脾阴虚糖尿病认知功能障碍的发病机制及滋补脾阴方药的调控作用研究
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摘要
目前，糖尿病（Diabetes Mellitus，DM）和阿尔茨海默病（Alzheimer’s disease，AD）的发病率正以惊人的速度增加，已成为许多国家重大的公共健康问题。据报道2010年全球糖尿病患者高达2.85亿，2030年将增加到4.39亿，其中95%为T2DM。我国糖尿病的发病率更是增长迅速，据统计中国的成人糖尿病患者已达9200万，糖尿病前期患者人数高达1.48亿，位居世界第二。随着对糖尿病研究的逐步深入，人们逐渐认识到糖尿病患者可出现认知功能障碍，甚至发生AD。流行病学研究也表明糖尿病患者罹患AD的风险更高。调查发现受试者人群中糖尿病患者形成痴呆的比例是普通人群的两倍，糖尿病患者可提前3年出现痴呆。对此，科学界提出了“糖尿病相关认知下降（diabetes-associated cognitive decline，DACD）”这一概念，或称为糖尿病认知功能障碍。
DACD的发病机制极其复杂，可能与脑胰岛素抵抗、脑血管及血管内皮功能损伤、氧化应激、非酶性蛋白糖基化、炎性反应以及钙离子稳态失衡等多种因素有关。虽然传统观念认为胰岛素的敏感器官主要是肝脏、脂肪以及骨骼肌等外周组织，但是在海马和皮质这些与学习记忆形成密切相关的部位也发现了胰岛素受体。虽然脑内胰岛素的来源尚不完全清楚，但已经证明来自外周循环的胰岛素可通过血脑屏障进入脑内，脑内也可内源性地产生胰岛素。胰岛素通过与脑内广泛分布的胰岛素受体结合激活PI3K/Akt通路，引导胰岛素信号向下传导。正常情况下，胰岛素与胰岛素受体结合后，引起胰岛素受体底物1（insulin receptor substrate1， IRS-1）的酪氨酸磷酸化增强，继而激活其下游磷脂酰肌醇3-激酶（phosphatidylinositol kinase-3， PI3K），诱导蛋白激酶B（protein kinase B， PKB/Akt）的磷酸化，这一途径中任一分子发生异常都可能阻碍胰岛素信号向下传导，引起胰岛素信号转导障碍，导致胰岛素效能下降，诱发胰岛素抵抗。
引起胰岛素抵抗的原因是多方面的。据报道能量代谢紊乱、氧化应激等因素都可能引起胰岛素信号转导障碍，诱发胰岛素抵抗。此外，内质网应激（endoplasmicreticulum stress，ERS）也可以通过引起JNK的活化抑制胰岛素信号转导。内质网(endoplasmic reticulum，ER)是位于细胞核附近的胞质区域，是哺乳动物细胞重要的Ca2+贮存器，同时也是蛋白质合成与翻译后修饰，多肽链正确折叠与装配的重要场所。ERS是细胞的一种应激反应过程。糖饥饿、钙平衡紊乱、糖基化抑制、二硫键合成减少时，内质网微环境改变，蛋白质折叠受到影响，导致大量未折叠或错误折叠的蛋白质堆积于内质网内，引起未折叠蛋白反应（unfolded proteinresponse，UPR）。但是，关于ERS在DACD发病机制中的作用尚未见报道。
DACD的确切病理变化尚不清楚。但已有研究表明DACD脑中病理变化与AD相似。T2DM患者脑组织存在淀粉样斑块和神经纤维缠结，淀粉样斑块的密度与T2DM病程密切相关。动物实验研究也证实糖尿病鼠不但存在学习、记忆障碍，其脑部神经元也出现了AD的特异性病理学改变： 淀粉样蛋白（β-amyloid peptide,Aβ）沉积及tau的异常磷酸化，及由其导致的神经元细胞退化。在自发性糖尿病大鼠脑组织中发现额皮质APP、β-分泌酶和Aβ蛋白表达增加，并在大鼠神经元胞浆中发现Aβ沉积增加。
有研究表明胰岛素信号转导障碍可以影响Aβ的产生和降解。胰岛素信号通路的下游效应器之一雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)作为自噬的抑制剂，在淀粉样蛋白β（Aβ）的代谢中发挥重要作用。胰岛素降解酶（insulindegrading enzyme, IDE）是Aβ的主要降解酶之一，它在降解胰岛素的同时可降解Aβ。
课题组前期研究发现，糖尿病大鼠及脾阴虚糖尿病大鼠海马中发生ERS，胰岛素信号通路关键分子IRS-1丝氨酸磷酸化水平增加，说明胰岛素信号转导受阻，糖尿病大鼠及脾阴虚糖尿病大鼠海马中发生了胰岛素抵抗。但是，胰岛素抵抗是否是由ERS诱导的仍需进一步研究。同时，我们发现糖尿病大鼠及脾阴虚糖尿病大鼠海马中总Aβ增加，虽然已有证据表明胰岛素与Aβ代谢之间密切相关，但是在我们的研究中Aβ增加是否与胰岛素信号转导障碍有关仍需进一步证实。因此，本研究在前期工作基础上，通过观察与学习记忆密切相关的皮质、与机体代谢密切相关的下丘脑中ERS及胰岛素信号通路的变化，结合体外实验，进一步深入探讨ERS与胰岛素信号转导障碍之间的关系，以及胰岛素信号转导障碍对Aβ代谢的影响。
目的：以脑内质网应激激活JNK诱导脑胰岛素信号转导障碍为切入点，探讨糖尿病认知功能障碍的发病机制；通过观察ZBPYR对内质网应激、胰岛素信号转导通路关键分子及Aβ的影响探讨ZBPYR的作用机制，明确ZBPYR的作用靶点，为从脾论治糖尿病认知功能障碍提供理论依据。通过中医学理论与现代医学的交融，丰富完善脾藏象理论及脾本质研究，为糖尿病认知功能障碍的防治提供新线索和新思路。
方法：将健康成年雄性SD大鼠随机分为空白对照组（cont）、糖尿病组（DM）、脾阴虚组（pi）、脾阴虚糖尿病组（piDM）和滋补脾阴方药组（ZBPYR），共5组。
1.在成功建立各种模型后，首先通过western blotting方法及RT-PCR方法观察各组大鼠皮质、下丘脑中内质网应激标志分子p-PERK、p-eIF2α、eIF2α、GRP78蛋白表达变化及GRP78mRNA水平变化，以验证各组大鼠皮质和下丘脑中是否发生内质网应激。
2.通过western blotting方法观察各组大鼠皮质、下丘脑中ERS与胰岛素信号通路桥梁分子JNK的变化及胰岛素信号转导通路关键分子p-IRS-1、IRS-1、p-Akt、Akt的变化，以验证各组大鼠皮质和下丘脑中是否发生胰岛素信号转导障碍。
3.以SH-SY5Y细胞为研究对象，给予内质网应激诱导剂衣霉素、毒胡萝卜素刺激诱导内质网应激的发生，然后再给予JNK抑制剂SP600125，观察胰岛素信号转导通路关键分子p-IRS-1、IRS-1、p-Akt、Akt的变化，以验证内质网应激是否可通过激活JNK诱导胰岛素信号转导障碍。
4.观察各组大鼠海马、皮质Aβ1-42及Aβ1-40的变化，及p-mTOR、mTOR、p-P70S6K、P70S6K、自噬标志分子LC3Ⅱ以及IDE的变化寻找Aβ1-42及Aβ1-40的变化的原因。
结果：
1. DM组、piDM组大鼠皮质、下丘脑p-PERK、p-eIF2α蛋白表达较cont组均增强（P<0.05），ZBPYR组较DM组、piDM组减弱（P<0.05）；DM组、piDM组GRP78蛋白表达及mRNA表达较cont组增加（P<0.05），ZBPYR组较DM组、piDM组减弱（P<0.05），eIF2α蛋白水平各组间差异无统计学意义。
2. DM组、piDM组大鼠皮质、下丘脑内质网应激标志分子p-IRE1α、p-JNK1、p-JNK2以及胰岛素信号转导通路关键分子p-IRS-1、p-Akt蛋白表达较cont组均增加（P<0.05），ZBPYR组较DM组、piDM组均减弱（P<0.05），IRS-1、Akt蛋白水平各组间差异无统计学意义。
3.体外实验结果显示Tm处理4h、Tg处理12h时p-IRE1α、p-JNK1、p-JNK2表达达到峰值，给予Tm处理4h或Tg处理12h时胰岛素信号转导通路中的p-IRS-1增加、再给予JNK抑制剂SP600125处理后抑制p-IRS-1的表达，p-Akt蛋白表达增加。
4. DM组、piDM组大鼠海马、皮质可溶性与不可溶Aβ1-42均高于cont组，ZBPYR降低了DM组和或piDM组海马、皮质可溶性与不可溶Aβ1-42的表达，DM组、pi组、piDM组大鼠皮质不可溶Aβ1-42均高于cont组，ZBPYR降低了DM组和piDM组皮质不可溶Aβ1-42的表达。各组大鼠海马、皮质p-mTOR、mTOR、p-P70S6K、P70S6K表达差异无统计学意义，但DM组、piDM组各组大鼠海马、皮质自噬标志分子LC3Ⅱ蛋白表达降低，IDE蛋白表达降低，而ZBPYR则抑制LC3Ⅱ以及IDE的降低。
结论：
1.内质网应激的发生是糖尿病、脾阴虚、脾阴虚糖尿病大鼠脑组织变化的特征之一，是诱导脑内胰岛素信号转导障碍的始动因素。滋补脾阴方药通过干扰PERK信号途径减轻脑内质网应激。
2.内质网应激通过激活JNK诱导胰岛素信号转导障碍促进糖尿病大鼠及脾阴虚糖尿病组大鼠认知功能障碍的发生。滋补脾阴方药通过调节内质网应激抑制JNK活化改善胰岛素信号转导障碍提高学习记忆能力。
3.脑组织中Aβ1-42增加可能是糖尿病大鼠、脾阴虚糖尿病大鼠认知功能障碍的主要病理变化，自噬及胰岛素降解酶可能参与调控Aβ1-42的降解。滋补脾阴方药能够减少Aβ1-42，其机制可能与调节自噬及IDE的表达有关。
At present, the incidence of Diabetes Mellitus(DM) and Alzheimer's disease (AD)is increasing. DM and AD have become major public health problems in manycountries. It was reported that the number of DM patients is up to439million in2030,which was285million to in2010, of which95%is T2DM. The incidence of DM inChina is growing rapidly, there are92million adults DM patients, the number ofpre-diabetes patients is148million, ranking second in the world. With the gradualdeepening of diabetes research, people realized that DM may be associated withcognitive impairment or AD. Epidemiological studies have shown that DM patients areeasier to suffer from AD. The survey found that the ratio of DM patients developingdementia in subjects population is doubled, compared with general population, the formof dementia of DM patients is three years in advance. In this regard, the scientificcommunity suggested a new conception termed diabetes-associated cognitive decline(DACD).
The pathogenesis of DACD is extremely complex, it may be associated withinsulin resistance in brain, cerebrovascular and vascular endothelial dysfunction,oxidative stress, non-enzymatic protein glycosylation, inflammation and calciumhomeostasis and other factors. Although the traditional concept is that the liver,skeletalmuscle and adipose tissue are insulin-sensitive organs, but it is worth noting that, insulinreceptor are found in the hippocampus and cortex which are closely related to learningand memory formation. Although the source of brain insulin is not yet entirely clear, ithas been proven that insulin from the peripheral circulation could enter the brainthrough the blood-brain barrier, insulin may be also produced in the brain. ThePI3K/Akt pathway is activated followed by insulin binding insulin receptor and inducing the insulin signaling. Under normal circumstances, insulin and insulin receptorbinding could enhance the level of tyrosine phosphorylation of insulin receptor substrate1(IRS-1), then activate downstream phosphatidylinositol3-kinase (PI3K), induceprotein kinase B (protein kinase B, PKB/Akt) phosphorylation. Any factor of thispathway disordered will induce insulin signaling transduction abnormally, leading toinsulin resistance.
Many reasons could induce insulin resistance. It is reported that the disorder ofenergy metabolism, oxidative stress and other factors may cause the insulin signalingtransduction abnormally, inducing insulin resistance. In addition, the endoplasmicreticulum stress (ERS) could inhibit insulin signaling by activating JNK in liver FAOcells. The endoplasmic reticulum (ER) is located in the cytoplasmic region near thenucleus, which is a organelle related with Ca2+storage, protein synthesis andpost-translational modification in mammalian cells. ERS is a cell stress responseprocess. Sugar starvation, calcium balance disorders, glycosylation inhibition, reductionof disulfide synthesis and the endoplasmic reticulum microenvironment changes couldaffect protein folding, resulting in the accumulation of unfolded or misfolded proteins inthe endoplasmic reticulum, causing unfolded protein response (UPR). However, the roleof ERS in DACD pathogenesis has not been reported.
The pathology of DACD is unclear. Studies have shown that the pathology ofDACD in brain is similar to AD. There was amyloid plaques and neurofibrillary tanglesin brain of T2DM patients. And density of amyloid plaque is associated with T2DMduration. Animal studies also confirmed that the diabetic rats not only express learningand memory dysfunction, Aβd eposition and tau phosphorylation which are AD-specificpathological changes were observed in brain of diabetic rats. The APP in frontal cortex,β-secretion enzymes and Aβ was increased in brain tissue of spontaneous diabetic rats,and Aβ deposition was also found in neurons.
It is reported that insulin signaling may affect the metabolism of Aβ. Mammaliantarget of rapamycin (mTOR) which is a downstream effector of insulin signalingpathway, as an inhibitor of autophagy, play important roles in the metabolism of Aβ.Insulin degrading enzyme (IDE) is a degrading enzyme for Aβ, studies have shown thatdegradation of insulin at the same time biodegradable Aβ.
Our group has found that the presence of ERS and including IRS-1serinephosphorylation increased which is a key molecule of insulin signaling pathway inhippocampus of diabetic rats and spleen Yin deficiency diabetic rats, suggesting insulin signalling transduction has been blocked, insulin resistance was induced inhippocampus of diabetic rats and spleen Yin deficiency diabetic rats. However, whetherthe insulin resistance is induced by the ERS still need to study. We also found total Aβexpression increased in hippocampus of diabetic rats and spleen Yin deficiency diabeticrats. There are evidences that insulin is closely related with metabolism of Aβ, but inour study whether Aβ increased is related with insulin signalling transduction barriersstill need to be confirmed. Therefore, based on previous work, we will observe ERS andthe insulin signaling pathway in cortex which is related to learning and memory andhypothalamus which is closely related to the metabolism, combineing with experimentsin vitro to further explore the relationship between ERS and insulin signallingtransduction obstacles, and the effects of insulin signalling transduction barriers on Aβ.
Objective：Using the endoplasmic reticulum stress activating JNK induced insulinsignaling transduction obstacles in brain as the starting point to explore the pathogenesisof diabetes-related cognitive decline; clearling the targets of ZBPYR through observingthe effects of ZBPYR on endoplasmic reticulum stress, key molecules of insulinsignaling pathway and Aβ, providing theoretical basis of treatment from spleen ofdiabetes-related cognitive decline. Through a blend of traditional Chinese medicinetheory and western medicine, enriched and improved the spleen theory of visceralmanifestation, providing new clues and new ideas for prevention and treatment ofdiabetes-related cognitive decline.
Methods： Healthy adult male SD rats were randomly divided into cont group,DM group, pi group, piDM group and ZBPYR group, a total of five groups.
1We first observed the protein expression of endoplasmic reticulum stress markersinculding p-PERK, p-eIF2α, eIF2α, GRP78and GRP78mRNA levels by westernblotting and RT-PCR in cerebral cortex, hypothalamus in a variety of models,, in orderto verify whether endoplasmic reticulum stress existed in the cortex and hypothalamusof rats.
2Then we observed the expression of JNK which is the bridge molecule of ERSand insulin signaling pathway, and insulin signaling transduction pathway criticalmolecules p-IRS-1, IRS-1, p-Akt, Akt in cortex, the hypothalamus of rats by westernblotting, to verify whether insulin signaling transduction obstacles occers in cortex andhypothalamus.
3The SH-SY5Y cell was treated with tunicamycin and thapsigargin which areendoplasmic reticulum stress inducer to induce endoplasmic reticulum stress. Then cell was treated with the JNK inhibitor SP600125.We observed the changes of keymolecules of insulin signaling transduction pathways including p-IRS-1, IRS-1andp-Akt, Akt to verify whether endoplasmic reticulum stress could induce insulinsignaling transduction barriers through activating JNK.
4We detected the changes of Aβ1-42and Aβ1-40in hippocampal and cortex, thenobserved the changes of p-mTOR, mTOR, p-P70S6K, P70S6K, LC3II which is amarker of autophagy and IDE to find the reason of changes of Aβ1-42and Aβ1-40.
Results：
1. The protein expression of p-PERK, p-eIF2α of DM group and piDM group ratsincreased than the cont group in cortex, hypothalamus (P <0.05), ZBPYR group reducedthan DM group, piDM group (P <0.05); GRP78protein expression and mRNAexpression of the DM group and piDM group of both increased than cont group (P<0.05). ZBPYR decreased the expression of GRP78than DM group and piDM group(P<0.05), the difference of protein levels of eIF2α in each group was not statisticallysignificant.
2. The proteins expression of the endoplasmic reticulum stress markers p-IRE1α,p-JNK1, p-JNK2, and critical molecular of insulin signaling pathway p-IRS-1increasedin DM group, piDM group than cont group (P <0.05), p-Akt decreased in DM groupand piDM group. The expression of p-IRE1α, p-JNK1, p-JNK2, p-IRS-1of ZBPYRgroup reduced than DM group, piDM group (P <0.05), the expression of p-Akt ofZBPYR group increased. The differences of protein levels of IRS-1, and Akt amonggroups was not statistically significant.
3. The expression of p-IRE1α, p-JNK1, p-JNK2was the strongest when Tmtreatment for4h, Tg treatment for12h in vitro, when expression of p-IRS-1increased.Cells were treated with JNK inhibitor SP600125inhibited the expression of the p-IRS-1,increased expression of p-Akt.
4. The soluble and insoluble Aβ1-42of DM group and piDM group were increasedthan cont group in hippocampus and cortex, ZBPYR reduced soluble and insolubleAβ1-42than DM group and, or piDM group in hippocampus and cortex. The corticalinsoluble Aβ1-42of DM group, pi group and piDM group were increased than cont group,ZBPYR reduced insoluble a Aβ1-42in cortical. The differences of protein levels ofp-mTOR, mTOR, p-P70S6K, P70S6K was not significant among groups inhippocampus and cortex, but the protein expression of LC3II which is autophagymarker of DM group and piDM group decreased in hippocampus and cortex, the protein expression of IDE also decreased, ZBPYR inhibited the reduction of LC3II and IDE.
Conclusions:
1.Endoplasmic reticulum stress in brain is one of the characteristics of diabetic rats,spleen deficiency rats and spleen Yin deficiency diabetic rats, which is initiating insulinsignaling transduction obstacles. ZBPYR could reduce endoplasmic reticulum stress byinterfering PERK signaling in brain.
2. Endoplasmic reticulum stress induced insulin signaling transduction barriersthrough the activating JNK, which could promote cognitive decline occur in diabeticrats and spleen Yin deficiency diabetic rats, ZBPYR could improve the ability oflearning and memory by improving insulin signaling which is regulated by JNKactivated by ERS.
3. Aβ1-42increased in brain is a major characteristic of diabetic rats and spleen Yindeficiency diabetic rats, which is degradated by autophagy and IDE. ZBPYR reducedAβ1-42through regulating the expression of autophagy and IDE.
DACD的发病机制极其复杂，可能与脑胰岛素抵抗、脑血管及血管内皮功能损伤、氧化应激、非酶性蛋白糖基化、炎性反应以及钙离子稳态失衡等多种因素有关。虽然传统观念认为胰岛素的敏感器官主要是肝脏、脂肪以及骨骼肌等外周组织，但是在海马和皮质这些与学习记忆形成密切相关的部位也发现了胰岛素受体。虽然脑内胰岛素的来源尚不完全清楚，但已经证明来自外周循环的胰岛素可通过血脑屏障进入脑内，脑内也可内源性地产生胰岛素。胰岛素通过与脑内广泛分布的胰岛素受体结合激活PI3K/Akt通路，引导胰岛素信号向下传导。正常情况下，胰岛素与胰岛素受体结合后，引起胰岛素受体底物1（insulin receptor substrate1， IRS-1）的酪氨酸磷酸化增强，继而激活其下游磷脂酰肌醇3-激酶（phosphatidylinositol kinase-3， PI3K），诱导蛋白激酶B（protein kinase B， PKB/Akt）的磷酸化，这一途径中任一分子发生异常都可能阻碍胰岛素信号向下传导，引起胰岛素信号转导障碍，导致胰岛素效能下降，诱发胰岛素抵抗。
引起胰岛素抵抗的原因是多方面的。据报道能量代谢紊乱、氧化应激等因素都可能引起胰岛素信号转导障碍，诱发胰岛素抵抗。此外，内质网应激（endoplasmicreticulum stress，ERS）也可以通过引起JNK的活化抑制胰岛素信号转导。内质网(endoplasmic reticulum，ER)是位于细胞核附近的胞质区域，是哺乳动物细胞重要的Ca2+贮存器，同时也是蛋白质合成与翻译后修饰，多肽链正确折叠与装配的重要场所。ERS是细胞的一种应激反应过程。糖饥饿、钙平衡紊乱、糖基化抑制、二硫键合成减少时，内质网微环境改变，蛋白质折叠受到影响，导致大量未折叠或错误折叠的蛋白质堆积于内质网内，引起未折叠蛋白反应（unfolded proteinresponse，UPR）。但是，关于ERS在DACD发病机制中的作用尚未见报道。
DACD的确切病理变化尚不清楚。但已有研究表明DACD脑中病理变化与AD相似。T2DM患者脑组织存在淀粉样斑块和神经纤维缠结，淀粉样斑块的密度与T2DM病程密切相关。动物实验研究也证实糖尿病鼠不但存在学习、记忆障碍，其脑部神经元也出现了AD的特异性病理学改变： 淀粉样蛋白（β-amyloid peptide,Aβ）沉积及tau的异常磷酸化，及由其导致的神经元细胞退化。在自发性糖尿病大鼠脑组织中发现额皮质APP、β-分泌酶和Aβ蛋白表达增加，并在大鼠神经元胞浆中发现Aβ沉积增加。
有研究表明胰岛素信号转导障碍可以影响Aβ的产生和降解。胰岛素信号通路的下游效应器之一雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)作为自噬的抑制剂，在淀粉样蛋白β（Aβ）的代谢中发挥重要作用。胰岛素降解酶（insulindegrading enzyme, IDE）是Aβ的主要降解酶之一，它在降解胰岛素的同时可降解Aβ。
课题组前期研究发现，糖尿病大鼠及脾阴虚糖尿病大鼠海马中发生ERS，胰岛素信号通路关键分子IRS-1丝氨酸磷酸化水平增加，说明胰岛素信号转导受阻，糖尿病大鼠及脾阴虚糖尿病大鼠海马中发生了胰岛素抵抗。但是，胰岛素抵抗是否是由ERS诱导的仍需进一步研究。同时，我们发现糖尿病大鼠及脾阴虚糖尿病大鼠海马中总Aβ增加，虽然已有证据表明胰岛素与Aβ代谢之间密切相关，但是在我们的研究中Aβ增加是否与胰岛素信号转导障碍有关仍需进一步证实。因此，本研究在前期工作基础上，通过观察与学习记忆密切相关的皮质、与机体代谢密切相关的下丘脑中ERS及胰岛素信号通路的变化，结合体外实验，进一步深入探讨ERS与胰岛素信号转导障碍之间的关系，以及胰岛素信号转导障碍对Aβ代谢的影响。
目的：以脑内质网应激激活JNK诱导脑胰岛素信号转导障碍为切入点，探讨糖尿病认知功能障碍的发病机制；通过观察ZBPYR对内质网应激、胰岛素信号转导通路关键分子及Aβ的影响探讨ZBPYR的作用机制，明确ZBPYR的作用靶点，为从脾论治糖尿病认知功能障碍提供理论依据。通过中医学理论与现代医学的交融，丰富完善脾藏象理论及脾本质研究，为糖尿病认知功能障碍的防治提供新线索和新思路。
方法：将健康成年雄性SD大鼠随机分为空白对照组（cont）、糖尿病组（DM）、脾阴虚组（pi）、脾阴虚糖尿病组（piDM）和滋补脾阴方药组（ZBPYR），共5组。
1.在成功建立各种模型后，首先通过western blotting方法及RT-PCR方法观察各组大鼠皮质、下丘脑中内质网应激标志分子p-PERK、p-eIF2α、eIF2α、GRP78蛋白表达变化及GRP78mRNA水平变化，以验证各组大鼠皮质和下丘脑中是否发生内质网应激。
2.通过western blotting方法观察各组大鼠皮质、下丘脑中ERS与胰岛素信号通路桥梁分子JNK的变化及胰岛素信号转导通路关键分子p-IRS-1、IRS-1、p-Akt、Akt的变化，以验证各组大鼠皮质和下丘脑中是否发生胰岛素信号转导障碍。
3.以SH-SY5Y细胞为研究对象，给予内质网应激诱导剂衣霉素、毒胡萝卜素刺激诱导内质网应激的发生，然后再给予JNK抑制剂SP600125，观察胰岛素信号转导通路关键分子p-IRS-1、IRS-1、p-Akt、Akt的变化，以验证内质网应激是否可通过激活JNK诱导胰岛素信号转导障碍。
4.观察各组大鼠海马、皮质Aβ1-42及Aβ1-40的变化，及p-mTOR、mTOR、p-P70S6K、P70S6K、自噬标志分子LC3Ⅱ以及IDE的变化寻找Aβ1-42及Aβ1-40的变化的原因。
结果：
1. DM组、piDM组大鼠皮质、下丘脑p-PERK、p-eIF2α蛋白表达较cont组均增强（P<0.05），ZBPYR组较DM组、piDM组减弱（P<0.05）；DM组、piDM组GRP78蛋白表达及mRNA表达较cont组增加（P<0.05），ZBPYR组较DM组、piDM组减弱（P<0.05），eIF2α蛋白水平各组间差异无统计学意义。
2. DM组、piDM组大鼠皮质、下丘脑内质网应激标志分子p-IRE1α、p-JNK1、p-JNK2以及胰岛素信号转导通路关键分子p-IRS-1、p-Akt蛋白表达较cont组均增加（P<0.05），ZBPYR组较DM组、piDM组均减弱（P<0.05），IRS-1、Akt蛋白水平各组间差异无统计学意义。
3.体外实验结果显示Tm处理4h、Tg处理12h时p-IRE1α、p-JNK1、p-JNK2表达达到峰值，给予Tm处理4h或Tg处理12h时胰岛素信号转导通路中的p-IRS-1增加、再给予JNK抑制剂SP600125处理后抑制p-IRS-1的表达，p-Akt蛋白表达增加。
4. DM组、piDM组大鼠海马、皮质可溶性与不可溶Aβ1-42均高于cont组，ZBPYR降低了DM组和或piDM组海马、皮质可溶性与不可溶Aβ1-42的表达，DM组、pi组、piDM组大鼠皮质不可溶Aβ1-42均高于cont组，ZBPYR降低了DM组和piDM组皮质不可溶Aβ1-42的表达。各组大鼠海马、皮质p-mTOR、mTOR、p-P70S6K、P70S6K表达差异无统计学意义，但DM组、piDM组各组大鼠海马、皮质自噬标志分子LC3Ⅱ蛋白表达降低，IDE蛋白表达降低，而ZBPYR则抑制LC3Ⅱ以及IDE的降低。
结论：
1.内质网应激的发生是糖尿病、脾阴虚、脾阴虚糖尿病大鼠脑组织变化的特征之一，是诱导脑内胰岛素信号转导障碍的始动因素。滋补脾阴方药通过干扰PERK信号途径减轻脑内质网应激。
2.内质网应激通过激活JNK诱导胰岛素信号转导障碍促进糖尿病大鼠及脾阴虚糖尿病组大鼠认知功能障碍的发生。滋补脾阴方药通过调节内质网应激抑制JNK活化改善胰岛素信号转导障碍提高学习记忆能力。
3.脑组织中Aβ1-42增加可能是糖尿病大鼠、脾阴虚糖尿病大鼠认知功能障碍的主要病理变化，自噬及胰岛素降解酶可能参与调控Aβ1-42的降解。滋补脾阴方药能够减少Aβ1-42，其机制可能与调节自噬及IDE的表达有关。
At present, the incidence of Diabetes Mellitus(DM) and Alzheimer's disease (AD)is increasing. DM and AD have become major public health problems in manycountries. It was reported that the number of DM patients is up to439million in2030,which was285million to in2010, of which95%is T2DM. The incidence of DM inChina is growing rapidly, there are92million adults DM patients, the number ofpre-diabetes patients is148million, ranking second in the world. With the gradualdeepening of diabetes research, people realized that DM may be associated withcognitive impairment or AD. Epidemiological studies have shown that DM patients areeasier to suffer from AD. The survey found that the ratio of DM patients developingdementia in subjects population is doubled, compared with general population, the formof dementia of DM patients is three years in advance. In this regard, the scientificcommunity suggested a new conception termed diabetes-associated cognitive decline(DACD).
The pathogenesis of DACD is extremely complex, it may be associated withinsulin resistance in brain, cerebrovascular and vascular endothelial dysfunction,oxidative stress, non-enzymatic protein glycosylation, inflammation and calciumhomeostasis and other factors. Although the traditional concept is that the liver,skeletalmuscle and adipose tissue are insulin-sensitive organs, but it is worth noting that, insulinreceptor are found in the hippocampus and cortex which are closely related to learningand memory formation. Although the source of brain insulin is not yet entirely clear, ithas been proven that insulin from the peripheral circulation could enter the brainthrough the blood-brain barrier, insulin may be also produced in the brain. ThePI3K/Akt pathway is activated followed by insulin binding insulin receptor and inducing the insulin signaling. Under normal circumstances, insulin and insulin receptorbinding could enhance the level of tyrosine phosphorylation of insulin receptor substrate1(IRS-1), then activate downstream phosphatidylinositol3-kinase (PI3K), induceprotein kinase B (protein kinase B, PKB/Akt) phosphorylation. Any factor of thispathway disordered will induce insulin signaling transduction abnormally, leading toinsulin resistance.
Many reasons could induce insulin resistance. It is reported that the disorder ofenergy metabolism, oxidative stress and other factors may cause the insulin signalingtransduction abnormally, inducing insulin resistance. In addition, the endoplasmicreticulum stress (ERS) could inhibit insulin signaling by activating JNK in liver FAOcells. The endoplasmic reticulum (ER) is located in the cytoplasmic region near thenucleus, which is a organelle related with Ca2+storage, protein synthesis andpost-translational modification in mammalian cells. ERS is a cell stress responseprocess. Sugar starvation, calcium balance disorders, glycosylation inhibition, reductionof disulfide synthesis and the endoplasmic reticulum microenvironment changes couldaffect protein folding, resulting in the accumulation of unfolded or misfolded proteins inthe endoplasmic reticulum, causing unfolded protein response (UPR). However, the roleof ERS in DACD pathogenesis has not been reported.
The pathology of DACD is unclear. Studies have shown that the pathology ofDACD in brain is similar to AD. There was amyloid plaques and neurofibrillary tanglesin brain of T2DM patients. And density of amyloid plaque is associated with T2DMduration. Animal studies also confirmed that the diabetic rats not only express learningand memory dysfunction, Aβd eposition and tau phosphorylation which are AD-specificpathological changes were observed in brain of diabetic rats. The APP in frontal cortex,β-secretion enzymes and Aβ was increased in brain tissue of spontaneous diabetic rats,and Aβ deposition was also found in neurons.
It is reported that insulin signaling may affect the metabolism of Aβ. Mammaliantarget of rapamycin (mTOR) which is a downstream effector of insulin signalingpathway, as an inhibitor of autophagy, play important roles in the metabolism of Aβ.Insulin degrading enzyme (IDE) is a degrading enzyme for Aβ, studies have shown thatdegradation of insulin at the same time biodegradable Aβ.
Our group has found that the presence of ERS and including IRS-1serinephosphorylation increased which is a key molecule of insulin signaling pathway inhippocampus of diabetic rats and spleen Yin deficiency diabetic rats, suggesting insulin signalling transduction has been blocked, insulin resistance was induced inhippocampus of diabetic rats and spleen Yin deficiency diabetic rats. However, whetherthe insulin resistance is induced by the ERS still need to study. We also found total Aβexpression increased in hippocampus of diabetic rats and spleen Yin deficiency diabeticrats. There are evidences that insulin is closely related with metabolism of Aβ, but inour study whether Aβ increased is related with insulin signalling transduction barriersstill need to be confirmed. Therefore, based on previous work, we will observe ERS andthe insulin signaling pathway in cortex which is related to learning and memory andhypothalamus which is closely related to the metabolism, combineing with experimentsin vitro to further explore the relationship between ERS and insulin signallingtransduction obstacles, and the effects of insulin signalling transduction barriers on Aβ.
Objective：Using the endoplasmic reticulum stress activating JNK induced insulinsignaling transduction obstacles in brain as the starting point to explore the pathogenesisof diabetes-related cognitive decline; clearling the targets of ZBPYR through observingthe effects of ZBPYR on endoplasmic reticulum stress, key molecules of insulinsignaling pathway and Aβ, providing theoretical basis of treatment from spleen ofdiabetes-related cognitive decline. Through a blend of traditional Chinese medicinetheory and western medicine, enriched and improved the spleen theory of visceralmanifestation, providing new clues and new ideas for prevention and treatment ofdiabetes-related cognitive decline.
Methods： Healthy adult male SD rats were randomly divided into cont group,DM group, pi group, piDM group and ZBPYR group, a total of five groups.
1We first observed the protein expression of endoplasmic reticulum stress markersinculding p-PERK, p-eIF2α, eIF2α, GRP78and GRP78mRNA levels by westernblotting and RT-PCR in cerebral cortex, hypothalamus in a variety of models,, in orderto verify whether endoplasmic reticulum stress existed in the cortex and hypothalamusof rats.
2Then we observed the expression of JNK which is the bridge molecule of ERSand insulin signaling pathway, and insulin signaling transduction pathway criticalmolecules p-IRS-1, IRS-1, p-Akt, Akt in cortex, the hypothalamus of rats by westernblotting, to verify whether insulin signaling transduction obstacles occers in cortex andhypothalamus.
3The SH-SY5Y cell was treated with tunicamycin and thapsigargin which areendoplasmic reticulum stress inducer to induce endoplasmic reticulum stress. Then cell was treated with the JNK inhibitor SP600125.We observed the changes of keymolecules of insulin signaling transduction pathways including p-IRS-1, IRS-1andp-Akt, Akt to verify whether endoplasmic reticulum stress could induce insulinsignaling transduction barriers through activating JNK.
4We detected the changes of Aβ1-42and Aβ1-40in hippocampal and cortex, thenobserved the changes of p-mTOR, mTOR, p-P70S6K, P70S6K, LC3II which is amarker of autophagy and IDE to find the reason of changes of Aβ1-42and Aβ1-40.
Results：
1. The protein expression of p-PERK, p-eIF2α of DM group and piDM group ratsincreased than the cont group in cortex, hypothalamus (P <0.05), ZBPYR group reducedthan DM group, piDM group (P <0.05); GRP78protein expression and mRNAexpression of the DM group and piDM group of both increased than cont group (P<0.05). ZBPYR decreased the expression of GRP78than DM group and piDM group(P<0.05), the difference of protein levels of eIF2α in each group was not statisticallysignificant.
2. The proteins expression of the endoplasmic reticulum stress markers p-IRE1α,p-JNK1, p-JNK2, and critical molecular of insulin signaling pathway p-IRS-1increasedin DM group, piDM group than cont group (P <0.05), p-Akt decreased in DM groupand piDM group. The expression of p-IRE1α, p-JNK1, p-JNK2, p-IRS-1of ZBPYRgroup reduced than DM group, piDM group (P <0.05), the expression of p-Akt ofZBPYR group increased. The differences of protein levels of IRS-1, and Akt amonggroups was not statistically significant.
3. The expression of p-IRE1α, p-JNK1, p-JNK2was the strongest when Tmtreatment for4h, Tg treatment for12h in vitro, when expression of p-IRS-1increased.Cells were treated with JNK inhibitor SP600125inhibited the expression of the p-IRS-1,increased expression of p-Akt.
4. The soluble and insoluble Aβ1-42of DM group and piDM group were increasedthan cont group in hippocampus and cortex, ZBPYR reduced soluble and insolubleAβ1-42than DM group and, or piDM group in hippocampus and cortex. The corticalinsoluble Aβ1-42of DM group, pi group and piDM group were increased than cont group,ZBPYR reduced insoluble a Aβ1-42in cortical. The differences of protein levels ofp-mTOR, mTOR, p-P70S6K, P70S6K was not significant among groups inhippocampus and cortex, but the protein expression of LC3II which is autophagymarker of DM group and piDM group decreased in hippocampus and cortex, the protein expression of IDE also decreased, ZBPYR inhibited the reduction of LC3II and IDE.
Conclusions:
1.Endoplasmic reticulum stress in brain is one of the characteristics of diabetic rats,spleen deficiency rats and spleen Yin deficiency diabetic rats, which is initiating insulinsignaling transduction obstacles. ZBPYR could reduce endoplasmic reticulum stress byinterfering PERK signaling in brain.
2. Endoplasmic reticulum stress induced insulin signaling transduction barriersthrough the activating JNK, which could promote cognitive decline occur in diabeticrats and spleen Yin deficiency diabetic rats, ZBPYR could improve the ability oflearning and memory by improving insulin signaling which is regulated by JNKactivated by ERS.
3. Aβ1-42increased in brain is a major characteristic of diabetic rats and spleen Yindeficiency diabetic rats, which is degradated by autophagy and IDE. ZBPYR reducedAβ1-42through regulating the expression of autophagy and IDE.
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