木犀草素对衰老模型大鼠记忆功能及凋亡相关蛋白的影响

Effects of hesperetin on memory function and apoptosis-related protein expression in senile model rats

目的探讨不同剂量的木犀草素对D-半乳糖致衰老大鼠记忆功能及凋亡蛋白的影响。方法SPF级雄性6~8周龄Wistar大鼠48只,按照随机数字表法分为对照组、模型组、木犀草素低剂量组(25 mg/kg)、中剂量组(50 mg/kg)、高剂量组(100 mg/kg)及维生素C组(100 mg/kg),每组8只。采用D-半乳糖(1000 mg/kg)皮下注射法制备大鼠衰老模型,同时使用木犀草素进行预防性灌胃治疗。Morris水迷宫实验评估小鼠学习记忆能力;透射电镜检测大鼠海马神经元形态;分光光度法检测检测大鼠大脑皮质中白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、总抗氧化能力(total antioxidant capacity,T-AOC)水平;RT-PCR检测miR-34a mRNA表达;Western blot技术检测沉默调节蛋白1(sirtuin 1,SIRT1)、B淋巴细胞瘤2(B-cell lymphoma-2,Bcl-2)、裂解caspase-3、p53、p21的表达水平。采用SPSS 22.0进行统计分析,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果(1)6组大鼠首次找到原平台时间差异有统计学意义(F=120.93,P<0.001),模型组大鼠首次找到原平台时间[(54.61±3.60)s]高于对照组[(10.54±4.27)s](P<0.05),木犀草素低、中、高剂量组大鼠首次找到原平台时间[(45.50±3.81)s,(37.46±2.94)s,(32.32±3.14)s]均低于模型组[(54.61±3.60)s](均P<0.05)。(2)6组大鼠大脑皮质中SOD、MDA、T-AOC、TNF-α、IL-1β和IL-6水平均差异具有统计学意义(F=281.636,75.119,208.228,38.999,28.428,52.767,均P<0.001)。模型组较对照组相比炎症因子和抗氧化指标异常,木犀草素中、高剂量组大鼠大脑皮质中SOD、T-AOC含量高于模型组(均P<0.05);MDA、TNF-α、IL-1β和IL-6水平低于模型组(均P<0.05)。(3)6组大鼠的miR-34a mRNA相对表达水平差异有统计学意义(F=81.439,P<0.001)。木犀草素不同剂量组大鼠海马组织中miR-34a mRNA表达水平均低于模型组(均P<0.05)。(4)6组大鼠海马组织中SIRT1、p53、p21蛋白表达差异具有统计学意义(F=159.946,38.342,123.608均P<0.001),木犀草素中、高剂量组p53、p21表达均低于模型组(均P<0.05),SIRT1蛋白表达均高于模型组(P<0.05)。(5)6组大鼠海马组织中Bcl-2、裂解caspase-3蛋白表达差异具有统计学意义(F=112.659,43.296,均P<0.05),木犀草素低、中、高剂量组Bcl-2表达[(0.24±0.04),(0.40±0.03),(0.48±0.05)]高于模型组[(0.09±0.06)](P<0.05),木犀草素低、中、高剂量组裂解caspase-3表达[(0.62±0.04),(0.61±0.09),(0.51±0.10)]低于模型组[(0.75±0.05)](P<0.05)。结论木犀草素可以缓解衰老模型大鼠脑组织氧化损伤,这可能与下调miR-34a/SIRT1/p53信号通路,减少细胞凋亡有关。

ObjectiveTo investigate the effect and mechanism of different doses of luteolin on memory function and apoptosis-related proteins of aging rats induced by D-galactose.MethodsForty-eight SPF-grade male Wistar rats aged 6-8 weeks were randomly divided into control group,model group,luteolin low-dose group(25 mg/kg),medium-dose group(50 mg/kg),high-dose group(100 mg/kg),and vitamin C group(100 mg/kg),with 8 rats in each group.D-galactose(1000 mg/kg)was subcutaneously injected to establish the aging rat model,while luteolin was used for preventive treatment.The Morris water maze test was used to evaluate the learning and memory abilities of the rats.Transmission electron microscopy was used to detect the morphology of hippocampal neurons in rats.Spectrophotometry was used to detect the levels of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD),malondialdehyde(MDA),and the total antioxidant capacity(T-AOC).RT-PCR was used to detect miR-34a mRNA expression.Western blot technique was used to detect the expression levels of silent regulator protein 1(SIRT1),B-cell lymphoma-2(Bcl-2),cleaved caspase-3,p53,and p21.Statistical analysis was performed using SPSS 22.0,and one-way ANOVA was used for multi-group comparison,followed by LSD-t test for further pairwise comparisons.Results(1)The differences in escape latency among the 6 groups of rats were statistically significant(F=120.93,P<0.001).The latency of first finding the platform location of the model group rats((54.61±3.60)s)was higher than that of the control group((10.54±4.27)s)(P<0.05).The latency of first finding the platform location of rats in the low,medium and high dosage groups of luteolin((45.50±3.81)s,(37.46±2.94)s,(32.32±3.14)s)was lower than that of the model group((54.61±3.60)s)(all P<0.05).(2)The differences of SOD,MDA,T-AOC,TNF-α,IL-1β,and IL-6 levels in the cerebral cortex of the 6 groups of rats were all statistically significant(F=281.636,75.119,208.228,38.999,28.428,52.767,all P<0.001).Compared with the control group,the model group showed abnormal levels of inflammatory factors and antioxidant indexes.In the medium and high dosage groups of luteolin,the SOD and T-AOC contents in the cerebral cortex of rats were higher than those in the model group(all P<0.05),while the levels of MDA,TNF-α,IL-1β,and IL-6 were lower than those in the model group(all P<0.05).(3)The differences in relative expression levels of miR-34a mRNA among the 6 groups of rats were statistically significant(F=81.439,P<0.001).The expression levels of miR-34a mRNA in the hippocampal tissues of rats in the luteolin treatment group were lower than those in the model group(P<0.05).(4)The differences in protein expression levels of SIRT1,p53,and p21 in the hippocampal tissues of the 6 groups of rats were statistically significant(F=159.946,38.342,123.608,all P<0.001).The expression levels of p53 and p21 in the medium and high dosage groups of luteolin were lower than those in the model group(all P<0.05),while the expression level of SIRT1 protein was higher than that in the model group(P<0.05).(5)The differences in protein expression levels of Bcl-2 and cleaved caspase-3 in the hippocampal tissues of the 6 groups of rats were statistically significant(F=112.659,43.296,both P<0.05).The expression levels of Bcl-2 in the low,medium,and high dosage groups of luteolin((0.24±0.04),(0.40±0.03),(0.48±0.05)pg/μg)were higher than those in the model group((0.09±0.06)μg)(P<0.05),while the expression levels of cleaved caspase-3 in the low,medium,and high dosage groups of luteolin((0.62±0.04),(0.61±0.09),(0.51±0.10)μg)were lower than those in the model group((0.75±0.05)μg)(P<0.05).ConclusionsLuteolin can alleviate cellular oxidative damage through downregulating the miR-34a SIRT1/p53 signaling pathway and reducing cell apoptosis.