【摘要】 目的探讨金钗石斛多糖对脑缺血-再灌注大鼠核因子κB(NF-κB)信号通路的调控机制。方法将90只SD大鼠按随机数字表法随机分为6组,分别为假手术组、模型组和金钗石斛多糖低剂量组(DL,50 mg/kg)、中剂量组(DM,100 mg/kg)、高剂量组(DH,200 mg/kg)以及尼莫地平组(10 mg/kg),每组15只,再按随机数字表法每组随机取5只供相应指标检测。采用大脑中动脉阻塞法建立SD大鼠局灶性脑缺血-再灌注模型,观察金钗石斛多糖对大鼠神经功能缺损(Bederson行为学评分)、脑含水量和脑梗死体积的改善作用。采用酶联免疫吸附法检测脑组织促炎因子肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)水平,采用实时荧光定量多聚酶链反应(RT-q PCR)检测小胶质细胞标记物BCL-2相关蛋白A1α(A1)和星形胶质细胞标记物胶质细胞纤维酸性蛋白(GFAP)mRNA转录水平,蛋白免疫印迹法检测NF-κB信号通路磷酸化抑制蛋白(IκBα)和胞核p65蛋白的表达水平。多组间计量资料比较采用单因素方差分析。结果 (1)6组间大鼠神经功能缺损评分、脑含水量、脑梗死体积、脑组织TNF-α和IL-1β水平、A1和GFAP mRNA转录水平以及磷酸化IκBα蛋白和胞核p65蛋白水平差异均有统计学意义(F值分别为22.24、8.699、33.89、19.26、27.53、109.5、15.28、66.86、41.63,均P<0.01)。(2)模型组大鼠神经功能缺损评分、脑含水量、脑梗死体积分别为(2.8±0.3)分、(86.1±3.8)%、(31.0±4.5)%,均明显高于假手术组(均P<0.01);脑组织TNF-α和IL-1β水平、A1和GFAP mRNA转录水平以及磷酸化IκBα蛋白和胞核p65蛋白水平较假手术组均明显增高,差异均有统计学意义(均P<0.01)。(3)DH组前三项分别为(1.5±0.5)分、(72.9±5.4)%、(17.5±4.1)%,与模型组比较(包括脑组织TNF-α和IL-1β水平、A1和GFAP mRNA转录水平以及磷酸化IκBα蛋白和胞核p65蛋白水平),差异均有统计学意义(均P<0.05);与尼莫地平组比较,各指标差异均无统计学意义(均P>0.05);与假手术组比较,除脑含水量、IκBα、胞核p65蛋白水平差异无统计学意义外(P>0.05),其余指标差异均有统计学意义(均P<0.05)。(4)DM组上述指标与模型组比较,仅IL-1β水平和磷酸化IκBα蛋白水平显著减少,其余指标差异无统计学意义(均P>0.05);与假手术组相比,各指标差异均有统计学意义(均P<0.01)。(5)DL组上述指标与模型组相比,差异均无统计学意义(均P>0.05);与假手术组相比,各指标差异均有统计学意义(均P<0.01)。结论高剂量金钗石斛多糖能减轻局灶性脑缺血-再灌注大鼠脑损伤,作用机制与抑制早期NF-κB信号通路激活有关。中、低剂量金钗石斛多糖减轻局灶脑缺血-再灌注炎性脑损伤的作用不明显。更多还原

【Abstract】 Objective To investigate the regulation mechanism of dendrobium nobile polysaccharides( DNP) for nuclear factor kappa B( NF-κB) signal pathway in focal cerebral ischemia/reperfusion rats.Methods According to the digital random method,90 SD rats were divided into 6 groups: sham operation,model,DNP low-dose( DL,50 mg/kg),moderate-dose( DM,100 mg/kg) and high-dose( DH,200 mg/kg),and nimodipine( 10 mg/kg) groups( n = 15 in each group),and according to the random number method,selecting 5 in each group for the corresponding index detection.A focal cerebral ischemia/reperfusion model in SD rats was induced by middle cerebral artery occlusion.The improvement effect of DNP on rat neurological deficit( Bederson behavioral score) and brain water content,and infarct volume were observed.Enzyme-linked immunosorbent assay( ELISA) was used to detect the levels of proinflammatory cytokine tumor necrosis factor-α( TNF-α) and interleukin-β( IL-1β) in brain tissue.Real-time fluorescence quantitative polymerase chain reaction( RT-q PCR) was used to detect microglial cell marker BCL-2-related protein A1α( A1) and astrocyte marker glial fibrillary acidic protein( GFAP) mRNA transcription levels.Western blot was used to detect the NF-κB signaling pathway phosphorylated IκBα protein and nuclear p65 protein expression levels.Single factor variance analysis was used to compare the measurement data among the groups.Results( 1) There were significant differences in the neurological deficit score,brain water content,cerebral infarction volume,brain tissue TNF-α and IL-1β levels,A1 and GFAP mRNA transcription levels and phosphorylated IκBα protein and nuclear p65 protein levels among the 6 groups( F = 22.24,8.699,33.89,19.26,27.53,109.5,15.28,66.86,and 41.63,respectively( all P < 0.01).( 2) The neurological deficit score,cerebral water content,and cerebral infarction volume in the model group were 2.8 ± 0.3,8 6.1 ± 3.8 %,and 3 1.0 ± 4.5 %,respectively.The TNF-α and IL-1 β levels,A1 and GFAP mRNA transcription levels in brain tissue,and phosphorylated IκBα protein and nuclear p65 protein levels were increased significantly compared with those in the sham operation group.There were significant differences( all P < 0.01).( 3) The above indices in the DH group were 1.5 ± 0.5,72.9 ± 5.4%,and 17.5 ± 4.1%,respectively.Compared with the model group( including TNF-α and IL-1β levels,A1 and GFAP mRNA transcription levels and phosphorylation of IκBα protein and nuclear p65 protein levels in brain tissue).There were significant differences( all P < 0.05).Compared with the nimodipine group,there were no significant differences( all P > 0.05).Compared with the sham operation group,except for there were no significant difference in brain water content,phosphorylated IκBα protein and nuclear p65 protein( P >0.05),there were significant differences in other indices( all P < 0.05).( 4) Compared with the model group,only IL-1β and phosphorylated IκBα protein levels were decreased significantly in the DM group,there were no significant differences in other indices( all P > 0.05).Compared with the sham operation group,there were significant differences in other indices( all P < 0.05).( 5) Compared with the model group,there were no significant differences in other indices in the DL group( P > 0.05).Compared with the sham operation group,there were significant differences in all indices( all P < 0.05).Conclusion Highdose DNP may reduce focal cerebral ischemia/reperfusion injury in rats.The mechanism of may be associated with the inhibition of activation of early NF-κB signaling pathway.The effects of low-and moderate-dose DNP on reducing inflammatory brain damage of focal cerebral ischemia/reperfusion may be not obvious.更多还原