The therapeutic effect and mechanism exploration of polydatin on acute spinal cord injury in mice

English Keywords:Polydatin  Spinal cord injury  Neurons  Autophagy  Apoptosis

English Abstract:

  【Abstract】 Objectives: To investigate the mechanism underlying the protective effects of polydatin(PD) against oxygen-glucose deprivation(OGD)-induced neuronal injury and acute spinal cord injury(SCI). Methods: The mouse hippocampal neuronal cell line HT22 was used in in vitro experiments. The cytotoxicity of PD was assessed using the CCK-8 assay: cells were treated with 0, 1, 3, 10, 30 and 100μM PD for 24h to determine the safe concentration range. After OGD modeling(24h), cells were treated with different concentrations of PD(0, 3, 10, and 30μM), and cell viability was measured via CCK-8 to identify the optimal protective concentration. Autophagy markers(LC3 and P62) were detected by immunofluorescence and Western blot(WB); Autophagosome formation was observed using transmission electron microscopy; And the apoptosis rate was evaluated by TUNEL staining. For in vivo studies, an acute SCI model was established in C57BL/6 mice using Allen′s impact method. Animals were divided into sham, SCI model, and PD treatment(20mg/kg) groups, with n=10 per group. Tissues were collected on 7d and 14d post-injury. Spinal cord pathology was examined by HE staining on 7d and 14d. Immunofluorescence was performed on 14d to evaluate glial scar formation(GFAP+), neuronal survival (MAP2+), cell proliferation (BrdU+), and autophagy level (LC3Ⅱ). Results: In vitro results showed that 1-100μM PD did not exhibit significant cytotoxicity toward HT22 cells. The optimal concentration of PD was determined to be 10μM after OGD induction. Compared with the OGD group, 10μM PD significantly enhanced cell viability after OGD[OGD group: (54.63±3.90)% vs OGD+PD(10μM) group: (84.35±1.38)%, P<0.05] and effectively attenuated autophagy activation, as evidenced by a decreased LC3Ⅱ/Ⅰ ratio(OGD group: 11.0±0.57 vs OGD+PD group: 3.50±0.28, P<0.05), increased P62 accumulation(OGD group: 0.55±0.04 vs OGD+PD group: 0.93±0.06, P<0.05), reduced number of autolysosomes, and significantly lower apoptosis rate[OGD group: (35.33±2.6)% vs OGD+PD group: (19.67±1.76)%, P<0.05]. In vivo, HE staining confirmed that pathological damage in spinal cord tissue was markedly alleviated in the PD-treated group compared with the SCI model group. Immunofluorescence results indicated that PD treatment inhibited fibrous scar formation(SCI group: 2.32±0.19mm2 vs PD group: 1.07±0.24mm2, P<0.05), reduced neuronal damage(SCI group: 1.72±0.28mm vs PD group: 0.93±0.12mm, P<0.05) and promoted cell proliferation[SCI group: (16.14±2.24)% vs PD group: (39.09±3.16)%, P<0.05], and downregulated LC3Ⅱ expression(SCI group: 62.81±5.25 vs PD group: 34.09±3.98, P<0.05). Conclusions: PD ameliorates neural damage by concurrently suppressing autophagy and apoptosis, providing a dual-pathway therapeutic strategy for SCI.