Studies On The Mechanism Of Brain Injury In Insulin Resistant Rats And The Protective Effect Of Lycopene

BackgroundUnder the influence of external environmental or genetic factors human body tissues may exhibit insulin resistance, which is mainly characterized by decreased insulin sensitivity, often accompanied by hyperinsulinemia and glucose utilization deficits. Insulin reduces blood glucose through its metabolic signaling pathway: insulin binds insulin receptor (IR) and insulin-like growth factor receptor 1(IGF-1R), then tyrosine kinase are activate, making the insulin receptor substrate phosphorylation and downstream substrate protein phosphorylation or dephosphorylation, thereby activating the key enzyme of insulin signaling pathway-phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/AKT), activation of AKT may reduce the activity of downstream glycogen synthase, then promotes glucose transport and glycogen synthesis, making the blood glucose decrease. Insulin resistance appears mainly when this pathway has obstacle.Previous studies mainly showed that the liver, skeletal, and adipose tissue were target organs which insulin played its role, and central nervous system (CNS) is thought no insulin. In recent years, studies of insulin in the brain is deepening, more and more studies suggest that insulin signaling molecules are widely distributed in the brain, while the expression of insulin signals can be detected in CNS. The challenge now is to identify in detail the signaling pathways used by insulin in the brain, as failure of those signals has been associated with brain disorders including AD. The signaling pathways are also the basis for the relationship between insulin resistance and brain function decline.Insulin resistance is thought to be an important risk factor of Alzheimer’s disease, studies have shown that Aβ deposition increased in insulin resistant patients, but the mechanism underlie the relationship between insulin resistance and relevant AD-like changes is still not clear. Oxidative stress play important roles in a variety of pathological conditions, including type 2 diabetes mellitus (T2DM) and AD. Advanced glycation end products (AGEs) are also involved in the pathological process of AD. AGEs may bind to its receptor (RAGE) and activate the core transcription factor of inflammation-nuclear factor-kappa B (NF-κB), NF-κB is involved in the regulation of many inflammatory cytokines expression, including tumor necrosis factor -a (TNF-a) and interleukin -1beta (IL-1β). AGEs/RAGE regulated inflammation is also involved in the development of AD and other neurodegenerative diseases, but the comprehensive mechanism of AGEs/RAGE, oxidative stress and inflammation in brain insulin resistance injury remains unclear.More and more reseaches are focus on the natual traditional medicine which is high effective and less toxic. This kind of drugs always had multiple target for therapy. As a member of carotenoid family, lycopene is an effective natural antioxidants, the antioxidant capacity of lycopene is around 47 times β-compared with carotene, and 100 times compared with vitamin E. Lycopene can cross the "blood-brain barrier" and plays its antioxidant, scavenging free radicals and other biological functions in CNS. In recent years, studies on lycopene in the central nervous system degenerative diseases continue to increase, studies have shown that long-term intake of lycopene can help improve cognitive function in the elderly population, lycopene in the central nervous system degenerative diseases prevention and treatment is of concern.Therefore, exploring the central insulin signal transduction molecules altered expression levels in peripheral insulin resistance model can identify the existence of central insulin resistance, then exploring the mechanisms of AGEs/RAGE, oxidative stress and inflammation in brain tissue in animal models of insulin resistance injury and the protective effect of lycopene can provide theoretical basis for revealing the complex pathogenesis of AD and other neurodegenerative diseases and select a valid therapeutic target, while studying natural medicine pleiotropic and multi-system, multi-target prevention and treatment Features can thereby provide real value to explore the effective prevention and treatment drug for cognitive deficits from the treasure house of natural medicine.ObjectiveObserve the changes of learning and memory abilities, insulin sensitivity and central insulin signaling pathway protein levels in fructose-drinking resistant rats and the intervention effect of lycopene; Observe the changes of AGEs/RAGE, anti-oxidant defense system, pro-inflammatory cytokines, PPARy expression, and cholinergic activity in fructose-drinking resistant rats and the intervention effect of lycopene; further explore the mechanism and treatment target of insulin resistance brain injury and AD; studying natural medicine pleiotropic and multi-system, multi-target prevention and treatment Features can thereby provide real value to explore the effective prevention and treatment drug for cognitive deficits from the treasure house of natural medicine.Methods1. Insulin resistance model establishment and groupsAfter adaptation for a week, the Six-eight week-old male Wistar rats were randomly divided into 4 groups (8 in each group). Group Ⅰ comprised of control rats were fed with normal drinking water; Group Ⅱ rats received 10% fructose solution in drinking water for 16 weeks to develop insulin resistance; Groups III rats were administered 10% fructose solution in drinking water for 16 weeks and lycopene (4 mg/kg; oral gavage) for the last 10 weeks of the 16-week period. Group IV rats received lycopene only (4mg/kg; oral gavage). Lycopene was dissolved in double distilled water with 5% Tween 80. Non-treated rats (Groups I and Ⅱ) were administered with the vehicle of lycopene at the same volume for the last 10 weeks.2. Detection of the characteristics in fructose-drinking ratsThe HOMA-IR index was measured at the end of the experiments for assessing insulin sensitivity. The FBG levels were determined by a glucose-oxidase biochemistry analyzer and FINS levels were measured by homogeneous phase competitive immunoradiometric assay with immunoreagent kit using GC-911c immunoradiometric counter. HOMA-IR was calculated as formula:HOMA-IR= FBG×FINS/22.5.3. Test of behaviorThe Morris water maze (MWM) test, which consisted of 5-day training (visible and invisible platform training sessions) and a probe trial on day 6. was used to evaluate the learning and memory ability of the rats. The platform was placed in the center of the 4 quadrants and remained there throughout the experiment. Each rat was individually trained in both visible-platform (days 1-2) and hidden-platform (days 3-5) versions. On day 6, the platform was removed and the probe trial started. The proportion of time and path that the rats had spent in the target quadrant, in which the hidden escape platform was previously located, was noted. In addition, the times of the rats swimming through the hidden escape platform were also noted.4. Determination of the central insulin signal molecules expressionWestern blot method for IR, IGF-1R, PI3K, p-AKT, and AKT proteins expression in rat hippocampus and cerebral cortex.5. Histopathological examinationHE staining for determining the changes of histomorphology in rat hippocampus and cerebral cortex.6. AGEs/RAGE determinationImmunohistochemistry for determining the AGEs content in rats plasma; RT-PCR determining the changes of RAGE mRNA in rats hippocampus and cerebral cortex.7. Oxidative production determinationROS levels were quantified via the 2’-7’-dichlorofluorescein-diacetate (DCFH-DA) assay; The malondialdehyde (MDA) content, a measure of lipid peroxidation (LPO), was assayed in the form of thiobarbituric acidreactive substances; Protein carbonal content (PCC), a marker of oxidized proteins, was measured spectrophotometrically.8. Anti-oxidant defense system determinationSpectrophotometric methods for determining the activities or content of SOD, CAT, GSH, and GPx in rats hippocampus and cerebral cortex.9. Pro-inflammatory cytokines determinationELISA methods for determining the expression of NF-κB, TNF-a, and IL-1β in rats hippocampus and cerebral cortex.10. PPARy expression determination Western blot method for determining the PPARy expression in rats hippocampus and cerebral cortex.11. Cholinergic activity determinationSpectrophotometric methods for determining the activities or content of AChE and ACh in rats hippocampus and cerebral cortex.12. Statistical analysisWe used SPSS 17.0 for the analysis. All results were presented as mean±SEM. Statistical significance was assessed with one-way analysis of variance (ANOVA) followed by Tukey’s test in the intergroup variation, except the statistical analysis for the acquisition phase of MWM test is repeated-measures ANOVA. Two-way ANOVAs were employed to determine the significance of differences when two factors were assessed. Statistical significance was considered at P<0.05.Results1. Changes of central insulin signaling in fructose-drinking insulin resistant animal models and the intervation effect of lycopene1.1 Lycopene improves insulin sensitivity in fructose-drinking insulin resistant ratsLevels of plasma insulin and HOMA-IR, but not plasma glucose, in fructose-drinking insulin resistant rats were significantly higher than those in control rats (38.8±5.6 mU/L,9.12±0.67 in fructose-drinking insulin resistant rats and 13.7±2.7 mU/L,3.05±0.44 in control rats, respectively, P<0.01). Lycopene treatment significantly reduced the levels of plasma insulin and HOMA-IR (19.1±4.5mU/L and 4.19±0.33, respectively, P<0.05 for both) compared to the level of the fructose-drinking insulin resistant rats. There were no significant difference in the levels of plasma insulin and HOMA-IR between control and lycopene-treated control rats (13.1±3.5 and 2.81±0.25, respectively, P>0.05).1.2 Lycopne improves central insulin signaling expression in fructose-drinking insulin resistant ratsThe protein expression of IR, IGF-1R, PI3K, and phosphorylated AKT (p-AKT) in hippocampus (P<0.05) and cerebral cortex (P<0.05) were significantly decreased in fructose-drinking insulin resistant rats, and rats in lycopene-treated fructose-drinking insulin resistant group revealed a significant higher IR, IGF-1R, PI3K, and p-AKT protein expression in hippocampus (P<0.05) and cerebral cortex (P<0.05) than that in fructose-drinking insulin resistant group. Lycopene-treated group showed a significant decrease for IR, IGF-1R, PI3K, and p-AKT protein expression in hippocampus (P<0.05) and cerebral cortex (P<0.05) compared with control rats. No significant difference of IR, IGF-1R, PI3K, and p-AKT protein expression was observed between control and lycopene-treated control rats (P>0.05).2. The protective effects of lycopene on the brain injury in fructose-drinking insulin resistant rats and the underling mechanism2.1 Lycopene improves cognitive ability in fructose-drinking insulin resistant ratsLearning and memory functions was assessed in Morris water maze test the in each group. Rats in each group exhibited a similar escape latency in 2-day visible-platform test, suggesting no differences in vision or basal motivation (.P>0.05). For 3-day spatial hidden-platform test, the fructose-drinking insulin resistant group rats showed a significant increase in escape latency as compared to those controls (control group and lycopene-treated control group; P<0.01), and the change observed in the fructose-drinking insulin resistance group was partially reversed by administration of lycopene (4 trials/day for 3 days, P<0.01). In the probe trial, fructose-drinking insulin resistant rats had substantially decreased time of swimming through the hidden escape platform compared with both control rats (5.62±1.02 and 14.6±0.19 respectively, P<0..01) and Lycopene-treated control rats (14.9±1.21 respectively, P<0.01). Lycopene-treated fructose-drinking insulin resistant rats had increased times of swimming through the hidden escape platform compared with fructose-drinking insulin resistant rats (10.3±1.01; 12.47±1.24 and 5.62±1.02, respectively, P<0.05). Accordingly, rats in fructose-drinking insulin resistant group showed decreased proportion of path and time through the hidden escape platform, which were in sharp contrast with control and lycopene-treated control rats (P<0.01), Lycopene-treated fructose-drinking insulin resistant rats showed increased proportion of path and time compared with fructose-drinking insulin resistant rats (P<0.05). Notably, there was no significant difference in all above-mentioned data between control and Lycopene-treated control groups (P>0.05), suggesting that lycopene had little or no effect on the cognitive ability of healthy rats.2.2 Lycopene improves brain pathology injury in fructose-drinking insulin resistant ratsIn the CNTL group and LYCO group, rare damaged neurons were seen in the hippocampus and temporal cortex, neurons are alignment and many, the nucleis are obvious. However, neuronal degeneration and karyopycnosis were easily observed in the hippocampus and temporal cortex. The situation of F+LYCO group is between the CNTL group and F group.2.3 The mechanism of lycopene protective effects on the brain injury in fructose-drinking insulin resistant rats2.3.1 Lycopene decreases AGEs/RAGE system in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsThe expression of AGEs protein, RAGE mRNA in hippocampus (P<0.05) and cerebral cortex (P<0.05) were significantly decreased in fructose-drinking insulin resistant rats, and rats in lycopene-treated fructose-drinking insulin resistant group revealed a significant higher expression of AGEs protein, RAGE mRNA in hippocampus (P<0.05) and cerebral cortex (P<0.05) than that in fructose-drinking insulin resistant group. Lycopene-treated group showed a significant decrease for, expression of AGEs protein, RAGE mRNA in hippocampus (P<0.05) and cerebral cortex (P<0.05) compared with control rats. No significant difference of AGEs protein, RAGE mRNA expression was observed between control and lycopene-treated control rats (P>0.05).2.3.2 Lycopene decreases oxidative production in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsROS overproduction, LPO, and PCC were assayed. Rats in fructose-drinking insulin resistant group showed significant higher level of ROS, LPO and carbonyl in both the hippocampus (P<0.05, P<0.01,P<0.01) and cerebral cortex (P<0.01, P<0.01, P<0.05) than control rats. Reduced levels of ROS, LPO, and carbonyl in both hippocampus (P<0.05, P<0.05,P<0.05) and cerebral cortex (P<0.05,P<0.05, P<0.01) were observed in lycopene treated fructose-drinking rats compared with fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant increase of ROS, LPO, and contents of carbonyl proteins in hippocampus (P<0.05,P<0.05,P<0.05) and cerebral cortex (P<0.05,P<0.05, P<0.05) as compared with control rats. There were no significant differences in the levels of the three oxidative damage markers for both brain regions between control and lycopene-treated control rats (P>0.05).2.3.3 Lycopene up-regulates anti-oxidant defense system in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsSOD, CAT and GPx activities of both hippocampus (P<0.01, P<0.05,P<0.05) and cerebral cortex (P<0.05, P<0.01, P<0.05) in fructose-drinking insulin resistant rats were significantly lower than those of control rats, significant increase in SOD, CAT and GPx activities of both hippocampus (P<0.01, P<0.05, P<0.05) and cerebral cortex (P<0.05,P<0.05, P<0.01) in lycopene-treated fructose-drinking insulin resistant rats were observed as compared to fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant decrease of SOD, CAT and GPx activities in hippocampus (P<0.01,P<0.05, P<0.05) and cerebral cortex (P<0.05,P<0.05, P<0.01) as compared with control rats. No difference in SOD, CAT and GPx activities was observed between control and lycopene-treated control rats (P>0.05). Results showed that the levels of GSH in hippocampus (P<0.05) and cerebral cortex (P<0.05) of fructose-drinking insulin resistant rats were significantly lower than those of control animals, and lycopene administration increased the levels of GSH in hippocampus (P<0.05) and cerebral cortex (P<0.05) compared with fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant decrease of GSH level in hippocampus (P<0.01) and cerebral cortex (P<0.05) as compared with control rats. No difference in GSH level was observed between control and lycopene-treated control rats (P>0.05).2.3.4 Lycopene down-regulates pro-inflammation cytokines in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsRats in fructose-drinking insulin resistant group showed significant higher levels of TNF-a, IL-1β,and NF-κB in both the hippocampus (P<0.05, P<0.05, P<0.01) and cerebral cortex (P<0.01, P<0.05,P<0.05) than control rats. Reduced levels of TNF-a, IL-1β and NF-kB in both hippocampus (P<0.05, P<0.05, P<0.01) and cerebral coxtex (P<0.05, P<0.01, P<0.05) were observed in lycopene-treated fructose-drinking rats compared with fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant increase of TNF-a, IL-1β,and NF-κB in hippocampus (P<0.05,P<0.05,P<0.05) and cerebral cortex (P<0.01, P<0.05, P<0.05) as compared with control rats. There were no significant differences in the levels of the three pro-inflammatory cytokines for both brain regions between control and lycopene-treated control rats (P>0.05).2.3.5 Lycopene up-regulates PPARy expression in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsThe protein expression of PPARy in hippocampus (P<0.05) and cerebral cortex (P<0.01) were demonstrated significant decreases in fructose-drinking insulin resistant rats, and rats in lycopene-treated fructose-drinking insulin resistant group revealed a significant higher PPARy protein expression in hippocampus (P<0.01) and cerebral cortex (P<0.05) than that in fructose-drinking insulin resistant group. Lycopene-treated group is likely to show significant decrease of PPARy protein expression in hippocampus (P<0.05) and cerebral cortex (P<0.01) as compared with control rats. No significant difference was observed between control and lycopene-treated control rats (P>0.05).2.3.6 Lycopene improves cholinergic activity in hippocampus and cerebral cortex of fructose-drinking insulin resistant ratsFructose-drinking insulin resistant group rats showed significantly higher AChE activity in the hippocampus (P<0.05) and cerebral cortex (P<0.01) than control rats. Reduced AChE activity in both brain regions in lycopene-treated fructose-drinking insulin resistant rats were observed in the hippocampus (P<0.01) and cerebral cortex (P<0.05) compared with fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant increase of AChE activity in hippocampus (P<0.05) and cerebral cortex (P<0.01) as compared with control rats. No significant difference in AChE activity was observed between control and lycopene-treated control rats (P>0.05). The ACh content in hippocampus (P<0.01) and cerebral cortex (P<0.01) were significantly decreased in fructose-drinking insulin resistant rats compared with that in the controls. Lycopene administration considerably increased ACh content compared to those in hippocampus (P<0.05) and cerebral cortex (P<0.05) of the fructose-drinking insulin resistant rats. Lycopene-treated group is likely to show significant decrease of ACh level in hippocampus (P<0.05) and cerebral cortex (P<0.05) as compared with control rats, and no significant difference was found in ACh level among both control group and the lycopene-treated control group (.P>0.05).Conclusion1. Fructose-drinking induced peripheral insulin resistance model has good repeatability and stability, which is an ideal model for insulin resistance mechanisms and intervention research. Lycopene can improve insulin sensitivity in insulin resistant rats.2. The abnormalities of central inuslin signaling pathway are in insulin resistant model, lycopene can improve the central insulin signaling. Enhanced IR, IGF1-R, PI3K, and PKB/AKT expression may be the important mechanism of the protect effect of lycopene in the insulin resistant brain.3. Cognitive function decline, decrease of AChE activity and ACh content exist in the hippocampus and cerebral cortex of insulin resistant rats, and lycopene can improve these effects.4. The decrease in the number of neuron cells, disordered arrangement, nuclear enrichment phenomenon, exist in the the hippocampus and cerebral cortex of insulin resistant rats, and lycopene can improve these effects.5. The increase in AGEs expression and RAGE mRNA expression exist in the the hippocampus and cerebral cortex of insulin resistant rats, and lycopene can improve these effects.6. The increase in oxidative product content exist in the the hippocampus and cerebral cortex of insulin resistant rats; The decrease in oxidative defense system content also exist in the the hippocampus and cerebral cortex of insulin resistant rats, and lycopene can improve these effects.7. The increase in TNF-a, IL-1β, and NF-κB levels exist in the the hippocampus and cerebral cortex of insulin resistant rats, lycopene can improve these effects.8. The decrease in PPARy expression exist in the the hippocampus and cerebral cortex of insulin resistant rat, lycopene can improve these effects. PPARγ may be effective target for AD and brain insulin resistance treatment. In conclusion, lycopne has pleiotropic feature and can be drugs for the prevention and treatment of degenerative diseases including AD.SignifanceCompared with our previous study, this present study enhance our understanding about the effect of insulin resistance on central insulin signal pathway, and then AGEs/RAGE system, oxidative stress, neuroinflammation, and cholinergic activity, and thus affect cognitive function, clarifies the realationship between it and AD. The study fully studying pleiotropic and multi-system, multi-target prevention and treatment Features of lycopenbe, thereby provide real value for lycopne in prevention and treatment of cognitive deficits.