荭草苷调节微管稳定以促进脊髓损伤后轴突再生

Axon regeneration promotion contributed by orientin’s regulation on microtubule stability in spinal cord injury

目的探究荭草苷对脊髓损伤的作用及潜在机制。方法建立氧糖剥夺模型及脊髓损伤大鼠模型,通过CCK-8检测荭草苷对原代神经元是否具有细胞毒性。在氧糖剥夺模型中,采用了实时荧光定量PCR、蛋白印迹、免疫荧光染色,并对脊髓损伤大鼠的脊髓切片进行免疫荧光实验,共同探究荭草苷对轴突再生以及微管稳定的影响。最后,通过BBB量表评分、斜板实验评分验证其促进脊髓损伤大鼠运动功能恢复的作用。结果 CCK-8检测表明,荭草苷在0~50μmol/L浓度之间对原代神经元无细胞毒性作用(P>0.05)。体外实验发现,荭草苷可显著上调Nefh、GAP43等轴突再生关键蛋白的mRNA和(或)蛋白表达,并使轴突长度增加,而且在上调乙酰化微管蛋白表达的同时降低酪氨酸化微管蛋白的表达,增加A/T比值(P<0.05,P<0.01)。脊髓切片的免疫荧光结果与体外实验相符。在脊髓损伤大鼠中,荭草苷亦明显增加了BBB与斜板实验评分(P<0.05,P<0.01)。结论荭草苷可调节微管稳定以激活轴突内源性生长潜力,并改善脊髓损伤大鼠运动功能,为脊髓损伤治疗提供了新策略。

AIM To investigate the function and underlying mechanism of orientin in spinal cord injury.METHODS Oxygen glucose deprivation(OGD)rat models and spinal cord injury rat models were established. CCK-8 assay was used to test whether orientin was cytotoxic to primary neurons. The real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining conducted on OGD rat models, and immunofluorescence experiments performed on spinal cord section of spinal cord injury rat models were to jointly reveal the effects of orientin on axon regeneration and microtubule stability. Finally, the role of orientin in promoting motor function recovery was verified by the BBB scale score and inclined plate test score in spinal cord injury rats.RESULTS The results of a CCK-8 assay revealed that orientin was not cytotoxic to primary neurons at concentrations of 0~50 μmol/L(P>0.05). The in vitro experiments showed that orientin up-regulated the mRNA and/or protein expressions of axon regeneration key proteins such as Nefh and GAP43, and increased the axon length. And while up-regulating the expression of ace-tubulin, it also down-regulated the expression of tyr-tubulin, thereby increasing the A/T ratio(P<0.05,P<0.01).The result of immunofluorescence analysis of spinal cord section were consistent with the outcome of the in vitro experiments. In spinal cord injury rats, orientin increased the scores of the BBB test and the inclined plate test(P<0.05,P<0.01).CONCLUSION Orientin can regulate microtubule stability to activate the potential endogenous growth of axon and improve motor function in spinal cord injury rats, highlighting the spinal cord injury treatment.