“糖脂清”调控自噬改善STZ诱导糖尿病模型小鼠认知功能的机制研究

Mechanism of Regulating Autophagy by TangZhiqing to Improve Cognitive Function of STZ-Induced Diabetic Mice

目的:观察“糖脂清”对糖尿病模型小鼠认知功能的影响以及对海马神经元的保护作用,并探讨其相关作用机制。方法:采用高脂高糖饲料联合链脲佐菌素(STZ)腹腔注射的方法诱导2型糖尿病小鼠模型,随机分为模型组,二甲双胍组和糖脂清高、中、低剂量组,造模成功后给予不同剂量“糖脂清”和二甲双胍干预12周。灌胃给药12周结束后,通过Morris水迷宫实验进行行为学检测;取全脑进行海马区HE染色;采用ELISA法检测各组小鼠血清中的Atg5、Atg12的表达水平;采用Western blot对各组小鼠的海马组织Beclin1、LC3、Atg5、Atg7等自噬相关蛋白的表达;运用Real-time PCR技术检测小鼠海马Atg5、Atg7 mRNA的表达情况。结果:水迷宫结果显示,与空白组比较,模型组小鼠穿越平台次数明显减少(P<0.01);与模型组相比较,糖脂清中、高剂量组以及二甲双胍组小鼠的穿越平台次数明显增加(P<0.05,P<0.01),糖脂清各剂量组及二甲双胍组的目标象限路程比明显上升(P<0.05,P<0.01),糖脂清低剂量组和高剂量组的目标象限时间比明显升高(P<0.01)。HE染色结果显示:模型组小鼠海马组织的CA1区和CA3区神经元细胞数量明显减少、细胞结构的破坏较多、细胞坏死情况等程度较深,糖脂清各剂量组及二甲双胍组小鼠的海马神经元破坏较模型组减少。ELISA结果显示:与空白组相比,模型组Atg5、Atg12含量明显减少(P<0.01),而糖脂清低剂量组血清中Atg5、Atg12的含量虽较空白组减少(P<0.05,P<0.01),但较模型组增加(P<0.01);与模型组相比较,各给药组的Atg5含量均明显升高(P<0.01);糖脂清低剂量组、高剂量组以及二甲双胍组的Atg12含量明显上升(P<0.01)。Western blot结果显示:与空白组比较,模型组的Beclin1、Atg5、Atg7、LC3蛋白表达水平均明显降低(P<0.01);与模型组比较,糖脂清高剂量组及二甲双胍组的Beclin1、LC3、Atg5、Atg7蛋白表达水平出现了不同程度的升高(P<0.05,P<0.01)。Real-time PCR结果显示:与空白组比较,模型组Atg5、Atg7 mRNA表达呈下降趋势,但结果分析后提示无统计学意义(P>0.05);与模型组相比较,糖脂清高剂量组Atg5 mRNA表达量明显升高(P<0.01)。结论“:糖脂清”可改善糖尿病模型小鼠的认知功能障碍,具体机制可能与调节Beclin1、LC3、Atg5、Atg7、Atg12等自噬相关蛋白的表达,促进自噬有关。

Objective:To observe the effect of Tangzhiqing(TZQ)on cognitive function of diabetes model mice and its protective effect on hippocampal neurons,and to explore its related mechanism.Methods:The type 2 diabetes mouse model was induced by intraperitoneal injection of high fat and high sugar diet combined with streptozotocin(STZ).The mice were randomly divided into model group,metformin group,and high,medium,and low dose groups of TZQ.After successful modeling,different doses of TZQ and metformin were given through gavage for 12 weeks.After 12 weeks of administration,behavioral testing was conducted using the Morris water maze experiment.HE staining was conducted for the hippocampus of the brain.ELISA method was used to detect the expression levels of Atg5 and Atg12 in the serum of mice in each group.Western blot was used to investigate the expression of autophagy related proteins,such as Beclin1,LC3,Atg5,and Atg7 in the hippocampus of the mice in each group.Real time PCR technology was employed to detect the expression of Atg5 and Atg7 mRNA in the hippocampus of the mice.Result:The water maze results indicated that compared with the blank group,the number of times the model group mice crossed the platform was significantly reduced(P<0.01).Compared with the model group,the number of times of mice crossing the platform significantly increased in high and medium TZQ dose groups and in metformin group(P<0.05,P<0.01).Target quadrant distance ratio increased significantly in all TZQ groups and metformin group(P<0.05,P<0.01),while decreased significantly in low and high dose TZQ groups(P<0.05,P<0.01).The HE staining results showed that the number of neurons in CA1 and CA3 regions of the hippocampus in the model group was significantly reduced,more cell structures were damaged,and the degree of cell necrosis was more severe.The damage of hippocampal neurons in TZQ and metformin groups was less than that in the model group.The ELISA results showed that compared with the blank group,the content of Atg5 and Atg12 in the model group was significantly reduced(P<0.01),while the content of Atg5 and Atg12 in the serum of the low dose TZQ group decreased compared to the blank group(P<0.05,P<0.01),but increased compared to the model group(P<0.01).Compared with the model group,Atg5 content in each treatment group was significantly high(P<0.01).The content of Atg12 increased significantly in TZQ low dose and high dose groups and metformin group(P<0.01).Western blot results showed that compared with the blank control group,the expression levels of Beclin1,Atg5,Atg7,and LC3 proteins in the model group significantly decreased(P<0.01),while compared with the model group,the expression levels of Beclin1,LC3,Atg5,and Atg7 proteins in the high dose TZQ group and metformin group increased to varying degrees(P<0.05,P<0.01).Real time PCR results showed that compared with the blank group,the mRNA expression of Atg5 and Atg7 in the model group showed a downward trend,but the analysis of the results showed no statistical significance(P>0.05).Compared with the model group,the Atg5 mRNA expression level in the high dose TZQ group was significantly increased(P<0.01).Conclusion:Tangzhiqing can improve the cognitive dysfunction of diabetes model mice.The mechanism may be related to regulating the expression of autophagy related proteins such as Beclin1,LC3,Atg5,Atg7,Atg12,and promoting autophagy.