摘要目的 从Wnt/β-连接蛋白信号通路探讨甘草酸二铵(DG)对重型颅脑损伤(STBI)大鼠中枢神经再生修复的影响.方法 按随机数字表法将72只雄性SD大鼠均分为正常组、STBI模型组、神经节苷脂(GA)治疗组和DG治疗组.采用改良的Feeney自由落体方法建立STBI大鼠模型;正常组不致伤.制模后6 h,GA治疗组和DG治疗组分别经尾静脉注射单唾液酸四己糖神经节苷脂钠注射液或DG注射液各0.78 mL,每日注射1次,连续注射7 d;正常组和STBI模型组则给予等量生理盐水.各组分别于给药后1、3、7 d取6只大鼠进行神经功能缺损评分(NSS);然后取腹主动脉血和脑组织,采用酶联免疫吸附试验(ELISA)检测血清脑源性神经营养因子(BDNF)、神经生长因子(NGF)含量;取海马齿状回颗粒下区(SGZ)组织,行苏木素-伊红(HE)染色后光镜下观察病理学改变;用实时定量反转录-聚合酶链反应(RT-qPCR)检测海马组织Wnt3a、β-连接蛋白、糖原合成酶激酶-3β(GSK-3β)及Axin的mRNA表达.结果 ①正常组大鼠无神经功能缺损表现,NSS评分为0分.STBI制模后1 d大鼠NSS评分即明显升高,之后随时间延长逐渐下降.加用GA或DG治疗后1 d大鼠NSS评分即显著低于模型大鼠(分:7.33±2.07、6.17±2.23比9.33±1.63,均P<0.01),且随时间延长呈逐渐下降趋势,至7 d时差异仍有统计学意义(分:2.67±0.82、1.00±0.00比6.17±2.23,均P<0.01),而且DG治疗组的NSS评分较GA治疗组降低更为显著.②正常组大鼠海马SGZ细胞层次、结构清晰,排列较为紧密整齐.STBI模型组大鼠SGZ神经细胞和组织于各个时间点均有不同程度的损伤与破坏.加用GA或DG治疗后大鼠神经组织损伤得以改善,并随时间延长逐渐好转,而DG的作用更为明显.③正常组海马组织Wnt3a、β-连接蛋白的mRNA几乎不表达,GSK-3β、Axin的mRNA表达较高,血清中BDNF和NGF含量很少.STBI制模后1 d,大鼠海马组织Wnt3a、β-连接蛋白的mRNA表达量及血清BDNF、NGF含量均明显升高,GSK-3β、Axin的mRNA表达量明显下降.加用GA或DG后1 d海马组织Wnt3a、β-连接蛋白的mRNA表达量及血清BDNF、NGF含量均较模型组明显升高,GSK-3β、Axin的mRNA表达量显著降低,且DG的作用较GA更为显著〔Wnt3a mRNA(2-ΔΔct):3.51±0.14比2.93±0.05,β-连接蛋白mRNA(2-ΔΔct):1.90±0.08比1.75±0.04,BDNF(ng/L):4.06±0.55比3.16±0.64,NGF(ng/L):9.53±1.08比7.26±0.43,GSK-3βmRNA(2-ΔΔct):0.75±0.01比0.79±0.01,Axin mRNA(2-ΔΔct):0.74±0.02比0.76±0.02,均P<0.05〕,并随时间延长呈逐渐升高或下降趋势,至7 d时差异仍有统计学意义.结论 DG能够促进STBI大鼠中枢神经再生修复,其机制可能与Wnt/β-连接蛋白信号通路介导的神经细胞增殖分化及海马SGZ神经组织的重建有关.

abstractsObjective To observe the effects of diammonium glycyrrhizinate (DG) on nerve regeneration repair in rats with severe traumatic brain injury (STBI) from the perspective of Wnt/β-catenin signaling pathway. Methods Seventy-two Sprague-Dawle (SD) male rats were randomly divided into normal group, STBI model group, ganglioside (GA) treatment group and DG treatment group. The STBI animal model was reproduced referring to modified Feeney free fall impact model. No injury was made in normal group. Six hours after modeling, monosialotetrahexosylganglioside sodium injection and DG injection were injected via tail vein of rats in GA treatment group and DG treatment group respectively, once a day for 7 days. Normal group and STBI model group were given the&nbsp;same amount of normal saline. Six rats in each group were sacrificed on the 1st, 3rd and 7th day after the challenge for neurological severity score (NSS), and then the blood of abdominal aorta was drawn and brain tissue was harvested. The contents of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in serum were detected by enzyme linked immunosorbent assay (ELISA). The pathological changes of sub-granular zone (SGZ) were observed under light microscope after hematoxylin eosin (HE) staining. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of Wnt3a, β-catenin, glycogen synthetase kinase-3β (GSK-3β) and Axin. Results ① There was no neurological deficit in the normal group and NSS was 0. NSS score of rats increased significantly on the first day after modeling, and then decreased gradually over time. NSS of the rats treated with GA and DG were significantly lower than that of the STBI model rats (score: 7.33±2.07, 6.17±2.23 vs. 9.33±1.63, both P < 0.01). Though NSS gradually decreased over time, the differences were still statistically significant on the 7th day (score: 2.67±0.82, 1.00±0.00 vs. 6.17±2.23, both P < 0.01), and NSS of DG treatment group was significantly lower than that of GA treatment group. ② In SGZ of rats, cells were arranged in a compact and orderly way in the normal group, but neurons and tissues were damaged and destroyed at different time points in the STBI model group. After either GA or DG treatment, the damage of nerve tissue was improved gradually over time, and the effect of DG was more obvious.③ In the normal group, the mRNA expressions of Wnt3a and β-catenin were almost not expressed, the mRNA expressions of GSK-3β and Axin were higher, and the contents of BDNF and NGF in serum were less. On the 1st day after STBI, the mRNA expressions of Wnt3a and β-catenin in hippocampus, the contents of BDNF and NGF in serum were significantly increased, and the mRNA expressions of GSK-3βand Axin were significantly decreased. The mRNA expressions of Wnt3a and β-catenin in the hippocampus and the contents of BDNF and NGF in serum were significantly higher than those in the model group 1 day after GA or DG was added, the mRNA expressions of GSK-3β and Axin were significantly decreased, and the effect of DG was more significant than that of GA [Wnt3a mRNA (2-ΔΔCt):3.51±0.14 vs. 2.93±0.05, β-catenin mRNA (2-ΔΔCt): 1.90±0.08 vs. 1.75±0.04, BDNF (ng/L): 4.06±0.55 vs. 3.16±0.64, NGF (ng/L): 9.53±1.08 vs. 7.26±0.43, GSK-3βmRNA (2-ΔΔCt): 0.75±0.01 vs. 0.79±0.01, Axin mRNA (2-ΔΔCt): 0.74±0.02 vs. 0.76±0.02, all P < 0.05]. It was gradually increasing or decreasing over time and the difference was still statistically significant up to the 7th day. Conclusion DG can promote the recovery of nerve function in rats with STBI, and its mechanism may be related to the regeneration of nerve cells proliferation and differentiation by Wnt/β-catenin signaling pathway and the reconstruction of nerve tissue in SGZ of hippocampus.