| ObjectiveAlzheimer’s disease (AD) is a central nervous system degenerative disease characterized by progressive cognitive dysfunction and behavioral impairment. Memory disorders, aphasia, disuse, agnosia, spatial ability, abstract thinking and calculation force damage, personality and behaviour change are the common clinical performance for AD. AD is the most common type of dementia, accounting for 60-80% of all dementia. However the "amyloid beta waterfall theory" and "tau protein theory" are widely accepted about the pathogenesis of AD, growing evidence suggests that AD is actually an abnormal metabolic disease showing brain glucose utilization and energy production obstacle. The metabolic abnormalities associated with abnormal insulin signaling pathway in the central nervous system, including the decrease of insulin in the brain, insulin resistance and signaling pathway defects. Abnormal insulin signaling pathways make the uptake and utilization of glucose in brain obstacle and energy production decline, leading to metabolic abnormalities, synaptic damage, and cognitive decline. These are important in the development process of AD. Therefore, we targeted with insulin signaling pathway in the central nervous system and glucose transporters, observed the changes of structure and quantity of neurons and synapses and the effect of cognitive function.Curcumin was used as the Chinese medicine control and donepezil as the western medicine control. Studies in our group found that curcumin regulated insulin signaling pathway and cerebral glucose metabolism, improved spatial memory ability in the APPswe/PS1dE9 double transgenic mice. Banxiaxiexin Tang makes the body strong, eliminates phlegm, clears heat, tonifies deficiency and reduces excess. In addition, drugs in banxiaxiexin tang are neural protective, such as the guanosine in pinellia, baicalin in radix scutellariae, berberine in rhizoma coptidis, saponins of ginseng and licorice glycosides. Guanine nucleoside, hereinafter referred to as guanosine, widely exists in a variety of medicinal plants, is also the main component of pinellia. Guanosine plays neuro-protective role relying on G protein coupled receptor (GPCR) and intracellular signal transduction pathways. But banxiaxiexin tang and guanosine which play neuro-protective role by regulating insulin signaling pathway and early cerebral glucose metabolism remain to be further discussed.Therefore, we observed the effect of insulin signaling pathway and cerebral glucose metabolism with banxiaxiexin tang and guanosine treatment for 3 months in the APPswe/PS1dE9 double transgenic mice AD model, analyzed the neural protective mechanism of banxiaxiexin tang and guanosine, looked for new drug targets with slowing down AD pathological changes and clinical symptoms.Materials and methods1. Using 3 months APPswe/PS1dE9 double transgenic mice75 and wild type C57BL/ 6J mice 15 for experimental animals. APPswe/PS1dE9 double transgenic mice were randomly divided into the model group (isopyknic solvent for intraperitoneal injection and lavage), the western medicine control group (donepezil 0.92 mg/kg/day lavage, isopyknic solvent for intraperitoneal injection), the banxiaxiexin tang group (banxiaxiexin tang 650 mg/kg/day lavage, isopyknic solvent for intraperitoneal injection), the Chinese medicine control group (curcumin 200 mg/kg/day lavage, isopyknic solvent for intraperitoneal injection), and the guanosine group (guanosine 16 mg/kg/day for intraperitoneal injection, isopyknic solvent for lavage). Each group has 15 APPswe/PS1dE9 double transgenic mice. Treating started at the beginning of 3 months and continued for 3 months. The normal group used wild type C57BL/6J mice (isopyknic solvent for intraperitoneal injection and lavage).2. Using Morris water maze and step-down test to observe the learning and memory ability and the memory retention, to evaluate the influence on cognitive function with banxiaxiexin tang and guanosine treatment for 6 months APPswe/PS1dE9 double transgenic mice.3. Using transmission electron microscopy (TEM) to observe synaptic numbers and ultrastructure of neurons and synapses with banxiaxiexin tang and guanosine treatment for 6 months APPswe/PS1dE9 double transgenic mice.4. Using immunohistochemistry and Western blot to observe PI3K, Akt in insulin signaling pathway and IRS2, Glutl, Glut3 in glucose metabolism with banxiaxiexin tang and guanosine treatment for 6 months APPswe/PS1dE9 double transgenic mice. At the same time, GPCR was also observed. We evaluated the effect of insulin signaling pathway and glucose metabolism with banxiaxiexin tang and guanosine treatment.Results1. Banxiaxiexin tang improved the spatial learning and memory ability and memory retention of APPswe/PS1dE9 double transgenic mice, guanosine improved their memory retention.Morris water maze results showed that the time of escape latency and distance of swimming were declined along with the increase of days. Starting from the first day, the escape latency time and swimming distance in the model group were increased significantly compared with the normal group (P<0.01). In the 3-5 days, the escape latency time in the western medicine control group were declined compared with the model group (P<0.05 or 0.01). In the 2-5 days, the swimming distance in the western medicine control group were decreased compared with the model group (P<0.05 or 0.01). From the fourth day on, the escape latency time in Chinese medicine control and banxiaxiexin tang groups were decreased compared with the model group (P<0.05 or 0.01). In the 2-5 days, the swimming distance in Chinese medicine control and banxiaxiexin tang groups were decreased compared with the model group (P<0.05 or 0.01). In the 3-5 days, the swimming distance in the guanosine group were decreased compared with the model group (P<0.05 or 0.01).We found that in space exploration experiment, the time of targeting quadrant in the model group was less than the normal group (P<0.01), the time of targeting quadrant in western and Chinese medicine control groups and banxiaxiexin tang group were longer than the model group (P<0.05).Step-down test reflected the memory retention. The latentperiod of step-down test in the model group was declined significantly compared with the normal group (P<0.01). Compared with the model group, the latentperiod in each drug group were significantly increased (P<0.01).2. Banxiaxiexin tang and guanosine increased the synaptic number in hippocampus of APPswe/PS1dE9 double transgenic mice, banxiaxiexin tang obviously improved the structure of neurons and synapses.Using TEM we found that the synaptic structure in hippocampus of normal mice was normal. The synaptic structure in the model group was abnormal, with unclear synaptic membranes, broadening synaptic cleft and swelling mitochondria. The synaptic number in the model group was significantly less than the normal group. Except the guanosine group, the synaptic number and structure in other drug groups were significantly improved compared with the model group. Compared with model group, the synaptic number in guanosine group was increased. Except for synapses, donepezil, curcumin as well as banxiaxiexin tang also improved the neuron structure in hippocampus of APPswe/PS1dE9 double transgenic mice.3. Banxiaxiexin tang adjusted key proteins in central insulin signaling pathway and glucose metabolism of the brain in APPswe/PS1dE9 double transgenic mice, guanosine improved the expression of IRS2 and GLUT 3 proteins.Immunohistochemical staining results showed that compared with the normal group, positive cells with PI3K, Akt and IRS2, GLUT 1 in the model mice hippocampus were significantly reduced (P<0.01). Donepezil, curcumin and banxiaxiexin tang increased the positive cells with PI3K, Akt and IRS2, GLUT 1 compared with the model group (P<0.01). Guanosine increased the positive cells with Akt and IRS2 (P<0.01), but didn’t affect the number of positive cells with PI3K and GLUT 1.Western-blot results showed that compared with the normal group, the content of Akt and GLUT 1, GLUT 3 in the model mice hippocampus were reduced (P<0.05 or 0.01). Donepezil, curcumin and banxiaxiexin tang increased the content of Akt and GLUT 1, GLUT 3 in APPswe/PS1dE9 double transgenic mice (P<0.05 or 0.01). Compared with the model group, guanosine increased the content of GLUT 1 and GLUT 3 (P<0.05 or 0.01), but didn’t affect the content of Akt.ConclusionBanxiaxiexin tang adjusted key proteins in central insulin signaling pathway and glucose metabolism of the brain, increased the synaptic number in the hippocampus and improved the structure of neurons and synapses and cognitive function. Guanosine improved the expression of IRS2 and GLUT 3 protein, increased the quantity of synapse in the hippocampus and improved the ability of memory retention. Guanosine didn’t affect the expression of PI3K. The results of Akt and GLUT 1 in immunohistochemical staining and Western-blot performed contradiction.
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