益髓灸对D-半乳糖衰老小鼠模型海马组织Klotho蛋白表达及胰岛素通路的影响

Effect of Yisui moxibustion on Klotho protein expression and insulin pathway of hippocampal tissues in aging mice model induced by D-galactose

目的探讨观察益髓灸对D-半乳糖衰老小鼠海马组织Klotho、胰岛素受体底物-1(IRS-1)、蛋白激酶B(Akt)、叉头框蛋白O3(FOXO3a)mRNA及蛋白表达的影响。方法将60只雄性昆明小鼠随机分为生理盐水组、模型组、足三里组、百会组、非穴位组,每组12只。模型组和各干预组小鼠给予D-半乳糖皮下注射建立衰老小鼠模型,生理盐水组注射等量的生理盐水。3个干预组于造模第13天开始于相应穴位进行艾灸,生理盐水组、模型组仅给予同等程度的捉抓。造模前后采用Morris水迷宫实验对5组小鼠进行行为测试。治疗结束后检测小鼠海马组织Klotho、IRS-1、Akt、FOXO3a mRNA及蛋白水平。结果 (1)造模后,模型组潜伏期较生理盐水组、百会组、足三里组延长(P<0.05),非穴位组潜伏期与百会组、足三里组比较,差异无统计学意义(P>0.05)。(2)与生理盐水组比较,模型组、非穴位组Klotho、IRS-1 mRNA及蛋白表达水平降低,Akt1、FOXO3a mRNA表达水平增高(P<0.05);与模型组比较,足三里组及百会组Klotho、IRS-1 mRNA表达水平增高,Akt1、FOXO3a mRNA表达水平降低(P<0.05)。(3)与生理盐水组比较,模型组海马组织Klotho、磷酸化IRS-1(p-IRS-1)、Akt、FOXO3a蛋白水平降低(P<0.05),IRS-1、磷酸化Akt(p-Akt)、磷酸化FOXO3a(p-FOXO3a)蛋白水平升高(P<0.05);与模型组比较,足三里组及百会组Klotho、p-IRS-1、Akt、FOXO3a蛋白水平升高(P<0.05),IRS-1、p-Akt、p-FOXO3a蛋白水平降低(P<0.05),而非穴位组p-Akt蛋白表达水平降低(P<0.05)。结论益髓灸的早期干预可能通过上调Klotho、IRS-1 mRNA水平,促进D-半乳糖衰老小鼠海马Klotho蛋白表达,激活IRS-1的丝氨酸抑制性位点,负性调控下游的Akt活性而下调其底物FOXO3a的磷酸化表达,从而发挥延缓脑衰老的作用。

Objective To explore the effect of Yisui moxibustion on Klotho,insulin receptor substrate 1(IRS-1),protein kinase B(Akt),forkhead box O3(FOXO3a)mRNA and protein expressions of hippocampal tissues in aging mice model induced by D-galactose.Methods Sixty male Kunming mice are randomly divided into normal saline group,model group,Zusanli group,Baihui group and nonacupoint group,with 12 mice in each group.The mice in the model group and each intervention group were given subcutaneous injection of D-galactose for establishing aging mice model,and in the normal saline group received normal sodium injection with the same dosage.On the 13th day of modeling,the three intervention groups underwent moxibustion at corresponding acupoints,while the normal saline group and model group were given scratch with the same degree.Behavior test using Morris water maze was conducted in the five groups before and after modeling.After the treatment finished,the expression levels of Klotho,IRS-1,Akt and FOXO3a mRNAs and proteins were detected in hippocampal tissues of the mice.Results (1)After modeling,the latent periods of the model group and the nonacupoint group were longer than that of the normal saline group,the Zusanli group or the Baihui group(P<0.05),and no statistically significant difference was found in the latent period between the nonacupoint group and the Baihui group,or between the nonacupoint group and the Zusanli group(P>0.05).(2)In the model group and nonacupoint group,the expression levels of Klotho and IRS-1 mRNAs and proteins decreased,and the expression levels of Akt1 and FOXO3a mRNAs increased compared to the levels in the normal saline group(P<0.05).Compared to the model group,the Zusanli group and Baihui group had higher expression levels of Klotho and IRS-1 mRNAs but lower expression levels of Akt1 and FOXO3a mRNAs(P<0.05).(3)In the model group,the levels of Klotho,phospho-IRS-1(p-IRS-1),Akt and FOXO3a proteins decreased(P<0.05),and the levels of IRS-1,phospho-Akt(p-Akt)and phospho-FOXO3a(p-FOXO3a)proteins increased compared to the levels in the normal saline group(P<0.05).Compared to the model group,the Zusanli group and Baihui group obtained higher levels of Klotho,p-IRS-1,Akt and FOXO3a proteins but lower levels of IRS-1,p-Akt and p-FOXO3a proteins(P<0.05),and the nonacupoint group had a lower expression level of p-Akt protein(P<0.05).Conclusion Anti-aging effect of Yisui moxibusition on the brain might relate to:Yisui moxibusition can probably up-regulate the Klotho and IRS-1 mRNA levels to promote the expression of Klotho protein in hippocampus of aging mice model induced by D-galactose,stimulate the activation of IRS-1 serine inhibition site,down-regulate its downstream Akt activity and thus down-regulate of phosphorylation expression of its substrate FOXO3a.