基于“肠道菌群-肠-脑轴”探讨半夏泻心汤改善大鼠糖尿病认知功能障碍作用机制

Exploration of the Action Mechanism of Banxia Xiexin Decoction for Diabetic Cognitive Dysfunction in Rats based on the“Intestinal Flora-Gut-Brain Axis”

【目的】观察半夏泻心汤对糖尿病认知功能障碍大鼠的治疗作用及机制。【方法】实验分为5组,即空白组,模型组,半夏泻心汤低、中、高剂量组和二甲双胍组,每组10只大鼠。除空白组,其余各组大鼠构建糖尿病认知功能障碍模型。半夏泻心汤低、中、高剂量组分别对应灌胃半夏泻心汤煎液0.425、0.85、1.70 g·kg-1·d-1,二甲双胍组灌胃二甲双胍0.30 g·kg-1·d-1,模型组及空白组大鼠均灌胃等体积的生理盐水。每日1次,连续28 d。给药结束后,检查大鼠空腹血糖值,Morris水迷宫试验评价大鼠认知功能,流式细胞术检测血清T淋巴细胞亚群,蛋白质免疫印迹法检测脑组织微管相关蛋白轻链3(LC3)、Beclin-1蛋白表达,16S-rDNA测序肠道菌群结构,实时定量聚合酶链反应(qRT-PCR)法检测脑组织蛋白激酶A(PKA)、环腺苷酸(cAMP)反应元件结合蛋白(CREB)m RNA表达。【结果】与空白组比较,模型组大鼠空腹血糖值升高,Morris水迷宫试验逃避潜伏期升高、穿越目标区次数减少(P<0.05),表明模型制备成功。半夏泻心汤低、中、高剂量组大鼠逃避潜伏期缩短、穿越目标区次数增加,空腹血糖值降低,CD8^(+)T细胞比例增加、CD4^(+)T细胞比例和CD4^(+)/CD8^(+)比值降低,LC3-Ⅱ/LC3-Ι比值和Beclin-1蛋白表达水平降低,肠道Chao 1和Shannon指数增加,厚壁菌门(Firmicutes)、乳酸杆菌属(Lactobacillus)、密螺旋体属(Treponema)丰度增加及拟杆菌门(Bacteroidetes)、普氏菌属(Prevotella)、瘤胃球菌属(Ruminococcus)丰度降低,脑组织PKA、CREB mRNA表达水平升高,与模型组比较,差异均有统计学意义(P<0.05),呈剂量依赖性,且疗效和二甲双胍组相当。【结论】半夏泻心汤可改善大鼠糖尿病认知功能障碍,其机制可能与调节肠道菌群,增强细胞免疫功能,并抑制神经细胞自噬、激活脑组织PKA-CREB信号通路进而保护神经功能有关。

Objective To observe the therapeutic effects and mechanism of Banxia Xiexin Decoction(BXD)for rats with diabetic cognitive dysfunction.Methods In the experiment there were 5 groups,namely,the blank group,the model group,the BXD low-,medium-and high-dose groups and the metformin group,with 10 rats in each group.Except the blank group,the rat model of diabetic cognitive dysfunction was established in the other groups.The rats in BXD low-,medium-and high-dose groups were given intragastric administration of BXD at 0.425、0.85、1.70 g·kg-1·d-1,respectively,the rats in metformin group were given intragastric administration of metformin at 0.30 g·kg-1·d-1,and the blank group and the model group were given intragastric administration of equal volume of normal saline.Once a day,the treatment lased for 28 days.After treatment,the fasting blood glucose values of the rats were detected,the cognitive function of the rats was evaluated by Morris water maze test,the serum T lymphocyte subsets were detected by flow cytometry,the protein expression of microtubuleassociated light chain 3(LC3)and Beclin-1 protein in brain tissues was deteted by Western Blot method,the structure of the intestinal flora was sequenced by 16S-rDNA analysis.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to determine the m RNA expression of protein kinase A(PKA),cyclic adenosine monophosphate(cAMP)response element binding protein(CREB)in brain tissues.Results The fasting blood glucose and escape latency in Morris water maze test of the rats in the model group were higher than those in the blank group,and the frequency of crossing the target area in the Morris test was lower than that in the blank group(P<0.05),indicating that the model was successfully prepared.The escape latency of BXD low-,medium-and high-dose groups was shortened,the frequency of crossing the target area was increased,the fasting blood glucose was decreased,the proportion of CD8^(+)T cells was increased,the proportion of CD4^(+)T cells and the CD4^(+)/CD8^(+)ratio were decreased,and the LC3-Ⅱ/LC3-Ιratio and Beclin-1 protein expression level were decreased,intestinal Chao 1 and Shannon index were increased,the abundance of Firmicutes,Lactobacillus and Treponema was increased and the abundance of Bacteroidetes,Prevotella and Ruminococcus was decreased,and the mRNA expression levels of PKA and CREB in brain tissues were increased,the differences being statistically significant as compared with those in the model group(P<0.05),in a dose-dependent manner,and the therapeutic effect was similar to that of the metformin group.Conclusion BXD has effects in improving cognitive dysfunction in rats with diabetes mellitus,and its mechanism may be related to the regulation of intestinal flora,enhancement of cellular immune function,and inhibition of neuronal autophagy,activation of PKA-CREB signaling pathway in brain tissues,thereby protecting the neurological function.