【目的】

糖尿病认知功能障碍（Diabetic Cognitive Impairment， DCI）是糖尿病慢性并发症，其确切的发病机制目前尚不清晰。DCI中“肾虚血瘀，髓海不充”证型最为常见，兔仙合剂（Tu-Xian decoction， TXD）有“补肾活血，益脑健智”之功。死亡相关蛋白激酶1（Death-associated protein kinase 1， DAPK-1）参与多种神经退行性病变。本课题通过代谢组学、体外培养与动物实验研究，探讨兔仙合剂是否通过抑制DAPK-1保护糖尿病大鼠认知功能。

【方法】

第一部分

采用超高效液相色谱-质谱联用(Ultra high-performance liquid chromatograph-mass spectrum， UHPLC-MS)非靶标代谢组学技术鉴定兔仙合剂主要成分。检索文献查阅TXD内主要成分是否具有调节DAPK-1的功能。

第二部分

从新生24小时SD大鼠提取原代海马神经元，采用淋巴细胞分离液密度梯度离心与差速贴壁法纯化后，将神经元分为4组：正常对照组（Control， Con，葡萄糖含量25mmol/L）、高糖组（High glucose， HG，葡萄糖含量50mmol/L）、兔仙合剂组（TXD，葡萄糖含量50mmol/L，设置0.1mg/mL、1.0mg/mL、5.0mg/mL、10.0mg/mL、50.0mg/mL 5个TXD浓度梯度）、DAPK-1抑制剂组（TC-DAPK6，葡萄糖含量50mmol/L，设置0.1μmol/L、1.0μmol/L、10.0μmol/L、20.0μmol/L 4个TC-DAPK6浓度梯度），分组干预神经元72小时后，采用CCK-8法测定海马神经元的相对活性。将神经元分为3组：正常对照组（Con）、高糖组（HG）、兔仙合剂组（TXD，采用上述CCK-8检测中神经元相对活性最高组浓度，即最适浓度干预），分组干预72小时后，采用蛋白免疫印迹法测定T-DAPK-1、P-DAPK-1（Ser308）表达水平。

第三部分

采用高糖高脂（High-sugar high-fat, HSHF）饮食联合35mg/Kg链脲菌素（Streptozotocin, STZ）建立糖尿病大鼠模型。随机将糖尿病大鼠分为：糖尿病（Diabetic， DM）组，DAPK-1抑制剂（TC-DAPK6）组、高剂量兔仙合剂（H-TXD， 12g/Kg）组、中剂量兔仙合剂（M-TXD， 6g/Kg）组、低剂量兔仙合剂（L-TXD, 3g/Kg）组。采用普通饲料喂养正常对照（Control， Con）组大鼠。分组干预大鼠12周后，Morris水迷宫检测大鼠认知功能，头颅核磁共振成像检测大鼠海马体积，苏木素伊红染色、尼氏染色检测大脑病理变化，蛋白质印迹法检测大鼠海马体Aβ、APP、P-APP、Tau、P-Tau（Thr231）、P-Tau（Ser396）、Caspase-3、T-DAPK-1、P-DAPK-1（Ser308）蛋白表达水平。免疫组化检测大鼠海马体Bcl-2表达水平。

【结果】

第一部分

经鉴定TXD含黄酮类、酚类、萜类、苯丙素类、生物碱类、脂肪酰类、有机酸类及其衍生物、糖类及其衍生物等908种已知化合物。

第二部分

1.  提取原代海马神经元，经神经元特异性烯醇化酶（Neuron-specific enolase，NSE）免疫荧光鉴定纯度达95％以上。

2. 分组干预72小时后，HG组神经元相对活性为（79.23±5.62）%/Con，各剂量兔仙合剂组神经元相对活性分别为：0.1mg/mL TXD组（79.83±4.02）%/Con；1.0mg/mL TXD组（100.60±7.59）%/Con；5.0mg/mL TXD组（116.03±6.99）%/Con；10.0mg/mL TXD组（93.53±2.78）%/Con；50.0mg/mL TXD组（92.17±5.07）%/Con。与Con组相比，HG组神经元相对活性降低（P＜0.01）。与HG组相比，1.0mg/mL TXD组（P＜0.01）、5.0mg/mL TXD组（P＜0.0001）神经元相对活性升高。

分组干预72小时后，各剂量DAPK-1抑制剂组神经元相对活性为：0.1μmol/L TC-DAPK6组（87.23±3.08）%/Con；       1.0μmol/L TC-DAPK6组（92.08±2.81）%/Con；10.0μmol/L TC-DAPK6组（      102.33±7.75）%/Con；20.0μmol/L TC-DAPK6组（85.80±6.35）%/Con。与HG组相比，10.0μmol/L TC-DAPK6组神经元相对活性升高（P＜0.01）。

3. 与Con组（（1.16±0.14）/β actin）相比，HG组神经元T-DAPK-1表达水平（（1.97±0.14）/β actin）升高（P＜0.05）。与Con组（（0.57±0.10）/T-DAPK-1）相比，HG组神经元P-DAPK-1（Ser308）表达水平（（0.29±0.07）/T-DAPK-1）下降（P＜0.05），表明DAPK-1活性升高。与HG组相比，5mg/mL TXD组T-DAPK-1（（1.83±0.16）/β actin）表达水平虽无显著差异（P＞0.05），但P-DAPK-1（Ser308）（（0.48±0.09）/T-DAPK-1）表达水平升高（P＜0.05），表明DAPK-1活性下降。

第三部分

1. 成功建立糖尿病大鼠模型。通过Morris水迷宫检测发现糖尿病大鼠表现出认知功能下降的行为学特点。在隐蔽平台实验中，与Con组（第3天：20.8±17.2s；第4天：12.9±6.0s；第5天：12.2±4.6s）相比，DM组第3天逃逸潜伏期（40.8±20.0s，P＜0.05）、第4天逃逸潜伏期（35.1±20.6s，P＜0.0001）、第5天逃逸潜伏期（28.1±21.0s，P＜0.05）延长。与Con组（第3天：461.4±493.9cm；第4天：234.9±172.2cm；第5天：223.1±141.6cm）相比，DM组第3天逃逸距离（936.4±571.1cm，P＜0.05）、第4天逃逸距离（852.6±629.8cm，P＜0.0001）、第5天逃逸距离（686.5±634.9cm，P＜0.01）延长；在探索试验中，与Con组（3.25±0.71次）相比，DM组大鼠穿越目标平台次数（1.17±0.41次）减少（P＜0.0001）；与Con组（28.7±3.6s）相比，DM组大鼠在目标平台象限内花费时间（17.9±3.9s）减少（P＜0.0001）。

2.  Morris水迷宫检测TXD和DAPK-1抑制剂（TC-DAPK6）干预对糖尿病大鼠空间学习、记忆能力影响：在隐蔽平台实验中，与DM组（第3天：40.8±20.0s；第4天：35.1±20.6s）相比，H-TXD组第3天（25.7±19.1s）、TC-DAPK6组第3天（25.6±18.7s）、TC-DAPK6组第4天（18.9±10.9s）逃逸潜伏期缩短（P＜0.05）；与DM组（第3天：936.4±571.1cm；第4天：852.6±629.8cm）相比，H-TXD组第3天（518.7±466.4cm）、H-TXD组第4天（436.3±369.4cm）、TC-DAPK6组第4天（399.6±287.6cm）逃逸潜伏期缩短（P＜0.05）。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间逃逸潜伏期与逃逸距离未见显著差异（P＞0.05）。

在空间探索试验中，与DM组（1.17±0.41次）相比，H-TXD组（3.17±0.75次, P＜0.0001）、M-TXD组（2.57±0.79次, P＜0.01）和TC-DAPK6组（2.67±0.52次, P＜0.01）穿越目标平台次数增加；与DM组（17.9±3.9s）相比，H-TXD组（25.1±3.3s, P＜0.01）、M-TXD组（23.4±2.6s, P＜0.05）和TC-DAPK6组（23.2±1.3s, P＜0.05）在目标平台象限内花费时间延长。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间穿越目标平台次数和在目标平台象限内花费时间未见显著差异（P＞0.05）。

3. 头颅核磁共振成像检测评估TXD、DAPK-1抑制剂（TC-DAPK6）干预对糖尿病大鼠海马体积影响：与Con组（147.13±5.24mm²）相比，DM组糖尿病大鼠的双侧海马体总体积（119.62±8.11mm²）减少（P＜0.05）。与DM组相比，H-TXD、M-TXD、L-TXD、TC-DAPK6干预对海马体体积改变无统计学意义（P＞0.05）。

4. 苏木素伊红染色、尼氏染色检测TXD、DAPK-1抑制剂（TC-DAPK6）干预对糖尿病大鼠海马体病理改变：与Con组相比，DM组大鼠病理切片显示海马体神经元分层不完整、细胞凋亡、肥大的星形细胞和颗粒变性。与DM组相比，各剂量TXD组和TC-DAPK6组大鼠大脑病理切片显示：海马结构更完整，分层更清晰，异常形态细胞更少。

与Con组（39.7±3.2个/FOV）相比，DM组（15.3±5.5个/FOV）大鼠海马体CA1区尼氏体数量减少（P＜0.001）。与DM组相比，H-TXD组（33.0±6.1个/FOV, P＜0.01）、M-TXD组（29.7±2.1个/FOV, P＜0.05）、L-TXD组（31.3±3.1个/FOV, P＜0.01）、TC-DAPK6组（30.0±4.0个/FOV, P＜0.05）大鼠海马体CA1区尼氏体数量增加。

5. 蛋白免疫印迹法检测TXD、DAPK-1抑制剂（TC-DAPK6）干预对糖尿病大鼠海马体蛋白表达影响：

与Con组（Aβ：0.14±0.05/β actin）相比，DM组大鼠海马体Aβ（0.53±0.15/β actin）表达水平增加（P＜0.01）。与DM组相比，H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组大鼠海马体Aβ表达水平无显著差异（P＞0.05）；H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体Aβ表达水平无显著差异（P＞0.05）。

与Con组（P-APP：0.15±0.05/APP）相比，DM组大鼠海马体P-APP（0.81±0.11/APP）表达水平增加（P＜0.001）。与DM组相比，H-TXD组（0.24±0.07/APP, P＜0.001）、TC-DAPK6组(0.35±0.08/APP, P＜0.01）大鼠海马体P-APP表达水平减少。与H-TXD组比，M-TXD组（0.55±0.17/APP）、L-TXD组（0.57±0.19/APP）大鼠海马体P-APP（Thr668）表达水平升高（P＜0.05）；TC-DAPK6组大鼠海马体P-APP（Thr668）表达水平无显著差异（P＞0.05）。

与Con组（P-Tau（Thr231）:0.44±0.24/Tau；P-Tau（Ser396）:0.27±0.15/Tau）相比，DM组大鼠海马体P-Tau（Thr231）（1.42±0.46/Tau，P＜0.01）、P-Tau（Ser396）（1.66±0.37/Tau，P＜0.0001）表达水平增加。与DM组相比，H-TXD组（P-Tau（Thr231）:0.51±0.10/Tau，P＜0.01；P-Tau（Ser396）:0.21±0.07/Tau，P＜0.0001）、M-TXD组（P-Tau（Thr231）:0.69±0.15/Tau，P＜0.05；P-Tau（Ser396）:0.46±0.25/Tau，P＜0.001）、L-TXD组（P-Tau（Thr231）:0.74±0.13/Tau，P＜0.05；P-Tau（Ser396）:0.52±0.18/Tau，P＜0.001）、TC-DAPK6组（P-Tau（Thr231）:0.41±0.18/Tau，P＜0.01；P-Tau（Ser396）:0.33±0.22/Tau，P＜0.001）大鼠海马体P-Tau表达水平减少。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体P-Tau表达水平无显著差异（P＞0.05）。

与Con组（0.50±0.15/β actin）相比，DM组大鼠海马体Caspase-3（1.14±0.27/β actin）表达水平增加（P＜0.01）。与DM组相比，H-TXD组（0.41±0.10/β actin，P＜0.001）、M-TXD组（0.51±0.11/β actin，P＜0.01）、L-TXD组（0.45±0.11/β actin，P＜0.01）、TC-DAPK6组（0.44±0.09/β actin，P＜0.01）大鼠海马体Caspase-3表达水平下降。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体Caspase-3表达水平无显著差异（P＞0.05）。

与Con组（0.55±0.14/β actin）相比，DM组糖尿病大鼠海马体T-DAPK-1（1.14±0.31/β actin）表达水平增加（P＜0.05）。与DM组相比，H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组大鼠海马体T-DAPK-1表达水平无显著差异（P＞0.05）。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体T-DAPK-1表达水平无显著差异（P＞0.05）。

与Con组（0.80±0.18/T-DAPK-1）相比，DM组大鼠海马体P-DAPK-1（Ser308）（0.14±0.05/T-DAPK-1）表达水平下降（P＜0.01），表明DAPK-1活性升高。与DM组相比，H-TXD组（0.59±0.09/T-DAPK-1，P＜0.05）、M-TXD组（0.52±0.18/T-DAPK-1，P＜0.05）、L-TXD组（0.55±0.15/T-DAPK-1，P＜0.05）、TC-DAPK6组（0.70±0.17/T-DAPK-1，P＜0.01）P-DAPK-1（Ser308）表达水平增加，表明DAPK-1活性下降。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体P-DAPK-1（Ser308）/T-DAPK-1表达无显著差异（P＞0.05）。

6. 免疫组织化学法检测TXD、TC-DAPK6干预对糖尿病大鼠海马体Bcl-2表达影响：与Con组（22.0±4.6个/FOV）相比，DM组大鼠海马体Bcl-2（6.3±1.2个/FOV）表达水平下降（P＜0.01）。与DM组相比，H-TXD组（24.3±3.1个/FOV，P＜0.01）、M-TXD组（22.0±3.6个/FOV，P＜0.01）、L-TXD组（18.0±5.0个/FOV，P＜0.05）、TC-DAPK6组（23.7±6.0个/FOV，P＜0.01）大鼠海马体Bcl-2表达水平升高。H-TXD组、M-TXD组、L-TXD组、TC-DAPK6组各组间大鼠海马体Bcl-2表达水平无显著差异（P＞0.05）。

【结论】

1.  DAPK-1参与糖尿病大鼠认知功能障碍的发生、发展。

2. TXD可降低高糖环境下海马神经元内DAPK-1的活性，提高神经元活性。TXD可通过降低糖尿病大鼠海马体的DAPK-1活性，下调P-APP、磷酸化Tau、Caspase-3表达水平，上调Bcl-2表达水平，从而保护海马神经元，保护糖尿病大鼠认知功能。与中、低剂量TXD组相比，在下调P-APP表达水平方面，高剂量TXD组效果最优。

【创新点】

本研究将DAPK-1引入DCI研究，观察到高糖下海马神经元及糖尿病大鼠海马体中DAPK-1的表达与活性升高，而抑制DAPK-1活性对高糖下海马神经元及糖尿病大鼠海马体具有保护作用。本研究发现TXD降低DAPK-1的活性从而保护海马神经元是TXD对糖尿病大鼠认知功能障碍的保护机制之一，为今后继续探索TXD防治DCI提供一定科研依据。

【Objective】

Diabetic Cognitive Impairment (DCI) is a chronic complication of diabetes, and its exact pathogenesis is still unclear. In DCI, the syndrome of "kidney deficiency and blood stasis, unhealthy brain" is the most common. Tu-Xian decoction (TXD) has the function of "tonifying kidney and blood circulation, benefiting brain and strengthening intelligence". Death-associated protein kinase 1 (DAPK-1) is involved in various neurodegenerative diseases. Through metabolomics, in vitro culture and animal experiments, this topic explores whether Tu-Xian decoction can protect cognitive impairment in diabetic rats by inhibiting DAPK-1.

【Methods】

Part 1

The main components of Tu-Xian decoction were identified by Ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) non-target metabolomics technology. Search the literature to check whether the main components in TXD have the function of regulating DAPK-1.

Part 2

The primary hippocampal neurons were isolated from newborn 24-hour SD rats, purified by lymphocyte separation medium density gradient centrifugation and differential adhesion method, and the neurons were divided into 4 groups: control group (Con, glucose content 25mmol/L), high glucose group (HG, glucose content 50mmol/L), Tu-Xian decoction group (TXD, glucose content 50mmol/L, set 0.1mg/mL, 1.0mg/mL, 5.0mg/mL, 10.0 mg/mL, 50.0mg/mL 5 TXD concentration gradients), DAPK-1 inhibitor group (TC-DAPK6, glucose content 50mmol/L, set 0.1μmol/L, 1.0μmol/L, 10.0μmol/L, 20.0μmol /L 4 concentration gradients of TC-DAPK6), the neurons were intervened in groups for 72 hours, and the relative activity of hippocampal neurons was measured by CCK-8 method. Divide the neurons into 3 groups: control group (Con), high glucose group (HG), and Tu-Xian decoction group (TXD, using the concentration of the group with the highest relative activity of neurons in the above CCK-8 test, that is, the optimal concentration intervention), after 72 hours of group intervention, the expression levels of T-DAPK-1 and P-DAPK-1 (Ser308) were determined by Western blotting.

Part 3

The diabetic rat model was established by high-sugar high-fat (HSHF) diet combined with 35 mg/Kg streptozotocin (STZ). Diabetic rats were randomly divided into: Diabetic (DM) group, DAPK-1 inhibitor (TC-DAPK6) group, high-dose Tu-Xian decoction (H-TXD, 12g/Kg) group, medium-dose Tu-Xian decoction (M-TXD, 6g/Kg) group, low-dose Tu-Xian decoction (L-TXD, 3g/Kg) group. Rats in the control (Con) group were fed with normal diet. After 12 weeks of group intervention, the cognitive function of the rats was detected by Morris water maze, the volume of the hippocampus of the rats was detected by cranial magnetic resonance imaging, the pathological changes of the brain were detected by hematoxylin and eosin staining and Nissl staining, and Aβ, APP, P-APP, Tau, P-Tau (Thr231), P-Tau (Ser396), Caspase-3, T-DAPK-1, P-DAPK-1 (Ser308) protein expression levels in the hippocampus of rats was detected by Western blotting. The expression level of Bcl-2 in rat hippocampus was detected by immunohistochemistry.

【Results】

Part 1

TXD was identified to contain 908 known compounds including flavonoids, phenols, terpenoids, phenylpropanes, alkaloids, aliphatic acylates, organic acids and their derivatives, sugars and their derivatives.

Part 2

1. The primary hippocampal neurons were isolated, and the purity was over 95% as identified by neuron-specific enolase (NSE) immunofluorescence.

2. After 72 hours of group intervention, the relative activity of neurons in the HG group was (79.23±5.62)%/Con, and the relative activity of neurons in each dose of Tu-Xian decoction group was:0.1mg/mL TXD group (79.83±4.02)%/Con; 1.0mg/mL TXD group (100.60±7.59)%/Con; 5.0mg/mL TXD group (116.03±6.99)%/Con; 10.0mg/mL TXD group (93.53±2.78)%/Con; 50.0mg/mL TXD group (92.17±5.07) %/Con. Compared with the Con group, the relative activity of neurons in the HG group decreased (P<0.01). Compared with the HG group, the relative activity of neurons in the 1.0mg/mL TXD group (P<0.01) and 5.0mg/mL TXD group (P<0.0001) increased.

After 72 hours of group intervention, the relative activity of neurons in each dose of DAPK-1 inhibitor group was: 0.1 μmol/L TC-DAPK6 group (87.23±3.08) %/Con; 1.0 μmol/L TC-DAPK6 group (92.08±2.81) %/Con; 10.0μmol/L TC-DAPK6 group (102.33±7.75) %/Con; 20.0μmol/L TC-DAPK6 group (85.80±6.35) %/Con. Compared with the HG group, the relative activity of neurons in the 10.0μmol/L TC-DAPK6 group increased (P<0.01).

3. Compared with Con group ((1.16±0.14)/β actin), the expression level of T-DAPK-1 in neurons of HG group ((1.97±0.14)/β actin) increased (P<0.05). Compared with Con group ((0.57±0.10)/T-DAPK-1), the expression level of P-DAPK-1 (Ser308) in neurons of HG group ((0.29±0.07)/T-DAPK-1) decreased (P<0.05), indicating that DAPK-1 activity increased. Compared with the HG group, there was no significant difference in the expression level of T-DAPK-1 ((1.83±0.16)/β actin) in the 5mg/mL TXD group (P>0.05), but P-DAPK-1 (Ser308) ((0.48±0.09)/T-DAPK-1) expression level in the 5mg/mL TXD group increased (P<0.05), indicating that DAPK-1 activity decreased.

Part 3

1. Successfully established a diabetic rat model. The behavioral characteristics of cognitive decline in diabetic rats were found by the Morris water maze test. In the hidden platform experiment, compared with the Con group (Day 3: 20.8±17.2s; Day 4: 12.9±6.0s; Day 5: 12.2±4.6s), the escape latency of the DM group on Days 3-5 ( Day 3: 40.8±20.0, P<0.05s; Day 4: 35.1±20.6s, P<0.0001; Day 5: 28.1±21.0s, P<0.05) increased ; compared with Con group (Day 3: 461.4±493.9cm; Day 4 : 234.9±172.2cm; Day 5: 223.1±141.6cm), the escape distance of the DM group on Days 3-5 (Day 3: 936.4±571.1cm, P<0.05; Day 4: 852.6±629.8cm, P<0.0001; Day 5: 686.5±634.9cm, P<0.01) increased. In the probe trial, compared with the Con group (3.25±0.71 times), the number of times of rats in the DM group crossed the target platform (1.17±0.41 times) decreased (P<0.05); Compared with Con group (28.7±3.6s), rats in DM group spent less time (17.9±3.9s) in the target platform quadrant (P<0.0001).

2. The Morris water maze was used to detect the effects of TXD and DAPK-1 inhibitor (TC-DAPK6) intervention on spatial learning and memory abilities of diabetic rats. In the hidden platform experiment, compared with the DM group (Day 3: 40.8±20.0s; Day 4: 35.1±20.6s), escape latency of H-TXD group on Day 3 (25.7±19.1s), TC-DAPK6 group on Day 3 (25.6±18.7s),TC-DAPK6 group on Day 4 (18.9±10.9s)  decreased (P<0.05); compared with the DM group (Day 3: 936.4±571.1cm; Day 4: 852.6±629.8cm), the escape latency of H-TXD group on Day 3 (518.7±466.4cm), H-TXD group on Day 4 (436.3±369.4cm), TC-DAPK6 group on Day 4 (399.6±287.6cm)  decreased (P<0.05). There were no significant differences in escape latency and escape distance among H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

In the probe trial, compared with the DM group (1.17±0.41 times), the times of crossing the target platform of the H-TXD group (3.17±0.75 times, P <0.0001), the M-TXD group (2.57±0.79 times, P<0.01) and the TC-DAPK6 group (2.67±0.52 times, P <0.01) increased; compared with the DM group (17.9±3.9s), rats in the H-TXD group (25.1±3.3s, P <0.01), the M-TXD group (23.4±2.6s, P <0.05) and the TC-DAPK6 group ( 23.2±1.3s, P <0.05) spent longer in the quadrant of the target platform. There were no significant differences in the number of crossing the target platform and the time spent in the quadrant of the target platform among H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

3. Brain magnetic resonance imaging was used to evaluate the effect of TXD and DAPK-1 inhibitor (TC-DAPK6) intervention on the hippocampal volume of diabetic rats: compared with the Con group (147.13±5.24mm²), the total hippocampus volume of diabetic rats in the DM group (119.62±8.11mm²) decreased (P<0.05). Compared with the DM group, H-TXD, M-TXD, L-TXD, and TC-DAPK6 intervention had no statistically significant changes in hippocampus volume (P>0.05).

4. Hematoxylin and eosin staining, Nissl staining to detect TXD, DAPK-1 inhibitor (TC-DAPK6) intervention in the pathological changes of the hippocampus of diabetic rats: compared with the Con group, the pathological sections of the rats in the DM group showed the distribution of neurons in the hippocampus, layer incompleteness, apoptosis, hypertrophic astrocytes, and granular degeneration. Compared with the DM group, the pathological sections of the brains of rats in each dose of TXD group and TC-DAPK6 group showed that the structure of the hippocampus were more complete, the layers were clearer, and the abnormal cells were less.

Compared with the Con group (39.7±3.2/FOV), the number of Nissl bodies in the CA1 area of rats in the DM group (15.3±5.5/FOV) decreased (P<0.001). Compared with the DM group, the number of Nissl bodies in the CA1 area of rats in the H-TXD group (33.0±6.1/FOV, P<0.01), the M-TXD group (29.7±2.1/FOV, P<0.05), the L-TXD group (31.3±3.1/FOV, P<0.01), TC-DAPK6 group (30.0±4.0/FOV, P<0.05) increased.

5. Western blotting to detect the effect of TXD and DAPK-1 inhibitor (TC-DAPK6) on protein expression in hippocampus of diabetic rats.

Compared with the Con group (Aβ: 0.14±0.05/β actin), the expression level of Aβ (0.53±0.15/β actin) in the hippocampus of rats in the DM group increased (P<0.01). Compared with the DM group, there were no significant differences in the expression level of Aβ in the hippocampus of rats in the H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05); There were no significant differences in the expression level of hippocampal Aβ among rats in the H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

Compared with the Con group (P-APP: 0.15±0.05/APP), the expression level of P-APP (0.81±0.11/APP) in the hippocampus of rats in the DM group increased (P<0.001). Compared with DM group, the expression level of P-APP in hippocampus of rats in H-TXD group (0.24±0.07/APP, P<0.001) and TC-DAPK6 group (0.35±0.08/APP, P<0.01) decreased. Compared with the H-TXD group, the expression levels of P-APP (Thr668) in the hippocampus of rats in the M-TXD (0.55±0.17/APP) and L-TXD (0.57±0.19/APP) groups increased (P<0.05), while the expression levels of P-APP (Thr668) of rats in the TC-DAPK6 group were not significantly different (P>0.05).

Compared with the Con group (P-Tau (Thr231): 0.44±0.24/Tau; P-Tau (Ser396): 0.27±0.15/Tau), the hippocampal P-Tau (Thr231) (1.42±0.46/Tau, P<0.01), P-Tau (Ser396) (1.66±0.37/Tau, P<0.0001) of the DM group rats increased. Compared with the DM group, the expression level of P-Tau in the hippocampus of the H-TXD group (P-Tau (Thr231): 0.51±0.10/Tau, P<0.01; P-Tau (Ser396): 0.21±0.07/Tau, P<0.0001), the M-TXD group (P-Tau (Thr231): 0.69±0.15/Tau, P<0.05; P-Tau (Ser396): 0.46±0.25/Tau, P<0.0001), L-TXD group (P-Tau (Thr231): 0.74±0.13/Tau, P<0.05; P-Tau (Ser396): 0.52±0.18/Tau, P<0.001 ), TC-DAPK6 group (P-Tau (Thr231): 0.41±0.18/Tau, P<0.01; P-Tau (Ser396): 0.33±0.22/Tau, P<0.001) decreased. There were no significant differences in the expression level of P-Tau in hippocampus among rats in H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

Compared with the Con group (0.50±0.15/β actin), the expression level of Caspase-3 (1.14±0.27/β actin) in the hippocampus of diabetic rats in the DM group increased (P<0.01). Compared with the DM group, the hippocampal Caspase-3 expression levels were decreased in the H-TXD (0.41±0.10/β actin, P<0.001), M-TXD (0.51±0.11/β actin, P<0.01), L-TXD (0.45±0.11/β actin, P<0.01) and TC-DAPK6 (0.44±0.09/β actin, P<0.01) groups of rats. There was no significant difference in the expression level of Caspase-3 in rat hippocampus among H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

Compared with the Con group (0.55±0.14/β actin), the expression level of T-DAPK-1 (1.14±0.31/β actin) in the hippocampus of rats in the DM group increased (P<0.05). Compared with the DM group, there were no significant differences in the expression level of T-DAPK-1 in the hippocampus of rats in the H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05). There were no significant differences in the expression level of T-DAPK-1 in rat hippocampus among H-TXD group, M-TXD group, L-TXD group and TC-DAPK6 group (P>0.05).

Compared with the Con group (0.80±0.18/T-DAPK-1), the expression level of P-DAPK-1 (Ser308) (0.14±0.05/T-DAPK-1) in the hippocampus of rats in the DM group decreased (P<0.05), indicating that increased DAPK-1 activity. Compared with DM group, the expression level of P-DAPK-1 (Ser308) in the hippocampus of rats in the H-TXD group (0.59±0.09/T-DAPK-1, P<0.05), M-TXD group (0.52±0.18/T-DAPK-1, P<0.05), L-TXD group (0.55±0.15/T-DAPK-1, P<0.05) TC-DAPK6 group (0.70±0.17/T-DAPK-1, P<0.01) increased, indicating that the  decreased DAPK-1 activity. There were no significant differences in the expression of P-DAPK-1 (Ser308)/T-DAPK-1 in hippocampus of rats among H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

6. Immunohistochemical detection of the effect of TXD and TC-DAPK6 on the expression of Bcl-2 in the hippocampus of diabetic rats: Compared with the Con group (22.0±4.6/FOV), the Bcl-2 level of the rats in the DM group (6.3±1.2/FOV) decreased (P<0.01). Compared with the DM group, the level of Bcl-2 in the hippocampus of H-TXD group (24.3±3.1/FOV, P<0.01), M-TXD group (22.0±3.6/FOV, P<0.01), L-TXD group (18.0±5.0/FOV, P<0.05), TC-DAPK6 group (23.7±6.0/FOV, P<0.01) rats increased. There were no significant differences in the expression of Bcl-2 in the hippocampus of diabetic rats among H-TXD group, M-TXD group, L-TXD group, and TC-DAPK6 group (P>0.05).

【Conclusion】

1. DAPK-1 is involved in the occurrence and development of cognitive impairment in diabetic rats.

2. TXD can reduce the activity of DAPK-1 in hippocampal neurons under high glucose environment, and increase the activity of neurons. TXD can protect hippocampal neurons and alleviate cognitive decline in diabetic rats by reducing DAPK-1 activity in the hippocampus of diabetic rats, down-regulating P-APP, phosphorylation Tau, and Caspase-3 levels, and up-regulating Bcl-2 levels. Compared with the medium and low dose TXD groups, the high dose TXD group had the best effect in down-regulating P-APP level.

【Innovation】

This study innovatively introduced DAPK-1 into DCI research, and observed that the activity and expression of DAPK-1 increased in hippocampal neurons under high glucose and diabetic rat hippocampus, and inhibiting the activity of DAPK-1 had significant effects on hippocampal neurons under high glucose and diabetic rats. This study found that TXD protects hippocampal neurons by reducing DAPK-1 activity, which is one of the protective mechanisms of TXD against cognitive impairment in diabetic rats, which lays the foundation for further digging into the scientific value of TXD in the future.