毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响

Effects of calycosin on neuronal autophagy and apoptosis in rats with spinal cord injury

目的探讨毛蕊异黄酮(CAL)调控PI3K/Akt信号通路对脊髓损伤(SCI)大鼠神经元自噬及凋亡的影响及其作用机制。方法32只成年SD大鼠随机分为假手术组(Sham组),模型组(SCI组)和CAL低、高剂量组,每组8只。采用改良Allen’s法建立大鼠中度SCI模型,术后CAL低、高剂量组立即分别予以20、40 mg/kg CAL腹腔注射,1次/d,连续7 d;Sham组和SCI组给予等量生理盐水。术后1、3和7 d,采用行为学评分(BBB)评估大鼠运动功能的恢复情况;术后7 d,尼氏染色检测大鼠脊髓前角运动神经元存活数量,Western blot检测p62、Beclin-1、微管相关蛋白1轻链3(LC3B)、磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)和活化型胱天蛋白酶-3(Cleaved-Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)表达,免疫荧光染色检测大鼠脊髓前角神经元LC3B的表达。结果与Sham组相比,SCI组大鼠BBB评分、神经元存活数量、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平降低,p62、Beclin-1、LC3BⅡ、LC3BⅡ/Ⅰ、Cleaved-Caspase-3、Bax水平升高(P<0.05);与SCI组相比,CAL低、高剂量组BBB评分、神经元存活数量、LC3BⅡ、LC3BⅡ/Ⅰ、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平升高,p62、Cleaved-Caspase-3、Bax水平降低(P<0.05)。结论CAL可通过激活PI3K/Akt信号通路促进SCI大鼠神经元自噬,抑制其凋亡,进而发挥神经保护作用。

Objective To explore the effect of calycosin(CAL)on neuronal autophagy and apoptosis in rats with spinal cord injury(SCI)by regulating PI3K/Akt signaling pathway and its mechanism.Methods A total of 32 male or female adult SD rats were randomly divided into the sham group,the SCI group,the CAL low(20 mg/kg)dose group and the CAL high(40 mg/kg)dose group with 8 rats in each group.The rat model of moderate SCI was established by modified Allen’s method.After successful modeling,rats were injected intraperitoneally immediately with different dosage of CAL or equal amount of saline once a day for 7 consecutive days.Basso,Beattie and Bresnahan(BBB)scores were used to evaluate the recovery of motor function of rats at 1,3 and 7 d after surgery.At 7 d after surgery,Nissl staining was used to detect the surviving number of motor neurons in anterior horn of spinal cord.Western blot assay was used to assess expression levels of p62,Beclin-1,microtubule-associated protein 1 light chain 3(LC3B),phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway,Cleaved-Caspase-3,B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)proteins.Immunofluorescence staining was used to measure the expression of LC3B in anterior neurons of spinal cord.Results Compared with the sham group,BBB scores,the surviving number of motor neurons and levels of Bcl-2,Bcl-2/Bax,p-PI3K,p-PI3K/PI3K,p-Akt and p-Akt/Akt were significantly decreased(P<0.05),and levels of p62,Beclin-1,LC3BⅡ,LC3BⅡ/Ⅰ,Cleaved-Caspase-3 and Bax were significantly increased in the SCI group(P<0.05).Compared with the SCI group,BBB scores,the survival of anterior horn motor neurons and levels of LC3BⅡ,LC3BⅡ/Ⅰ,Bcl-2,Bcl-2/Bax,p-PI3K,p-PI3K/PI3K,p-Akt and p-Akt/Akt were increased in the CAL low dose group and CAL high dose group,and levels of p62,Cleaved-Caspase-3 and Baxcould were significantly decreased(P<0.05).Conclusion CAL could promote autophagy and inhibit apoptosis of neurons through activating PI3K/Akt signaling pathway,thereby conferring a protective role following SCI in rats.