The Protective Effect And Mechanism Of Esculentoside A On Neurons Of Spinal Cord Injury In Rats

Objective:To investigate the impact of esculentoside A（EsA）on the oxidative stress and neuronal apoptosis of rats with spinal cord injury（SCI）,this study aims to elucidate the neuroprotective effects of EsA and explore its underlying mechanisms.Methods:A rat model of spinal cord injury（SCI）was induced using the PHI-SH method.The treatment group received intraperitoneal injection of EsA（10 mg/kg）once daily following SCI,while the SCI group received the same dose and frequency of physiological saline.This study aimed to evaluate the recovery of motor function,spinal cord tissue injury,neuronal cell survival,and changes in oxidative stress indicators in rats after SCI.In addition,in vitro experiments were conducted using an H2O2-induced BV2 cell model and its supernatant coculture system with primary neuronal cells to detect ROS production,levels of oxidative stress indicators,and apoptosis of primary neuronal cells in BV2 cells.Results:（1）The SCI rat model was established using the PHI-SH method.（2）Behavioral assessments revealed that the BBB score in the EsA treatment group was significantly higher than that in the SCI group at 4w,5w,and 6w（P<0.05）.In the 6th week after injury,the EsA treatment group showed a significantly lower walking error rate than the SCI group（P<0.05）.Additionally,footprint analysis showed a higher score in the EsA treatment group at the 6th week after injury（P<0.05）.（3）Histological staining results showed that,after spinal cord injury,the area of the injured cavity was significantly smaller in the EsA treatment group than in the SCI group at-2mm,-1mm,0mm,and 1mm from the injury center.Furthermore,myelination retention in the treatment group was significantly greater than that in the SCI group（P<0.01）.（4）Western blot（WB）results demonstrated that the expression of Caspase3 and BCL2-Associated X（Bax）in the injured spinal cord tissue was significantly decreased while the expression of anti-apoptotic protein B-cell lymphoma-2（Bcl-2）was significantly increased in the EsA treatment group（P<0.01）.Nissl staining showed that compared with the SCI group,the number of anterior horn motor neurons 3mm and 4mm from the injury center in the EsA treatment group was significantly increased（P<0.01）.Immunofluorescence staining revealed that the number of Neu N⁺Caspase3⁺cells in the EsA treatment group was significantly lower than that in the SCI group 1 week after spinal cord injury（P<0.05）.（5）Oxidative stress indicators showed that EsA treatment significantly decreased the concentration of H2O2 in injured spinal cord tissue compared to the SCI group at 12,24,and 3 days after spinal cord injury（P<0.05）.The concentration of MDA in the injured spinal cord tissue was significantly lower in the EsA treatment group than the SCI group at 24hours,3 days,and 7 days after spinal cord injury（P<0.05）.In addition,the activities of antioxidant enzymes GSH-PX and SOD in the injured spinal cord tissue were significantly higher in the EsA treatment group than the SCI group at 24 hours,3 days,and 7 days after spinal cord injury（P<0.05）.（6）EsA treatment significantly increased the number of CD68⁺Nrf2⁺cells in the injured spinal cord tissue compared to the SCI group 1 week after spinal cord injury（P<0.05）.In addition,WB detection showed that the expression of Nrf2,HO1,and NQO1 proteins in the EsA treatment group was significantly higher than that in the SCI group（P<0.05）.（7）EsA significantly inhibited the accumulation of ROS in BV2 cells induced by H₂O₂（P<0.05）and decreased the production of MDA in BV2 cells（P<0.05）.Moreover,EsA significantly increased the activity of SOD（P<0.05）.However,the protective effect of EsA was antagonized by ML385（P<0.05）.（8）EsA treatment effectively increased the expression levels of Nrf2,HO1,and NQO1 proteins,but this protective effect was antagonized by ML385（P<0.05）.（9）Conditioned medium treated with EsA significantly increased the activity of primary neuronal cells,while the conditioned medium treated with ML385 reversed the effect of EsA（P<0.05）,as measured by the Cell Counting Kit 8（CCK8）test.（10）The conditioned medium treated with EsA significantly inhibited the expression of proapoptotic proteins Caspase3 and Bax and promoted the expression of anti-apoptotic protein Bcl-2（P<0.05）,as shown by the WB assay.In addition,the conditioned medium treated with EsA significantly inhibited the apoptosis of primary neuronal cells（P<0.05）.Nonetheless,ML385 could antagonize the protective effect of EsA（P<0.05）.Conclusion:EsA demonstrated the ability to enhance motor function,decrease the extent of spinal cord tissue damage,and promote myelin retention in rats with SCI.Furthermore,EsA effectively suppressed the oxidative stress response and neuronal cell apoptosis during SCI.The neuroprotective effects of EsA are achieved through the activation of the Nrf2/HO1 signaling pathway.