黄芪甲苷对小鼠神经母细胞瘤细胞神经突起回缩的影响及机制研究

The effect and mechanism of astragalosideⅣon lysophosphatidic acid-induced neurite retraction of N1E-115 cells

目的观察黄芪甲苷对溶血磷脂酸(LPA)诱导的小鼠神经母细胞瘤细胞(N1E-115)神经突起回缩的影响,探讨其作用机制。方法将N1E-115细胞按随机数字表法分为空白组、模型组及0、40、80μg/ml黄芪甲苷组。0、40、80μg/ml黄芪甲苷组分别以20、40、80μg/ml黄芪甲苷干预24 h,空白组及模型组不做药物干预,每组以无血清培养12 h,模型组及黄芪甲苷各浓度组以40μmol/L的LPA干预10 min。于倒置显微镜观察并拍照,采用Image J软件计数N1E-115细胞神经突起数量;采用免疫荧光染色法检测细胞RAS同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶2(ROCK2)、p-ROCK2、磷酸化肌球蛋白轻链2(p-MLC2)蛋白表达;荧光实时定量PCR法检测RhoA、ROCK2 mRNA水平;Western blot法检测RhoA、ROCK2、p-MLC2、肌球蛋白轻链2(MLC2)蛋白表达。结果与20μg/ml黄芪甲苷组比较,40、80μg/ml黄芪甲苷组神经突起回缩抑制率升高(P<0.05);与模型组比较,20、40、80μg/ml黄芪甲苷组RhoA、p-ROCK2、p-MLC2蛋白平均荧光强度及40μg/ml黄芪甲苷组ROCK2蛋白平均荧光强度降低(P<0.05或P<0.01);20、40、80μg/ml黄芪甲苷组RhoA mRNA[(0.89±0.09)、(0.41±0.01)、(0.09±0.03)比(1.50±0.01)]、ROCK2 mRNA[(0.89±0.09),(0.14±0.01),(0.20±0.01)比(1.62±0.17)]水平降低(P<0.05或P<0.01);20、40、80μg/ml黄芪甲苷组ROCK2蛋白[(0.75±0.06,0.57±0.02,0.66±0.01)比(1.08±0.02)]、p-MLC2蛋白[(1.72±0.03)、(1.40±0.04)、(1.29±0.03)比(2.19±0.11)]、MLC2蛋白[(1.13±0.02)、(0.68±0.03)、(0.75±0.03)比(1.60±0.03)]及40、80μg/ml黄芪甲苷组RhoA蛋白[(0.35±0.01)、(0.40±0.03)比(0.57±0.08)]表达降低(P<0.05或P<0.01)。结论黄芪甲苷可阻止LPA诱导的神经突起回缩,促进受损神经再生,其机制可能与下调RhoA-ROCK2通路RhoA、ROCK2、p-ROCK2、p-MLC2、MLC2蛋白表达,抑制神经生长锥崩溃有关。

Objective To observe the effect of astragalosideⅣon lysophosphatidic acid(LPA)-induced neurite retraction of N1E-115 cells and its potential mechanism.Methods N1E-115 cells were divided into blank group,model group,the low,medium and high dose groups of astragalosideⅣ.The blank group and model group was not intervened by astragaloside;while the low,medium and high dose groups were treated with 20,40 and 80μg/ml astragalosideⅣfor 24 h.Each group was cultured with serum-free medium for 12 h.The model group and astragalosideⅣgroups were intervened by 40μmol/L LPA for 10 min.Each group was observed and photographed with the inverted microscope,and the number of neurites in N1E-115 cells was counted by Image J software.The fluorescence expression of recombinant ras homolog gene family member A(RhoA),rho associated coiledcoil protein kinase 2(ROCK2),phospho-rho associated coiledcoil protein kinase 2(p-ROCK2)and phospho-myosin light chain 2(p-MLC2)proteins was detected by immunohistochemistry.Real-time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expression levels of RhoA and ROCK2;the protein expression levels of RhoA,ROCK2,p-MLC2 and myosin light chain 2(MLC2)were detected by Western blotting.Results Compared with 20μg/ml astragalosideⅣgroup,the inhibition rate of neurite retraction in 40 and 80μg/ml astragalosideⅣgroups increased(P<0.05).Compared with model group,the average fluorescence intensity of RhoA,p-ROCK2,p-MLC2 in 20,40,80μg/ml astragalosideⅣgroups and the ROCK2 average fluorescence intensity in 40μg/ml astragalosideⅣgroup were decreased(P<0.05,P<0.01);the expression of RhoA mRNA(0.89±0.09,0.41±0.01,0.09±0.03 vs.1.50±0.01)and ROCK2 mRNA(0.89±0.09,0.14±0.01,0.20±0.01 vs.1.62±0.17)decreased in 20,40,80μg/ml astragalosideⅣgroups(P<0.05,P<0.01);the ROCK2 protein(0.75±0.06,0.57±0.02,0.66±0.01 vs.1.08±0.02),p-MLC2 protein(1.72±0.03,1.40±0.04,1.29±0.03 vs.2.19±0.11),MLC2 protein(1.13±0.02,0.68±0.03,0.75±0.03 vs.1.60±0.03)in 20,40,80μg/ml astragalosideⅣgroups and the RhoA protein(0.35±0.01,0.40±0.03 vs.0.57±0.08)in 20,40μg/ml astragalosideⅣgroups were decreased(P<0.05,P<0.01).Conclusion AstragalosideⅣcan prevent LPA-induced neurite retraction and promote damaged nerve regeneration.The mechanism may down-regulae the protein expression levels of RhoA,ROCK2,p-ROCK2,p-MLC2 and MLC2 in RhoA-ROCK2 signaling pathway,and inhibite nerve growth cone collapse.