Based On The Theory Of "toxin Impairing Brain Collaterals" In The Mechanism Of Alzheimer's Disease

1. BackgroundAlzeimer’s disease (AD) is the most common one of dementia, regarding cognitive dysfunction as the main mlinical manifestations. Modern medical research in this field demonstrates that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in brains, disorder of Aβ clearance, cholinergic neuron damage, oxidative stress, brain inflammatory cascade, gene mutation, and so forth. Although researchers have produced a variety of drugs aimed at improving cholinergic neuronal function, oxidative stress and other aspects of AD, there are not effective ways to treat AD, especially in the aspect of brain injury recovery. However, in the direction of TCM theory, our previous research showed that AD belongs to the category of ’collateral disease’, and ’toxin hurts brain collaterals’ is the key point in its pathogenesis. It is also realized that brain microvascular endothe lial cells (BMECs) are not only the substance exchange barrier of blood and tissue, but also have important incretion function. Our researches pay more attention to BMECs rather than neurons, which is a meaningful probe to guide Traditional Chinese Medicine mordernation in brain diseases.2. PurposeIn this study, with the guidance of collateral disease theory, we take the main material basis of brain collaterals system-BMECs as the breakthrough point of our research, choose Tongluojiunao injection(TLJN)withthe function of detoxification and smoothing collaterals as intervening drugs, mainly explore the dynamic changes of Aβprotein transportersin ADimitated BMECs andchanges in thetransportermediatedspecific intracellular signal pathway, elucidate both the transporter change and endothelial cells injury and the biological effect between TLJN and vascular endothelial protection and explain the targets of TLJN to BMECs from the molecular level. At last, provide an in vitro experimental basis for "establishingthe toxin hurtingbraincollaterals"pathogenesis hypothesis in Alzheimer’s disease and the clinical application of TLJN.3. MethodsThe experiments are divided into4parts, the first part is to observe the protective effect of Jiedu Tongluo drugs on AD imitated rat brain; The second part is to investigate time course changes between cerebral vessels and neuronal injury in the AD imitated rat and the intervention effect of TLJN; The third part is to investigate the effect of Aβ1-42on the amyloid beta protein transporter and the intervention effect of TLJN; The fourth part is to investigate the AD imitated brain microvascular endothelial cells in PI3K/Akt/Caspase-3 intracellular signal pathway and the intervention effect of TLJN.The experimental plans as followed:the first part:condensed Aβ1-42was injected into both hippocampus of rats to establish AD imitated animal model, animal experiments were divided into five groups, respectively, control group (normal control group), sham group (sham operation group), model group (model group), TLJN group (TLJN injection group) and DNP group (positive drug group), behavior of rats were detected by Morris water maze after drug administration for7days,14days and21days, characteristic pathological changes were observed in Alzheimer’s disease brains (ELISA method for detecting the content of Aβ1-42in brain tissue, Western Blot method to detect the expression of P-tau, HE staining was chosen for neuron injury). The second part:Western Blot method and ELISA method were used to observe the expressions of both vWF and NSE in protein levels in cerebral tissues and blood in AD imitated rats of3detecting period of time. The third part:in vitro culture of AD imitated rat brain micro vascular endothelial cell, from the level of protein and mRNAto study brain damage in AD rat, the expression changes of specific amyloid beta protein transporters LRP-1and RAGE in AD imitated micro vascular endothelial cells in different period of time, detection of oxidative stress reaction by spectrophotometry (MDA content, SOD activity, Cat activity, GSH-Px activity), the viability of endothelial cells were detected by MTT method. The fourth part:through the cultivation of the AD imitated rat brain micro vascular endothelial cells to detect the main signal molecules change of LRP-1mediated PI3K/Akt/Caspase-3signal pathway induced by Abetal-42with the method of Western Blot and the regulating effect of TLJN on the signal pathway.4. Results①In the observation of the animal experimental range, by the Morris water maze test, we found that the behavior of AD rats changed significantly, demonstrated by the prolonged escape latency, increased swimming total distance, increased target quadrant stay time and reduced number of wear stage. In addition, the morphology of AD rats also changed significantly, showing different degrees of cell shrinkage, nuclear condensation in hippocampal neurons, karyolysis and nuclear fragmentation phenomenon:Tau protein hyperphosphorylation and increased Aβ1-42content in the whole brain tissue. After the administration of TLJN. behavior of AD rats, brain tissue structure and function were improved to some extent.②In this experiment, the vWF protein level in blood and brain in AD rats compared with sham operation group in three period of time were increased significantly, while the NSE protein in brain tissue and the blood in AD rats increased significantly after14days. In TLJN group, compared with model group, vWF and NSE protein decreased significantly in14days, showing the beneficial effects on AD imitated rats.③In the observation of cells experimental range, LRP-1and RAGE protein expressions in the AD imitated endothelial cells showed the dynamic change, presenting the elevated expression of LRP-1in Oh,3h,6h and12h, four period of time, but decreased in24h,36h,48h and72h four period of time. As for mRNA expression of LRP-1, increased at Oh,3h and6h three time points, decreased in the subsequent time points. Expression of RAGE in both protein and mRNA levels increased gradually in all8time points; LRP-1/RAGE protein ratio showed no statistical differences in Oh,3h,6h and12h four time points, but significantly decreased in the following four period of time; however, the ratio of LRP-1/RAGE in mRNA level showed no change. After TLJN administration, the expression of RAGE both in protein and mRNA levels were decreased in all8time points; as for the regulation of LRP-1expression, TLJN could down regulate the protein expression of LRP-1, but showed no effect on the expression of LRP-1in mRNA level. In AD imitated endothelial cells, intracellular oxidative stress (MDA content) increased at all detected8time points, antioxidant enzymes (SOD, Cat, GSH-Px activity) activity decreased gradually in the whole process, after the administration of TLJN, intracellular oxidative stress was decreased, the activities of antioxidant enzymes were increased.④In cellular experiments, in Aβ1-42induced AD imitated RMECs, LRP-1protein expression decreased, P-tau protein expression decreased, Caspase-3protein expression increased, inducing endothelial cell apoptosis; with the administration of TLJN, the expression of LRP-1increased, the expression of P-tau protein increased, the expression of Caspase-3decreased, reducing cell apoptosis; after the interaction of both TLJN and inhibitor-LY200942in Aβ1-42induced endothelial cells, the expression of LRP-1increased, the expression of P-tau protein reduced, and Caspase-3protein expression decreased, alleviating endothelial cell apoptosis.5. Conclusion①TLJN as the representative of the decoction with the function detoxification and smoothing collaterals can reduce t cognitive impairment, improve the characteristic pathological injury of brain in AD imitated rats.②In the AD imitated rats and rats micro vascular endothelial cell models, the cerebral microvessels are prior to the injury of neurons, due to break of the dynamic balance between LRP-1and RAGE, Aβ1-42were transported decreased and increased aggregation in brains, via LRP-1mediated PI3K/Akt/Caspase-3signal pathway, Caspase-3expression was increased, promoting the apoptosis of endothelial cells and increasing the damage to the neurons in AD imitated rats, eventually led to the cognitive dysfunction. ③After TLJN was administrated, it could recover the dynamic balance between LRP-1and RAGE, with the help of compound target in decoction, inhibited the expression of Caspase-3, reduced the apoptosis of endothelial cells, with the regulation to microvascular endothelial cells, achieve the purpose of improving the damaged neurons, to a certain extent for the Alzheimer’s disease"toxin damaging brain collaterals" pathogenesis hypothesis establishment provides experimental evidence in vitro.