content in spinal cord tissue was significantly reduced at 6h,12h,1d and 3d after injury when compared with control groups,the MDA content was significantly reduced at 1d,3d,and 7d after injury.SOD viability was significantly increased in the3d and 7d post-injury treatment groups,and GSH-PX viability was significantly enhanced in the 12h,1d,3d and 7d treatment groups（P<0.05）.（7）RT-q PCR and Western Blot results showed that the expression of proinflammatory factors was significantly reduced in the spinal cord tissue compared with the control group（P<0.05）.（8）Immunofluorescence staining showed that the number of M1 cells was significantly decreased and the number of M2 cells was significantly increased when compared with the control group（P<0.05）.（9）Western Blot results found that bergenin increased PPARγand RXRαexpression and reduced NF-κB p65phosphorylation,while the PPARγspecific antagonist GW9662 decreased PPARγand RXRαexpression and increased phosphorylation of NF-κB p65（P<0.05）.Conclusion:（1）Bergenin promotes the histopathological repair and motor function recovery of the rat spinal cord after SCI,promotes the survival of neuronal cells,and has a neuroprotective effect on spinal cord injury.（2）Bergenin may have antioxidant,anti-inflammatory effects and immune regulation effects in SCI.Bergenin can reduce the generation of oxidation products in spinal cord tissue,increase antioxidant enzyme activity,reduce the expression of pro-inflammatory factors,and promote M1 to M2polarization.（3）Bergenin may play a protective role in SCI through the agonistic PPARγsignaling pathway.