Zibu Piyin Recipe Improve Insulin Signaling Reduce The Expression Of β Amyloid Peptide1-42Through Regulating ERS In Brain

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.