Study Of The Correlation Between VLCFA And Aβ Production In The Brains Of Type 2 Diabetic Rats

Objective: Alzheimer’s disease(AD) is a common chronic neurodegenerative disease, characterized by a progressive loss of memory and cognitive function. A primary pathological feature of AD is the deposition of extracellular amyloid β-protein(Aβ) into senile plaques. Aβ is the major pathogenic factor of AD. AD is not only a common disease of the elderly, but also a disease with high incidence among diabetes mellitus(DM) patients. This undoubtedly worsens the situation of diabetes mellitus patients. Therefore, it is urgent to clarify the causes of high incidence of AD in patients with diabetes mellitus, and find the intervention measures.Based on the following two facts:(1) The levels of very long chain fatty acids(VLCFA, ≥C22) have been reported to be increased in the brain of AD patients. VLCFA has lipotoxicity to neurons, which leads to the increase of cell reactive oxygen species(ROS) and the enhancement of oxidative stress. Oxidative stress has been well recognized to be one of the major mechanisms of increased Aβ production.(2) Diabetes is a glycolipid metabolic disturbance disease. We assumed that, in the condition of diabetes, lipid metabolism disorder resultes in the accumulation of VLCFA in the brain and then causes oxidative stress, which increases abnormally Aβ production and eventually leads to cognitive impairment, that is the occurrence of diabetic AD.In order to confirm this hypothesis, in this study, a high-fat diet combined with a small dose STZ-induced type 2 diabetic rats model was used as subject, to observe the learning and memory ability of type 2 diabetic rats and the brain Aβ production, the oxidative stress level and the VLCFA change. This research is aim to discuss the effect of VLCFA on Aβ production in the brains of type 2 diabetic rats and to provide new experimental evidence for the prevention and treatment of diabetic AD.Methods:1 Experimental AnimalsThe brain tissue of type 2 diabetic rats prepared by Hou Lianguo et al. in our laboratory was used as materials. The rats were divided into two groups: control group(Con) and diabetes mellitus group(DM).2 Measurement of learning and memory ability by Morris water mazePlace navigation test and spatial probe test were conducted by Morris water maze(Jiliang, Shanghai). The average escape latency, the crossing times and the latency to original platform were recorded respectively to evaluate the learning and memory ability of rats.3 Determination of the content of brain Aβ40 and BACE1 by ELISAELISA kits(IBL, Japan) were used to detect the content of brain Aβ40 and BACE1. The content of Aβ40 was expressed by pg/mg protein and BACE1 by ng/μg protein.4 Analysis of APP and BACE1 m RNA expression by real time RT-PCROMEGA total RNA extraction kit was used to extract total RNA from the brain tissue of rats. APP, BACE1 m RNA levels were assayed by real time RT-PCR with 18 S r RNA as an internal.5 Analysis of APP protein level by immunohistochemistryImmunohistochemical staining of paraffin sections of the brain tissue was performed to assay the APP protein expression.6 Analysis of brain VLCFA by gas chromatographFatty acid was extracted from the brain tissue of rats. Gas chromatograph(GC-2010 Plus, SHIMADZU, Japan) was used to detect the content of various VLCFA.7 Detection of the content of malondialdehyde(MDA) by TBAThiobarbituric acid(TBA) colorimetry method was used to determine the content of MDA in the tissues, which was expressed by nmol/mg prot.8 Detection of NOX4, p47 and HO-1 protein levels by Western blotTotal protein was extracted from brain tissue of rats. NOX4, p47 andHO-1 protein levels in the brain of rats were assayed by Western blot.9 Statistical analysisAll data were expressed as the means ± SD. All statistical analysis was performed using SPSS 17.0 software. The escape latency was analysed using a repeated measures ANOVA. Other data were analysed using Student’s t-test for two-group comparisons. Statistical significance was set at P < 0.05.Results: 1 DM rats exhibit impaired learning and memoryThe results of Morris water maze showed that, compared with Con group, although the escape latency of rats in DM group was expanded on the first day, there were no significant difference on the other days. The crossing times were significantly reduced and the latency to original platform was significantly prolonged(P < 0.01) in DM group compared with Con group, indicating that the learning and memory ability of type 2 diabetic rats was impaired. 2 DM rats exhibit increased Aβ production 2.1 The content of brain AβThe results of ELISA showed that the content of Aβ40 in Con and DM groups were 4.87 ± 0.71 pg/mg protein and 6.22 ± 0.81 pg/mg protein respectively. The content of Aβ40 in the brain of DM group was significantly higher than that in Con group(P < 0.01) indicating the increase of brain Aβ in diabetic rat. 2.2 The expression of brain APPThe results of RT-PCR showed that the m RNA expression level of APP in the brain of DM group(0.046 ± 0.014) was significantly higher than that in Con group(0.025 ± 0.007, P < 0.001).The results of immunohistochemistry showed that the number of APP positive cells and color depth in the brain of DM group were significantly increased than that in Con group. Image analysis showed that the average optical density of APP positive cells in the brain of rats in DM group was increased significantly than that in Con group(P < 0.001).These results suggested that the m RNA and protein levels of APP were increased in the brains of diabetic rats. 2.3 The expression of brain BACE1The results of RT-PCR showed that the m RNA expression level of BACE1 in the brain of DM group(0.912 ± 0.038) was significantly higher than that in Con group(0.661 ± 0.118, P < 0.001).The results of ELISA showed that the protein level of BACE1 in the brain of DM group(0.95 ± 0.09 ng/μg protein) was significantly increased than that in Con group(0.78 ± 0.06 ng/μg protein, P < 0.01).These results suggested that the m RNA and protein expression of BACE1 were increased in the brains of diabetic rats.The above results showed that the β-secretase pathway of APP processing was up-regulated in the brains of diabetic rats, which led to the increase of Aβ production and caused AD-like pathological changes. 3 DM rats exhibit increased C26:0The results of gas chromatograph showed that the level of C26:0 in the brain of DM group(44.90 ± 13.94 μmol/ g tissue) was significantly higher than that in Con group(18.38 ± 6.29 μmol/ g tissue, P < 0.001). The levels of C22:0 and C24:0 were no significant differences between DM and Con groups(P > 0.05). It indicated that the level of C26:0 was increased in the brains of diabetic rats. 4 The level of C26:0 is positively correlated with the content of Aβ in the brains of diabetic ratsA correlation analysis showed that there were positive correlations between the level of brain C26:0 and the content of Aβ(r = 0.557, P = 0.016). It indicated that the increased level of C26:0 was related to the increase of Aβ production in the brains of diabetic rats. 5 DM rats exhibit enhanced oxidative stressThe results of TBA showed that the content of MDA in the brain of DM group(2.19 ± 0.27 nmol/mg protein) was significantly increased than that in Con group(1.83 ± 0.19 nmol/mg protein)(P < 0.05).The results of Western blot showed that the protein levels of oxidative stress markers NOX4,p47 and HO-1 in the brain of DM group were significantly higher than that in Con group.It indicated that oxidative stress was enhanced in the brains of diabetic rats. 6 The level of C26:0 is positively correlated with the content of MDA in the brains of diabetic ratsA correlation analysis showed that there were positive correlations between the level of brain C26:0 and the content of MDA(r = 0.504, P = 0.033). It indicated that the increased level of C26:0 was related to the enhanced oxidative stress in the brains of diabetic rats.Conclusions:1 The cognitive dysfunction is associated with the enhanced oxidative stress resulted from the increased C26:0 in the brains of type 2 diabetic rats.2 The increased level of Aβ correlates to the up-regulation of APP and BACE1 caused by oxidative stress.