延龄草皂苷对脑缺血大鼠梗死灶周围皮层少突胶质细胞GSK-3/β-catenin/CRMP2信号表达的影响

Effects of Total Saponin of Trillium tschonoskii Maxim.on Expression of GSK-3/β-catenin/CRMP2 Signal in Oligodendrocytes in Peri-infarct Cortex of Rats with Cerebral Ischemia

目的观察延龄草皂苷(TSTT)对脑缺血大鼠梗死灶周围皮层少突胶质细胞GSK-3/β-catenin/CRMP2信号表达的影响,探讨其改善脑缺血损伤的作用机制。方法采用线栓法制备局灶性脑缺血大鼠模型,将大鼠随机分为假手术组、模型组、TSTT 65 mg/kg组、TSTT 33 mg/kg组和金纳多组,各给药组灌胃相应药物,连续15 d。HE染色观察脑组织病理变化,LFB染色观察脑组织神经纤维损伤,免疫荧光染色检测梗死灶周围皮层2′,3′-环核苷酸-3′-磷酸二酯酶(CNPase)、蛋白聚糖(NG2)、糖原合成酶激酶-3β(GSK-3β)、β-连环蛋白(β-catenin)表达,实时荧光定量PCR检测梗死灶周围皮层GSK-3、β-catenin、脑衰蛋白反应调节蛋白-2(CRMP2)基因表达。结果与假手术组比较,模型组大鼠患侧脑组织明显坏死,神经细胞大量死亡,胞体皱缩,单位面积神经细胞数量显著减少(P<0.01),神经纤维丢失、排列混乱,髓鞘中有大量空泡,神经纤维相对积分光密度显著减少(P<0.01);与模型组比较,TSTT 65、33 mg/kg组大鼠神经细胞形态有所改善,单位面积神经细胞数量显著增加(P<0.05,P<0.01),神经纤维排列较有序,髓鞘中空泡较少,神经纤维相对积分光密度显著增加(P<0.01)。免疫荧光染色结果显示,与假手术组比较,模型组大鼠梗死灶周围皮层CNPase阳性表达显著降低,NG2阳性表达显著升高(P<0.01),NG2/GSK-3β、NG2/β-catenin、CNPase/GSK-3β、CNPase/β-catenin阳性细胞显著增加(P<0.01);与模型组比较,TSTT 65、33 mg/kg组大鼠梗死灶周围皮层CNPase、NG2表达显著升高,CNPase/GSK-3β、NG2/GSK-3β阳性细胞显著减少,CNPase/β-catenin、NG2/β-catenin阳性细胞显著增加(P<0.05,P<0.01)。实时荧光定量PCR结果显示,与假手术组比较,模型组大鼠梗死灶周围皮层GSK-3αmRNA表达显著升高(P<0.05),β-catenin、CRMP2 mRNA表达显著降低(P<0.05,P<0.01);与模型组比较,TSTT 65 mg/kg组大鼠梗死灶周围皮层GSK-3α、GSK-3βmRNA表达显著降低,β-catenin和CRMP2 mRNA表达显著升高(P<0.05)。结论TSTT可减轻脑缺血大鼠脑组织损伤,保护少突胶质细胞,促进少突胶质前体细胞存活,其机制可能与下调GSK-3表达,上调β-catenin、CRMP2表达有关。

Objective To investigate the effects of total saponin of Trillium tschonoskii Maxim.(TSTT)on the expression of GSK-3/β-catenin/CRMP2 signal in oligodendrocytes in peri-infarct cortex of cerebral ischemia rats;To explore its mechanism of improving cerebral ischemia injury.Methods Rat model of middle cerebral artery occlusion was established with a suture method.The rats were randomly divided into sham-operation group,model group,Ginaton group and TSTT 65 and 33 mg/kg groups.The medicated groups received gavage with corresponding drugs for 15 consecutive days.HE staining was used to observe the histopathologic changes in brain tissue,LFB staining was used to observe the damage of nerve fibers,immunofluorescence staining was conducted to observe the changes of 2',3'-cyclic nucleotide 3'-phosphodiesterase(CNPase),proteoglycan(NG2),glycogen synthase kinase-3β(GSK-3β)andβ-catenin in peri-infarcted cortex,RT-qPCR was used to detect GSK-3,β-catenin and CRMP2 gene expression in peri-infarcted cortex.Results Compared with the sham-operation group,the brain tissue of the affected side in the model group was necrotic,nerve cells died in large numbers,cell bodies shrunk,the number of nerve cells per unit area significantly reduced(P<0.01),nerve fibers were lost and disordered,there were a large number of vacuoles in the myelin sheath,and the relative integrated optical density of nerve fibers significantly reduced(P<0.01).Compared with the model group,the morphology of nerve cells was improved in TSTT 65 and 33 mg/kg groups,the number of nerve cells per unit area significantly increased(P<0.05,P<0.01),the nerve fibers were arranged in order,the number of myelin hollow vacuoles was low,and the relative integrated optical density of nerve fibers significantly increased(P<0.01).Immunofluorescence staining results showed that compared with the shamoperation group,the expression of CNPase significantly decreased and the expression of NG2 significantly increased(P<0.01),the number of NG2/GSK-3β,NG2/β-catenin,CNPase/GSK-3βand CNPase/β-catenin positive cells in periinfarcted cortex significantly increased in model group(P<0.01).Compared with the model group,the expressions of CNPase and NG2 in peri-infarcted cortex significantly increased in TSTT 65 and 33 mg/kg groups,the number of CNPase/GSK-3βand NG2/GSK-3βpositive cells decreased,the number of CNPase/β-catenin and NG2/β-catenin positive cells increased significantly(P<0.05,P<0.01).RT-qPCR results showed that compared with the sham-operation group,the expression of GSK-3αmRNA in peri-infarcted cortex significantly increased in the model group(P<0.05),while the expressions ofβ-catenin and CRMP2 mRNA significantly decreased(P<0.05,P<0.01);compared with the model group,the expression of GSK-3αand GSK-3βmRNA in peri-infarcted cortex significantly decreased in TSTT 65 mg/kg group,and the expression ofβ-catenin and CRMP2 mRNA significantly increased(P<0.05).Conclusion TSTT can reduce brain tissue damage,protect oligodendrocytes,and promote the survival of oligodendrocyte precursor cells in rats with cerebral ischemia.Its mechanism may be related to downregulating the expression of GSK-3 and up-regulating the expressions ofβ-catenin and CRMP2.