虎杖苷对急性脊髓损伤小鼠的治疗作用及机制探究

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

中文关键词:  虎杖苷  脊髓损伤  神经元  自噬  凋亡

英文关键词:Polydatin  Spinal cord injury  Neurons  Autophagy  Apoptosis

中文摘要:

  【摘要】 目的：探讨虎杖苷（polydatin，PD）对糖氧剥夺（oxygen-glucose deprivation，OGD）诱导的神经元损伤及急性脊髓损伤（spinal cord injury，SCI）的保护作用机制。方法：体外实验以小鼠海马神经元细胞系HT22为研究对象。采用CCK-8法检测PD的细胞毒性：设置0、1、3、10、30、100μM共6个PD浓度组，处理24h后检测细胞活力，确定安全浓度范围；在OGD造模（24h）同时，分别加入不同浓度PD（0、3、10、30μM）干预，CCK-8法检测细胞活力，筛选最佳保护浓度。通过免疫荧光、Western blot（WB）检测自噬标志物LC3和P62蛋白表达；透射电镜观察自噬小体形成；TUNEL染色评估细胞凋亡率。体内实验采用C57BL/6小鼠，建立Allen′s打击法制备急性SCI模型。动物分为假手术组、SCI模型组、PD治疗组（20mg/kg），每组n=10。给药7d、14d取材，HE染色观察脊髓组织7d、14d病理变化；免疫荧光检测损伤14d胶质瘢痕（GFAP+）、神经元存活（MAP2+）、细胞增殖（BrdU+）及自噬水平（LC3Ⅱ）。结果：体外实验结果显示，1~100μM浓度的PD对HT22细胞未表现出明显的细胞毒性作用。OGD造模后选定10μM作为最佳给药浓度，与OGD组相比，10μM PD处理可显著提高OGD后的细胞活力[OGD组：（54.63±3.90）% vs OGD+PD（10μM）组：（84.35±1.38）%，P<0.05]，并有效减缓自噬活化进程，具体表现为LC3Ⅱ/Ⅰ比值下降（OGD组：11.0±0.57 vs OGD+PD组：3.50±0.28，P<0.05）、P62蛋白积累增加（OGD组：0.55±0.04 vs OGD+PD组：0.93±0.06，P<0.05）、自噬溶酶体数量减少，同时细胞凋亡率显著降低[OGD组：（35.33±2.6）% vs OGD+PD组：（19.67±1.76）%，P<0.05]。体内实验中，HE染色结果证实，PD治疗组脊髓组织的病理损伤较SCI模型组明显减轻；免疫荧光检测显示，PD治疗可抑制纤维瘢痕形成（SCI组：2.32±0.19mm2 vs PD组：1.07±0.24mm2，P<0.05），减少神经元损伤（SCI组：1.72±0.28mm vs PD组：0.93±0.12mm，P<0.05），以及促进细胞增殖[SCI组：（16.14±2.24）% vs PD组：（39.09±3.16）%，P<0.05]，并下调LC3Ⅱ蛋白的表达水平（SCI组：62.81±5.25 vs PD组：34.09±3.98，P<0.05）。结论：PD通过抑制自噬和减少凋亡，改善神经损伤，其双重调控机制为SCI治疗提供了潜在新策略。

英文摘要:

  【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.