中药海马对脊髓损伤后内质网应激反应GRP-78/PERK/ATF-4信号通路的影响

Effect of Hippocampus kelloggi on GRP-78/PERK/ATF-4 signaling pathway of endoplasmic reticulum stress response after spinal cord injury

目的观察中药海马对葡萄糖调节蛋白78(glucose regulated protein 78,GRP-78)/蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)/激活转录因子4(activate transcription factor-4,ATF-4)信号通路的影响,探讨其改善脊髓损伤的相关机制。方法将36只大鼠按随机数字表法分为假手术组、模型组和海马组,每组12只。假手术组仅行椎板切除术,模型组及海马组制备脊髓损伤模型。术后海马组大鼠灌胃海马提取液10 ml/kg,连续干预14 d。采用大鼠脊髓损伤行为学评分(Basso Beattie Bresnahan,BBB)评价大鼠肢体运动功能,采用尼氏染色观察神经元形态,采用Western blot检测脊髓GRP-78、p-PERK和ATF-4蛋白表达,采用RT-qPCR检测GRP-78 mRNA和ATF-4 mRNA水平,采用ELISA法检测Caspase-3、Caspase-12含量,采用TUNEL染色检测细胞凋亡情况。结果与模型组比较,海马组术后第7、9、11、14天,BBB评分升高(P<0.05);海马组大鼠脊髓GRP-78[(0.49±0.06)比(0.74±0.03)]、p-PERK[(0.63±0.04)比(0.81±0.06)]和ATF-4[(0.51±0.06)比(0.69±0.05)]蛋白相对表达降低(P<0.05),GRP-78 mRNA[(0.54±0.05)比(0.63±0.06)]和ATF-4 mRNA[(0.61±0.06)比(0.78±0.04)]相对表达降低(P<0.05),脊髓组织Caspase-3、Caspase-12含量降低(P<0.05),海马组细胞凋亡率降低(P<0.05)。结论中药海马通过抑制GRP-78/PERK/ATF-4信号通路调控脊髓损伤后内质网应激反应,从而促进神经元修复。

Objective To observe the effect of Hippocampus kelloggi on GRP-78/PERK/ATF-4 signal pathway and explore its mechanism on improving spinal cord injury.Methods A total of 36 SD rats were randomly divided into sham operation group,model group and hippocampus group with 12 rats in each group.Only laminectomy was performed in the sham operation group.The spinal cord injury model was prepared in the model group and hippocampus group.Rats in the hippocampus group were given 10 ml/kg Hippocampus kelloggi extract by gavage for 14 days.Basso Beattie Bresnahan(BBB)score was used to evaluate the motor function of the limbs.The neuron morphology was observed by Nissl staining.The expression of GRP-78,p-PERK and ATF-4 proteins were detected by Western blot,the expression of GRP-78 and ATF-4 mRNAs was detected by qPCR,Caspase-3 and Caspase-12 were detected by ELISA,and the apoptosis was detected by TUNEL.Results Compared with the model group,the BBB score of hippocampal group increased on the 7th,9th,11th and 14th day after operation(P<0.05).For hippocampus group,the relative expression of GRP-78(0.49±0.06 vs.0.74±0.03),p-PERK(0.63±0.04 vs.0.81±0.06)and ATF-4(0.51±0.06 vs.0.69±0.05)protein were significantly decreased(P<0.05),GRP-78 mRNA(0.54±0.05 vs.0.63±0.06)and ATF-4 mRNA(0.61±0.06 vs.0.78±0.04)were significantly decreased(P<0.05),the content of Caspase-3 and caspase-12 were significantly decreased(P<0.05),and the apoptosis rate of hippocampal group was significantly decreased(P<0.05).Conclusion Hippocampus kelloggi can regulate the stress response of the endoplasmic reticulum after spinal cord injury by inhibiting GRP-78/PERK/ATF-4 signaling pathway to promote the repair of neurons.