Effect And Mechanism Of Huang-Lian-Jie-Du Decoction (HLJDD) On Alzheimer- Like Hyperphosphorylation Of Tau In Hippocampus Of Rats With Type 2 Diabetes

T2DM is a common metabolic disease of pathological characteristics with insu-lin resistance and insulin secretion defecting. Along with the effective intervention and treatment of diabetic vascular complications, and extending of the survival time of patients, brain has became another important target organs of sugar metabolism function while cognitive impairment and dementia has become another complication of T2DM patients. T2DM is a high risk factor for Alzheimer’s disease (AD), which may be related to its abnormal insulin levels and disorder of glucose metabolism. So, how to stop or delay the process of development of T2DM to AD has become the re-search focus and hot spot. This research uses Huang-lian-Jie-du Decoction (HLJDD) as treating drug, and T2DM rats model with STZ injection as the carrier to observe the change of the rats’learning and memory ability, brain tissue morphology, PI3K-PKB/Akt signaling pathway and glucose metabolism, and discuss the effect and pos-sible mechanism of HLJDD in the treatment of T2DM concurrent AD.Purposes:Through the establishment of T2DM rats model and treatment of HLJDD, study the effect of HLJDD on Alzheimer’s disease-like hyperphosphorylation of tau protein in hippocampus of T2DM rats, and explore the possible mechanism.Method:Establishing T2DM rats model by 4 weeks with "high sugar, high fat and 2 times STZ injection", and after 8 weeks intervention with HLJDD, observing the gen-eral conditions of rats (mental activity, weight,12 hours eating and drinking, blood glucose and insulin levels),and pathological and morphological changes of HE dyeing brain tissue; observing the level of total tau protein and phosphorylation of several tau protein sites (Ser 199、Ser202、Thr231、Ser396) in hippocampus of rats with Western blot; detecting the expression of key protein of in PI3K-Akt/PKB signaling pathway in hippocampus of T2DM rats with Western blot and IHC, including Insulin Receptor α/β, Akt, and GSK-3β.Injecting LiCl in backpart of hippocampus of T2DM rats to restrain the activity of GSK-3β, with 8 weeks intervention of HLJDD, then observing the expression of GSK-3β, total tau protein and phosphorylation of part tau protein sites(Serl99、Ser202. Thr231、Ser396) in hippocampus of LiCl rats with Western blot and IHC, detecting the level of GLUT3 and O-GlcNAc glycosylation of tau in T2DM rats with Western blot and IHC.Results:1. The influence of HLJDD on general conditions of rats① The rats of CTL had a good state of mind, and their weight increased gradu-ally, having a stable eating and drinking, while the rats of T2DM had a bad state of mind and were reluctant to move, having more eating, drinking and urine; Other rats had a state of mind better than T2DM, ate and drank a little less than the model group, however it was still more than the CTL group.② Compared with CTL group, the fasting insulin, fasting blood-glucose, insulin resistance index and the random blood glucose of T2DM rats obviously increased (P<0.01). Compared with T2DM group, the fasting insulin, fasting blood-glucose, insulin resistance index and the random blood glucose of the rats treated with HLJDD decreased obviously. There was no significant difference among the random blood glucoses of T2DM group and all HLJDD groups.2. The affection of HLJDD on behavior change of rats in Morris water maze① Place navigation test:The average escape latent period in T2DM group was significantly longer than in CTL group (P<0.01); the average escape latent period in each treatment group was significantly shorter than T2DM group (P<0.05, P<0.01).② Spatial probe test:Compared with CTL group, the number of platform escape, the time of residence in effective area and swimming distance in active area were sig-nificantly reduced (P<0.05, P<0.01). Compared with T2DM group, the times of resi-dence in effective area in all of HLJDD groups increased (P<0.01), there were no sta-tistically significant difference among all groups. Except the L-HLJDD group, the times of residence in effective area of other treatment groups were longer than T2DM group(P<0.05); compared with T2DM group, the swimming distances of all HLJDD groups in active area prolonged (P<0.05, P<0.01).3. The influence of HLJDD on the HE coloration of the brain tissue of ratsThe structure hippocampus neuronal cells of CTL rats were complete. The num-ber, size and cell membrane of pyramidal cells were normal. The nucleolus of pyram-idal cells were big and round, and showed complete membrane and obvious nucleolus. The pyramidal cells layer of T2DM rats became thinner, sparse and disordered, and the number of neurons decreased, many of which were pycnosis. Others showed tu-mefaction, light pigmentation in karyons and vacuolization. There were many spon-giocytes and vacuolation near pyramidal cells layer. The pathological change signifi-cantly relieved after treatment with HLJDD.4. The influence of HLJDD on the tau protein phosphorylation of the hippocampus of ratsThere was no difference about the expression of tau-5 in hippocampus of rats in the test of Western blot. The level of phosphorylation of sites in Ser199, Ser202, Thr231, Ser396 was higher than CTL group (P<0.05,P<0.01). Compared with T2DM group, the expression of PS 199, PS202, PT231 and PS396 obviously decreased in the hippocampus of rats of HLJDD groups (P<0.05,P<0.01), except the PT231 of M-HLJDD group.5. The influence of HLJDD on insulin signaling pathway① T2DM rats:1) There was no significant difference about expression of IRa and IRβ protein in cerebral cortex of rats among all groups. 2) There was no signifi-cant difference among all groups in total Akt and total GSK-3β protein in hippocam-pus of rats by Western blot; Compared with CTL group, the expression of P-Akt and P-GSK-3β protein in hippocampus of T2DM group significantly decreased (P<0.01). Compared with T2DM group, except H-HLJDD group, the expression of P-Akt and P-GSK-3β protein in hippocampus in other treatment groups significantly increased (P<0.05,P<0.01), there was no significant difference among all groups. Except H-HLJ DD group, the expression of P-GSK-3β protein in hippocampus of rats in all treat-ment groups are significantly higher than in T2DM group (P＜0.05).3) There was no significant difference of total GSK-3(3 protein in CA1 region of hippocampus by IHC. Compared with CTL group, the phosphoralation level of P-GSK-3β on Ser9 site in the hippocampal CA1 region of T2DM group significantly decreased (P<0.01).Compared with CTL group, the expression of P-GSK3p protein in CA1 region of hippocampus significantly increased (P<0.01).② LiCl rats:1) There was no significant difference about the intake of water and food in 12 hours, fasting insulin, fasting glucose, and insulin resistance index between LiCl group and T2DM group, which were higher than all treatment groups (P＜0.01, P＜0.05). There was no significant difference among all HLJDD groups.2) There was no significant difference about total GSK-3β protein in hippocampus of rats among all groups by Western blot and IHC; Compared with CTL group, the ex-pression of P-GSK-3β protein in hippocampus of T2DM group significantly de-creased (P＜0.05); Compared with T2DM group, the expression of P-GSK-3β protein in hippocampus of LiC1 group significantly increased(P<0.01); Except H-HLJDD group, the expression of P-GSK- 3β protein in hippocampus of rats in all treatment groups are significantly higher than in T2DM group (P＜0.05).3) There was no sig-nificant difference about tau-5 in hippocampus of rats among all groups by Western blot; Compared with CTL group, the expression of PS 199, PS202, PT231, PS396 protein in hippocampus of T2DM group significantly increased especially PS396 (P＜0.05, P＜0.01); Except PS396 in M-HLJDD group and PS202 in H-HLJDD, the expression of PS 199, PS202, PT231 and PS396 protein in hippocampus of rats in all treatment groups were significantly lower than that in T2DM group (P＜0.05, P＜0.01).6. The affection of HLJDD on the metabolism of glucose① GLUT3:Compared with CTL group, the level of GLUT3 in hippocampus of T2DM group obviously reduced (P＜0.01); Compared with T2DM group, the level of GLUT3 obviously increased in hippocampus of rats of all treatment groups (P＜0.01), except H- HLJDD group.② O-GlcNAc glycosylation of tau:The expression of RL2 in hippocampus of T2DM group was obviously more than that of CTL group in Western blot (P＜0.05); Compared with T2DM group, the expression of RL2 in hippocampus of all treatment groups obviously increased (P＜0.05). The positive protein expression of RL2 in CA1 region of hippocampus was obviously less than that of CTL group(P<0.01); Com-pared with T2DM group, the expression of RL2 in all treatment groups obviously in-creased (P<0.01), and there was no obvious difference among all treatment groups.Conclusion:1. After building the legal system for T2DM rats model by 4 weeks with "high sugar, high fat+2 times STZ intraperitoneal injection", animals had typical diabetes symp-toms with high level of blood sugar and low mortality. It is a preparation method of diabetic rats model with high success rate and high stability.2. HLJDD can effectively improve the T2DM model rats’learning and memory abil-ity with good educational effect, has good effect on reinstating neuronal degenerative changes of neuron cells and inhibiting excessive phosphorylation of tau protein in hippocampus in T2DM rat.3. HLJDD has good control effect on PI3K-Akt/PKB signal pathway of T2DM model, which is not the single way to inhibit tau protein hyperphosphorylation.4. HLJDD can increase the expression of GLUT3 and Tau protein O-GlcNAc glyco-sylation in hippocampus of T2DM rats, and negatively adjust tau protein phosphory-lation modification.