二甲双胍联合多奈哌齐对糖尿病大鼠海马小胶质细胞活化和认知功能的影响

Effects of metformin combined with donepezil on the activation of hippocampal microglia and cognitive function in diabetic rats

目的探讨二甲双胍联合多奈哌齐对糖尿病大鼠海马小胶质细胞活化、犬尿氨酸通路和认知功能的影响。方法将血糖值≥11.1 mmol·L^(-1)的Goto-Kakizaki(GK)大鼠随机分为3组:模型组、二甲双胍组、联合组;另选正常Wistar大鼠作为正常组。二甲双胍组以76.5 mg·kg^(-1)二甲双胍干预,联合组给予76.5 mg·kg^(-1)二甲双胍+3 mg·kg^(-1)多奈哌齐干预,正常组和模型组给予等量纯净水,连续4周。以Morris水迷宫法检测大鼠在连续5 d的学习和记忆能力,免疫荧光双标法检测海马吲哚胺2,3双加氧酶(IDO)+离子钙结合衔接分子(Iba-1)蛋白表达(平均阳性表达率),实时荧光定量-PCR法检测大鼠海马犬尿氨酸3-单加氧酶(KMO)和喹啉酸(QUIN)基因表达(2-ΔΔCt值)。结果正常组、模型组、二甲双胍组和联合组第5天的逃避潜伏期为(17.67±6.12),(36.42±8.30),(26.79±6.31)和(23.17±7.22)s;这4组的空间探索时间为(34.33±7.74),(17.04±6.42),(19.29±8.57)和(26.50±7.18)s;这4组的IDO+Iba-1蛋白阳性率为(17.26±4.10)%,(52.25±9.72)%,(46.58±8.86)%和(34.83±6.81)%;这4组的KMO基因相对表达量为1.05±0.18,3.37±0.44,2.71±0.47和2.21±0.29;这4组的QUIN基因相对表达量为1.08±0.20,4.10±0.44,3.63±0.35和2.74±0.47。上述指标,模型组与正常组比较,差异均有统计学意义(P<0.05,P<0.01);联合组与模型组比较、或联合组与二甲双胍组比较,差异均有统计学意义(P<0.05,P<0.01)。结论二甲双胍联合多奈哌齐改善糖尿病GK大鼠认知功能的作用机制可能与有效调节海马IDO/KMO/QUIN信号通路有关,进而减少小胶质细胞过度活化。

Objective To explore the effects of metformin combined with donepezil on hippocampal microglia cell viability,kynurenine pathway and cognitive function in diabetic rats.Methods Goto-Kakizaki(GK)rats were randomly divided into three groups with blood glucose level≥11.1 mmol·L^(-1):model group,metformin group,and combination group.Wistar rats were used as normal group.The metformin group was treated with 76.5 mg·kg^(-1) metformin,the combination group was treated with 76.5 mg·kg^(-1) metformin and 3 mg·kg^(-1) donepezil,the normal group and model group were given the same amount of purified water for 4 weeks.Learning and memory ability of rats in each group were detected by Morris water maze for 5 d.The indoleamine 2,3 dioxygenase(IDO)and ionized calcium binding adapter molecule(Iba-1)proteins expression(average positive expression rate)was detected by immunofluorescence double-labeling method.The rat hippocampal kynurenine 3-monooxygenase(KMO)and quinolinic acid(QUIN)gene expression(2-ΔΔCt value)were detected by quantitative real-time PCR.Results The escape latency of the normal group,model group,metformin group and combination group on the 5th day were(17.67±6.12),(36.42±8.30),(26.79±6.31),(23.17±7.22)s,respectively;the space exploration time in the 4 groups were(34.33±7.74),(17.04±6.42),(19.29±8.57),(26.50±7.18)s,respectively;the positive rate of IDO+Iba-1 protein in hippocampus in the 4 groups were(17.26±4.10)%,(52.25±9.72)%,(46.58±8.86)%and(34.83±6.81)%,respectively;the relative expression of KMO mRNA in the 4 groups were 1.05±0.18,3.37±0.44,2.71±0.47,2.21±0.29,respectively;the relative expression of QUIN mRNA in the 4 groups were 1.08±0.20,4.10±0.44,3.63±0.35,2.74±0.47,respectively.For the above indicators,the difference between the model group and the normal group was statistically significant(P<0.05,P<0.01);the combination group was compared with the model group or the combination group compared with the metformin group,the difference was statistically significant(P<0.05,P<0.01).Conclusion The mechanism of metformin combined with donepezil in improving the cognitive function of diabetic GK rats may be related to the effective regulation of the hippocampal IDO/KMO/QUIN signaling pathway,thereby reduce the excessive activation of microglia.