烙灸疗法对大鼠脊髓损伤后神经修复及Wnt信号通路的影响

Effect of cauterizing moxibustion on nerve repair after spinal cord injury and Wnt signaling pathway in rats

目的 基于Wnt信号通路探讨烙灸疗法对大鼠脊髓损伤后神经修复的影响。方法将60只雄性SD大鼠随机分为假手术组、模型组、烙灸组、西药组、Wnt抑制剂组和Wnt抑制剂+烙灸组,每组10只。除假手术组外,其余组大鼠采用撞击T9椎板方法建立脊髓损伤模型。烙灸组于建模后24 h在损伤部位局部进行烙灸,1次/d,每次5 min;西药组于建模后0.5 h给予20 mg/kg醋酸泼尼松注射,之后按照4.9 mg/(kg·d)继续干预;Wnt抑制剂组于建模后0.5 h在损伤部位皮下注射40μg/(kg·d)的Wnt通路抑制剂IWR-1;Wnt抑制剂+烙灸组于建模后0.5 h在损伤部位皮下注射40μg/(kg·d)的IWR-1,然后进行烙灸。各组均连续干预28 d。采用BBB运动评定量表和斜板试验评估大鼠运动能力,HE染色观察脊髓组织病理学形态,免疫组化法和Western blot法检测脊髓组织中巢蛋白(Nestin)、黏附连接蛋白(β-catenin)和神经胶质纤维酸性蛋白(GFAP)、糖原合成激酶-3(GSK-3β)表达情况。结果 随术后时间延长,除Wnt抑制剂组的BBB评分和斜板角度无明显变化外,其余各造模组大鼠的BBB评分和斜板角度均呈递增趋势;其中烙灸组和西药组大鼠术后第7,14,21,28天的BBB评分均明显高于同期模型组、Wnt抑制剂组、Wnt抑制剂+烙灸组(P均<0.05),术后第21,28天的斜板角度均明显高于同期模型组、Wnt抑制剂组、Wnt抑制剂+烙灸组(P均<0.05)。HE染色显示模型组、Wnt抑制剂组大鼠脊髓严重损伤,细胞坏死,神经元数量明显减少;烙灸组、西药组大鼠脊髓损伤明显轻于模型组、Wnt抑制剂组、Wnt抑制剂+烙灸组。Nestin、β-catenin、GFAP和GSK-3β在脊髓的白质和灰质中都有表达;与假手术组比较,模型组、Wnt抑制剂组和Wnt抑制剂+烙灸组脊髓组织中Nestin和GFAP蛋白表达量均明显升高(P均<0.05),β-catenin和GSK-3β蛋白表达量均明显降低(P均<0.05);与模型组比较,西药组和烙灸组脊髓组织中Nestin、β-catenin、GSK-3β蛋白表达量均明显升高而GFAP蛋白表达量均明显降低(P均<0.05),Wnt抑制剂+烙灸组β-catenin蛋白表达量明显升高(P<0.05)。结论 烙灸疗法可通过激活Wnt信号通路促进脊髓损伤大鼠神经修复。

Objective It is to investigate the effect of cauterizing moxibustion on nerve repair in rats after spinal cord injury based on Wnt signaling pathway.Methods Sixty male SD rats were randomly divided into 6 groups:sham operation group,model group,cauterizing moxibustion group,western medicine group,Wnt inhibitor and Wnt inhibitor+cauterizing moxibustion group,with 10 rats in each group.Except for the sham operation group,the rats in the other groups were used to establish spinal cord injury models by impacting T9 vertebral plate.The cauterizing moxibustion group was treated with cauterizing moxibustion at local injury site at 24 h after modeling,once daily,5 min per time;the western medicine group was treated with 20 mg/kg prednisone acetate by injection at 0.5 h after modeling,and then the treatment was continued with 4.9 mg/(kg·d);the Wnt inhibitor group was given Wnt pathway inhibitor IWR-140μg/(kg·d)by subcutaneous injection at 0.5 h after modeling;the Wnt inhibitor+cauterizing moxibustion group was given Wnt pathway inhibitor IWR-140μg/(kg·d)by subcutaneous injection at 0.5 h after modeling,then treated with cauterizing moxibustion.All the groups were continuously treated for 28 days.The motor ability of rats in each group was evaluated by weekly BBB motor scale and oblique plate test,the pathological morphology of spinal cord tissue was observed by HE staining,the expressions of nestin,β-catenin,glial fibrillary acidic protein(GFAP),glycogen synthetic kinase-3(GSK-3β)in spinal cord tissue were detected by immunohistochemistry and Western blotting.Results With the extension of postoperative time,the BBB scores and slant plate angles of rats in all modeling groups showed an increasing trend,except for the BBB scores and slant plate angles of rats in the Wnt inhibitor group,the BBB scores rats on the 7th,14th,21st,and 28th days after the operation in the cauterizing moxibustion group and western medicine group were significantly higher than those at the same period in the modeling group,Wnt inhibitor group,and Wnt inhibitor+cauterizing moxibustion group(all P<0.05),the oblique plate angles on the 21st and 28th postoperative days were significantly higher than those at the same period in the model group,Wnt inhibitor group,and Wnt inhibitor+cauterizing moxibustion group(all P<0.05).HE staining showed that the spinal cord of rats in the model group and Wnt inhibitor group was severely injured,with cell necrosis and a significant decrease of the number of neurons;the spinal cord injury of rats in the cauterizing moxibustion group and western medicine group was significantly lighter than that in the model group,Wnt inhibitor group and Wnt inhibitor+cauterizing moxibustion group.Nestin,β-catenin,GFAP and GSK-3βwere expressed in both white and gray matter of the spinal cord;compared with the sham operation group,the expressions of Nestin and GFAP proteins in the spinal cord tissues of the model group,Wnt-inhibitor group and Wnt-inhibitor+cauterizing moxibustion group were all significantly increased(all P<0.05),and protein expressions ofβ-catenin and GSK-3βproteins were all significantly decreased(all P<0.05);compared with the model group,the expressions of Nestin,β-catenin and GSK-3βprotein in the spinal cord tissues of the western medicine group and cauterizing moxibustion group were significantly higher and that of GFAP protein was significantly lower(all P<0.05),the expression ofβ-catenin protein in the Wnt inhibitor+cauterizing moxibustion group was significantly higher(P<0.05).Conclusion Cauterizing moxibustion therapy can promote the recovery of nerve function of rats with spinal cord injury via activating Wnt signaling pathway.