| Objective:Diabetes mellitus(DM) is one of metabolic disorders with chronic blood glucose increased.It is the result of the lack of insulin secretion and (or) the role of insulin. Type 2 diabetes mellitus is associated with insulin resistance. Diabetes mellitus can cause multi-system damages, including hearts, kidneys, nerves, retina, blood vessels and other tissues and organs .Diabetic encephalopathy(DE) is one of chronic complications of diabetes mellitus. Reflected that learning and memory decline.Some researches have shown that there is a close relationship between the endoplasmic reticulum stress(ERS) with the diabetes mellitus. Endoplasmic reticulum stress is the response of the cell when the stress is actived. The environmental changes of endoplasmic reticulum such as sugar starvation, disorders of calcium balance, inhibition of glycosylation and reduction of disulfide bond's synthesis could affect the folding of protein, resulting in a large number of accumulation of unfolded or misfolded protein in the endoplasmic reticulum,then unfolded protein response (UPR) is induced. UPR signaling pathways elicit the molecular chaperone glucose regulated protein 78 (GRP78), and then GRP78 was separated from inositol-requiring enzyme(IRE1), protein kinase-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). It leads to the oligomerization and autophosphorylation of PERK ,and then induces the phosphorylation of eIF2α.The process could reduce the synthesis of new proteins and prevent the accumulation of proteins. The phosphorylation of PERK and eIF2αindicates that endoplasmic reticulum stress is provoked. Diabetic encephalopathy belongs to the complications of XiaoKe in the Traditional Chinese Medicine.The location of this disease is brain, referring to liver, kidney, heart and spleen. The nature of the disease is root deficiency and branch excess with yin deficiency induced dryness and heat.The spleen provides the materiel basis of the acquired postnatal, which is the source of growth and development of qi and blood.Qi and blood is the material basis of life. Spleen deficiency induces the disorder of transportion and transformation,leading to nutrient and body fluid distributing abno- mally.This is the main reason of XiaoKe.On the other side, the deficiency of qi and blood induces lack of nutrients in the brain,leading to DE. Nouri- shing spleen yin recipe(Zibu Piyin Recipe,ZBPYR), created by Professor Libin Zhan, could reinforce spleen and nourish spleen yin. To research the effects of ZBPYR on ERS in the hippocampus of spleen-yin deficiency diabetic encephalopathy rats ,we established spleen-yin deficiency diabetic encephalopathy rat models.We Observed the changes of behavior of the rat models treated by ZBPYR,and investigated the effects of ZBPYR on the proteins which are related with ERS by Western Blotting.We want to found the new target of ZBPYR in the protein level,and provide a new clue for the treatment of DE.Methods: 1. Healthy adult male Sprague Dawley( SD) rats were divided randomly into control group, spleen-yin deficiency group ,diabetic encephalopathy group, spleen-yin deficiency diabetic encephalopathy group and spleen-yin deficiency diabetic encephalopathy +ZBPYR group, four rats each group.2. After the acclimatization, the control group and spleen-yin deficiency group were feed with foundational diet. Other groups were feed with high fat diet.4 weeks later,the rats which were feed with high fat diet are injected with low dose STZ(30mg/kg) to establish diabetes mellitus models. The rats whose the radom blood glucose was more than 16.7mmol/L were identified as diabetes mellitus models after 72h. We detected the level of fasting serum insulin(FSI),oral glucose tolerance tes(tOGTT)and insulin tolerance test(ITT)after intraperitoneal injection with 1% STZ on the 4th,8th,12nd day respectively.Spleen-yin deficiency diabetic encephalopathy model rats were induced by defatigation,hungry-full diet and drenching the drug of damaged yin.ZBPYR group were given by the intervention of ZBPYR in 16 days and decapitated for collecting Hippocampus.3. Morris water maze test and step-down test were used to evaluate the behavioral changes.4. Using Western blotting method to detect protein expression of GRP78,PERK,phospho-PERK,eIF2α,phospho-eIF2αin rat hippocampus.Results:1. After injection of STZ,the blood glucose of the rats of diabetes mellitus were higher than nondiabetes rats(P<0.01) In the oral glucose tolerance test,the blood glucose of diabetes mellitus rats were higher than nondiabetes rats on every time point,and presented impaired oral glucose tolerance (P<0.01), in the insulin tolerance test, the rate of decline in blood glucose of diabetes rats were more slowly than nondiabetes rats,indicating impaired insulin tolerance.Fasting serum insulin levels were similar between diabetes rats and non-diabetes rats(P>0.05).2. The spleen-yin deficiency group presented endogenous heat symptomes caused by yin-deficiency,such as the increment of water drink- ing(P<0.01),obvious elevation of the rectal temperature(P<0.05). The results of Morris water maze test and step-down test showed that the learning and memory ability of the diabetic encephalopathy group and the spleen-yin deficiency diabetic encephalopathy group were reduced signi- ficantly(P<0.05), but that of the ZBPYR treatment group were increased significantly(P<0.05).3. Western Blotting results indicated the levels of GRP78, phospho- PERK,phospho-eIF2αin hippocampus of the diabetic encephalopathy group and spleen-yin deficiency diabetic encephalopathy group were significantly increased ,and that of spleen-yin deficiency diabetic encephalopathy +ZBPYR group was signigicantly decreased.Total protein levels of PERK and eIF2αwere not changed.Conclusion:1. The impaired learning and memory ability of spleen-yin deficiency diabetic encephalopathy may relate to the initiating of the PERK signaling pathway of ERS.2. The high expressions of GRP78, phospho-PERK and phospho-eIF2αwere involved in the pathogenesis of spleen-yin deficiency diabetic ence- phalopathy 3. ZBPYR may improve the learning and memory ability of spleen-yin deficiency diabetic encephalopathy rats by affecting on the PERK signaling pathway of ERS.4. ZBPYR may improve the learning and memory ability of spleen-yin deficiency diabetic encephalopathy rats by inhibiting the expressions of GRP78,phospho-PERK and phospho-eIF2α... |
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