| Objective Isofraxidin（IF）intervenes in rat spinal cord injury（SCI）model and BV2 cell inflammatory injury model to explore the neuroprotective effect of isofraxidin on spinal cord injury.Methods 1.8 animals were randomly selected as the sham-operated group（Sham group,N.S）,and the remaining animals were used for model replication.The spinal cord was clamped using ophthalmic forceps to prepare an acute SCI rat model,in which only laminectomy without spinal cord clamping was performed in the Sham group.32 SCI rats were randomly divided into the model group（SCI group N.S）,IF low-dose group（L group,IF 10 mg/kg）,IF medium-dose group（M group,IF 20 mg/kg）,and IF highdose group（H group,IF 40 mg/kg）,8 animals in each group.All animals were administered by isovolumetric gavage,once a day,for 14 days.During the IF intervention,BBB scores were performed on days 1,3,5,7,and 14 to evaluate the recovery of motor function in both hind limbs of the rats.After 14 days,spinal cord were taken for the following indexes: HE staining to observe pathological histological changes,Nissler staining to observe the survival status of neurons,TUNEL staining to observe the apoptosis of tissue cells,The levels of superoxide dismutase（SOD）,catalase（CAT）and malondialdehyde（MDA）were measured by kits,the levels of interleukin-1β（IL-1β）,interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）and interleukin-10（IL-10）were measured by ELISA,the levels of The protein expression levels of TLR4,My D88,IκBα,p-IκBα and NF-κB p65 were detected by Western blot.2.CCK-8 assay was used to detect the cytotoxicity of different concentrations of IF on BV2 cells.A BV2 cell inflammation model was constructed using lipopolysaccharide（LPS）.IF was pretreated and cell viability changes in BV2 inflammation cell model were observed by CCK-8 method,the levels of IL-1β,IL-6,TNF-α and IL-10 in cell culture supernatant were detected by ELISA.Results 1.（1）BBB scores were statistically different between SCI group and Sham group at all time periods（P<0.001）;the hind limb motor ability of rats in each IF dose group recovered at 3,5,7 and 14 days after surgery,and the BBB scores increased to different degrees in each dose group compared with SCI group（P<0.05,P<0.01,or P<0.001）;（2）Spinal cord pathology histomorphological observations revealed that after IF intervention,HE staining results showed that the structural morphology of damaged spinal cord tissues was improved,and the number of cavities was reduced in groups M and H.Nissier staining results showed that the number of Nissier vesicles in spinal cord tissues was increased;TUNEL staining results showed that the number of apoptotic cells was reduced;（3）After IF intervention,MDA levels in spinal cord tissues were reduced（P<0.01 or P<0.001）and the activities of SOD and CAT increased（P<0.01 or P<0.001）;（4）IF intervention decreased the levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α（P<0.01 or P<0.001）and increased the levels of antiinflammatory factor IL-10（P<0.05 or P<0.01）in spinal cord tissues;（5）Western blot results showed that compared with SCI group,the expression of TLR4,My D88,p-IκBαand NF-κB p65 was down-regulated in spinal cord tissues of M and H groups（P<0.05,P<0.01,or P<0.001）;the expression of IκBα was up-regulated in spinal cord tissues of H group（P<0.01）,but the phosphorylation level of p-IκBα/IκBα was down-regulated（P<0.05）.2.（1）IF had no significant effect on the cell viability of normal BV2 cells in the concentration range of 14 μg/m L and below,while 16 μg/m L showed cytotoxicity;（2）0.4 μg/m L LPS was an appropriate concentration for establishing the inflammation model of BV2 cells;（3）IF concentrations at 4,6,8,and 10 μg/m L had increased cell viability in the inflammation model of BV2 cells,but were not statistically significant;（4）ELISA results showed that the levels of anti-inflammatory factor IL-10 in the BV2 cell inflammation model were increased（P<0.05 or P<0.01）and the levels of proinflammatory factors IL-1β,IL-6 and TNF-α were decreased（P< 0.05 or P<0.01）after IF intervention.Conclusions 1.IF can effectively improve the tissue structure of SCI rat spinal cord,increase neuronal survival,reduce apoptosis,decrease oxidative stress injury,and promote the recovery of motor function in the hind limbs of SCI rats.2.IF showed antiinflammatory effect in both SCI model rats and BV2 cell inflammation model,and its mechanism may be related to the inhibition of TLR4/My D88/NF-κB pathway. |
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