甘草酸调控水通道蛋白4表达影响创伤性脑损伤后神经元活性的研究

Regulation of aquaporin 4 expression by glycyrrhizin acid affects neuronal activity after traumatic brain injury

目的探究甘草酸对创伤性脑损伤(TBI)大鼠神经元损伤的作用,以及其可能机制。方法将大鼠随机分为假手术组、模型组、对照组和低、中、高剂量实验组,每组20只。假手术组仅进行颅脑开孔处理;其余5组大鼠用体外冲击法建立TBI模型。于建模后的0、24、48 h,低、中、高剂量实验组分别腹腔注射10、50、100 mg·kg^(-1)甘草酸溶液,对照组腹腔注射2 mg·kg^(-1)尼莫地平;假手术组和模型组在同样时间点腹腔注射等体积磷酸盐缓冲溶液。用脑水肿和改良的神经功能损害评分法(mNSS)评价大鼠神经功能障碍程度,用细胞凋亡染色法评估大鼠脑组织神经元凋亡率,用蛋白质印迹法检测凋亡相关蛋白和水通道蛋白4(AQP4)的表达水平。结果中、高剂量实验组和对照组、模型组、假手术组的mNSS评分分别为(6.98±0.82)、(5.28±0.37)、(5.91±0.52)、(13.28±1.59)和(0.36±0.01)分,脑水肿程度分别为(63.27±10.33)%、(60.09±9.38)%、(66.86±9.91)%、(85.92±11.93)%和(52.17±8.53)%,神经元凋亡率分别为(6.81±0.73)%、(5.39±0.25)%、(5.87±0.62)%、(15.13±3.29)%和(2.56±0.03)%,B-细胞淋巴瘤因子2(Bcl-2)蛋白相对表达水平分别为0.49±0.06、0.68±0.15、0.62±0.03、0.13±0.03和0.95±0.13,Bcl-2关联X蛋白相对表达水平分别为0.61±0.08、0.55±0.17、0.39±0.09、0.92±0.19和0.16±0.02,AQP4蛋白相对表达水平分别为0.69±0.15、0.38±0.03、0.47±0.09、0.86±0.13和0.13±0.09;模型组的上述指标与中、高剂量实验组和对照组相比较,在统计学上差异均有统计学意义(均P<0.05)。结论甘草酸能够减轻创伤性大鼠脑损伤的水肿程度和神经功能障碍,抑制神经元的损伤和凋亡,其作用机制可能与抑制AQP4表达有关。

Objective To explore the effects of glycyrrhizic acid(GA)on neurons injury in traumatic brain injury(TBI)rats and the possible mechanism.Methods The rats were randomly divided into shamoperation group,model group,control group and experimental-L,-M,-H groups,with 20 rats each group.The sham-operation group was only treated with craniotomy;the other 5 groups were used to establish TBI models by extracorporeal shock method.At 0,24 and 48 h after modeling,the experimental-L,-M,-H groups were intraperitoneally injected with 10,50 and 100 mg·kg^(-1)GA solution,respectively;control group was intraperitoneally injected with 2 mg·kg^(-1)nimodipine;sham-operation and model groups were intraperitoneally injected with the equal volume of phosphate buffered solution.The degree of neurological dysfunction was evaluated by cerebral edema and modified neurological severity score(m NSS).The apoptosis rates of neurons in rat brain tissue was evaluated by apoptosis staining.Western blot was used to analyze the expression levels of apoptosis-related proteins and aquaporin 4(AQP4)protein.Results The m NSS scores of experimental-M,-H groups,control group,model group,sham-operation group were(6.98±0.82),(5.28±0.37),(5.91±0.52),(13.28±1.59)and(0.36±0.01)points;the degrees of brain edema were(63.27±10.33)%,(60.09±9.38)%,(66.86±9.91)%,(85.92±11.93)%and(52.17±8.53)%;the apoptosis rates of neurons were(6.81±0.73)%,(5.39±0.25)%,(5.87±0.62)%,(15.13±3.29)%and(2.56±0.03)%;the relative expression levels of B cell lymphoma 2(Bcl-2)protein were 0.49±0.06,0.68±0.15,0.62±0.03,0.13±0.03 and 0.95±0.13;the relative expression levels of Bcl-2 associated X protein were 0.61±0.08,0.55±0.17,0.39±0.09,0.92±0.19 and0.16±0.02;the relative expression levels of AQP4 protein were 0.69±0.15,0.38±0.03,0.47±0.09,0.86±0.13and 0.13±0.09,respectively.There were statistically significant differences in the above indexes between the model group and the experimental-M,-H groups and control group(all P<0.05).Conclusion GA is able to reduce the brain edema degree and neurological dysfunction in TBI rats,and inhibit neuronal damage and apoptosis,and the mechanism of action may be associated with the inhibition of AQP 4 expression.