血糖波动对糖尿病大鼠海马Tau蛋白磷酸化的影响

The influence of blood glucose fluctuation on hippocampal tau protein phosphorylation of diabetic rats

目的 观察血糖波动对糖尿病大鼠海马Tau蛋白磷酸化（p-Tau）（ser202位点）的影响，探讨血糖波动对糖尿病大鼠海马Tau蛋白的影响及其分子机制的研究。方法 雄性SD大鼠随机数字表法分为正常对照组（NC组）、糖尿病组，糖尿病组大鼠腹腔注射链脲佐菌素60 mg/kg诱导成糖尿病模型后，随机数字表法分为2组：糖尿病血糖波动组（DF组），人为诱导血糖波动；糖尿病血糖持续组（DC组）。NC组生理盐水腹腔注射。8周末，测各组大鼠体重、血糖、HbA1C、胰岛素，免疫组化法观察海马p-Tau（ser202）的表达，Western印迹和图像分析法检测海马p-Tau的表达，实时定量聚合酶链反应（RT-PCR）法检测海马糖原合成激酶3β（GSK-3β）mRNA相对表达。结果 （1）血糖情况：DC组和DF组大鼠血糖波动大于NC组，2组每日血糖平均水平、每日平均血糖的标准差（SDBG）、最大血糖波动幅度（LAGE）均较NC组明显升高，差异具有统计学意义（均P〈0.05）；与DC组相比，DF组SDBG、LAGE升高，差异具有统计学意义（均P〈0.05），HbA1C含量、胰岛素水平差异无统计学意义（均P〉0.05）。（2）与NC组比，DC组与DF组大鼠海马p-Tau含量升高（均P〈0.05），大鼠海马GSK-3β mRNA水平升高（均P〈0.05）；与DC组比，DF组大鼠海马p-Tau含量升高（P〈0.05），大鼠海马GSK-3β mRNA水平升高（P〈0.05）。结论 糖尿病大鼠1天2次定时给予葡萄糖腹腔注射并错时给予胰岛素皮下注射能成功构建糖尿病血糖波动模型；血糖波动使糖尿病大鼠海马p-Tau水平显著增加，机制可能是上调GSK-3β的表达。

Objective To investigate the influence of blood glucose fluctuation on ser202 phosphorylation sites of tau protein （p-Tau） in hippocampus of diabetic rats ; to explore the possible mechanism of blood glucose fluctuation impacting on tau protein byperphosphorylation. Methods Healthy male Sprague Dawley rats were randomly divided into normal control group （NC group ） and diabetes group. After diabetic rats model was established, all the diabetic rats were randomly divided into diabetic continuous hyperglycemia group （ DC group ） and diabetic blood glucose fluctuant group （ DF group ）. Rats in DF group were given glucose solution intraperitoneal injection twice at regular time everyday. 30 minutes after each intraperitoneal injection, insulin subcutaneously injections were given. Rats in the NC group and DC groups were given the same volume of saline subcutaneous injection. Specimens were collected in 8 weeks, the levels of p-Tau and total tau in rat hippocampus were detected by immunohistochemical staining and Western blotting. The immunoreactive positive products were analyzed by image analysis system. Glycogen synthase kinase-3 β （ GSK-3 β） mRNA was detected by reahime PCR. Results （ 1 ） Blood glucose fluctuation of rats in DC and DF group were greater than NC group, And the mean blood glucose, standard deviation of mean blood glucose （ SDBG）, and large amplitude of glycemic excursion （LAGE） levels were increased significantly compared to NC group, the difference has statistical significance （all P〈0.05 ）. Compared with DC group, SDBG and LAGE levels of DF group were higher （ both P〈0.05 ）. HbAlc and insulin levels were no difference （P〉0.05）. （2） Compared with NC group, the hippoeampal p-Tau level of DC group and DF group were increased （P 〈 0. 05 ） ; Compared with DC group, the hippoeampal p-Tau expression of DF group was increased （ P 〈 0.05 ）. Compared with DC group, a higher hippocampal GSK-3β mRNA level was found in DF group （P 〈 0. 05 ）. Conclusions On the basis of diabetes animal model, giving glucose solution intraperitoneal injection and insulin subcutaneously injeetion 30 minutes later twice at regular time everyday could establish experimental model of diabetic blood glucose fluctuation. Blood glucose fluctuation may aggravate the diabetic rats hippocampal p-Tau. The possible mechanism seems to be an up regulation of the GSK-3β.