【摘要】 目的：探讨芒柄花素对大鼠慢性脊髓损伤（spinal cord injury,SCI）后神经功能恢复的影响及作用机制。方法：选取24只SPF级8～9周龄SD雄性大鼠，体质量为（327.29±15.19） g，随机分为假手术组（Sham组）、模型组（Vehicle组）和芒柄花素组，每组8只。Vehicle组和芒柄花素组采用水凝胶压迫法建立大鼠慢性SCI模型。芒柄花素组造模后第1天经口灌胃芒柄花素溶液(以30 mg·kg^-1标准)，持续给药28 d，每天1次。各组分别于造模后1、7、14、21、28 d行大鼠Basso-Beattie-Bresnahan（BBB）评分、前肢抓力和转棒检测以评估大鼠肢体运动功能。造模28 d后，心脏灌注取材检测脊髓组织内质网应激及神经元凋亡，采用苏木素-伊红染色观察SCI情况，尼氏染色观察神经元存活数量，采用蛋白质免疫印迹法（Western blot,WB）检测Bcl-2相关X蛋白（Bcl-2-associated X protein,Bax）、B淋巴细胞瘤-2基因（B-cell lymphoma-2,Bcl-2）蛋白及凋亡相关蛋白，使用免疫荧光染色观察内质网应激通路相关蛋白表达。结果：芒柄花素组术后21 d的BBB评分及第3、7、21天抓力测试优于Vehicle组，差异均有统计学意义（P<0.05）。HE和Nissl染色结果显示，芒柄花素组神经细胞存活数量优于Vehicle组，差异有统计学意义（P<0.05）。WB结果显示，芒柄花素组较Vehicle组Bcl-2表达上调，Bax、C/EBP同源蛋白（C/EBP homologous protein,CHOP）、磷酸化蛋白激酶R样内质网激酶（phosphorylated protein kinase R-like endoplasmic reticulum kinase,p-PERK）、磷酸化真核翻译起始因子2α（phosphorylated eukaryotic initiation factor 2α,p-eIF2α）蛋白表达下调，差异均有统计学意义（P<0.05）。免疫荧光结果显示，芒柄花素组较Vehicle组神经元p-PERK、激活转录因子6（activating transcription factor 6,ATF6）蛋白表达下降，差异均有统计学意义（P<0.05）。结论：芒柄花素可减轻大鼠慢性SCI后的病理变化，改善行为学评分，促进脊髓神经功能恢复。其具体机制可能与抑制内质网应激信号通路介导的坏死性凋亡有关。更多还原

【Abstract】 Objective To explore effect and mechanism of formononetin on recovery of neurological function after chronic spinal cord injury（SCI） in rats. Methods Twenty four SPF male SD rats,aged from 8 to 9 weeks old,weighted（327.29±15.19） g,were selected and randomly divided into sham operation group（Sham group）,model group（Vehicle group）,and formononetin group,with 8 rats in each group. Vehicle group and formononetin group were established chronic SCI models in rats by hydrogel compression. On the first day after modeling in formononetin group,formononetin officinalis solution was continuous administered orally(with a standard of 30 mg·kg^-1) for 28 days,once a day. Basso-Beattie-Bresnahan（BBB） score,forelimb grip strength and rotating bar test of rats were performed in each group at 1,7,14,21 and 28 days after modeling respectively to evaluate the limb motor function. Twenty eight days after modeling,cardiac perfusion samples were collected to detect endoplasmic reticulum stress and neuronal apoptosis in spinal cord tissue. Hematoxylin eosin staining was used to observe SCI,Nissl staining was used to observe number of surviving neurons,and Western blot（WB） was used to detect Bcl-2-associated X protein（Bax）,B-cell lymphoma-2（Bcl-2） proteins and apoptosis related proteins. The expression of endoplasmic reticulum stress pathway related proteins was observed by immunofluorescence staining. Results Formononetin group were better than Vehicle group in BBB score on the 21 st day after operation and grip test on the 3 rd,7 th and 21 st days,there were statistically differences（P<0.05）. The results of HE and Nissl staining showed the number of surviving nerve cells in formononetin group were better than those in Vehicle group,and the difference were statistically significant（P<0.05）. WB results showed the expression of Bcl-2 was upregulated,and protein expressions of Bax,C/EBP homologous protein（CHOP）,phosphorylated protein kinase R-like endoplasmic reticulum kinase（p-PERK）,and phosphorylated eukaryotic initiation factor 2α（p-eIF2α） in formononetin group were down regulated compared with Vehicle group,the differences were statistically significant（P<0.05）.Immunofluorescence showed the protein expressions of p-PERK and activating transcription factor 6（ATF6） in neurons in formononetin group decreased compared with Vehicle group,and the differences were statistically significant（P<0.05）. Conclusion Formononetin could alleviate pathological changes after chronic SCI in rats,improve behavioral scores,and promote the recovery of spinal cord nerve function. The specific mechanism may be related to the inhibition of necrotic apoptosis mediated by endoplasmic reticulum stress signaling pathway.更多还原