| ObjectiveIn the past decades,the incidence of overweight and obesity in China has been on the rise and has become a serious public health problem.In recent years,a series of evidences have shown that body mass index(BMI)≥30 kg/m2 is associated with the development of neurodegenerative diseases such as Alzheimer’s disease(AD).The typical pathological features of AD are the massive loss of amyloid β-peptide(AP),neurofibrillary tangles(NFTs)and cholinergic neurons in the brain,which are mainly manifested as the decline of learning ability and memory loss.With the deepening of the research on the relationship between AD and obesity,more and more studies have shown that patients with early AD occur brain insulin resistance and glucose metabolism disorders.Persistent hyperinsulinemia caused by obesity will damage blood brain barrier(BBB),resulting in abnormal insulin transport.According to those recent studies in a rat model,chronic HFD consumption for 8 weeks only induced peripheral insulin resistance but did not initially cause brain insulin resistance.However,the extension of HFD consumption to 12 weeks could lead to the development of brain insulin resistance.Insulin-degrading enzyme(IDE)can not only degrade insulin,but also the only protease that can degrade Aβ in the body.However,Aβ has a lower affinity with IDE than insulin.Under hyperinsulinemia,excess insulin would competes with Aβ for IDE,and decrease the AP degradation and facilitate its accumulation in the brain.As an alternative to ozone-depleting solvents(ODS),1-bromopropane(1-BP)is widely used in industry.With the increase of 1-BP production and its wide application,the number of contact people has increased year by year.Current research indicates that 1-BP has a neurotoxicity.Since the application of 1-BP,cases of 1-BP never poisoning have been reported continuously.1-BP can activate glial cells which resident in brain,and triggering an inflammatory response.When stimulated,microglia cells rapidly undergo "Amoebus-like" changes and secrete proinflammatory factors such as interleukin-1β(IL-1β),interleukin-10(IL-10),and transforming growth factor leukocytes-β(TGF-β).Then,microglia recruited astrocytes in the form of "crosstalk".The interaction between astrocytes and microglia increases,eventually leading to neuronal damage and induced neurodegenerative diseases.Methods1.Grouping and processing of experimental animals:48 male SPF Wistar male rats weighing 70-90g.After acclimation for 1 week,the rats were randomly divided into a control group(Con),1-bromopropane exposure group(1-BP),high-fat diet group(HFD),high-fat diet+1-bromopropane exposure group(HFD+1-BP),12 rats in each group.During the experiment,the Con group and the 1-BP exposure group were fed with chow diet,and the HFD group and the HFD+1-BP group were given high-fat diet(D12451,with a fat supply ratio of 45%).The weight and food consumption of rats were measured once every week.After 6th weeks,the rats in 1-BP exposed groups were treated 1-BP exposure,with the feeding pattern as before and till to the end of the experiment.1-BP was dissolved in corn oil and orally administered at 100 mg/kg.bw,the chosen dose did not result in the obvious CNS damages.The rats in the control group and single HFD group,were given an equivalent volume of corn oil.2.Sucrose prefer test(SPT):SPT was conducted in the 1st week of the formal experiment to detect the behavior and preference of rats.At the 16th week,the SPT experiment was conducted again to compare whether the results of the two SPT experiments had changed.3.Glucose tolerance test(GTT)and insulin tolerance test(ITT):At the 6th week,blood was collected from the tail vein for GTT and ITT.The interval between the two tests was 5 days.After fasting for 6 hours,the blood glucose was detected with the blood of tail vein.At the 15th week,the tail vein was bled for GTT experiment and ITT experiment again,the interval between the two experiments was 5 days.4.Morris Water maze(MWM)experiment:The MWM was carried out at the 16th week.During the MWM,both escape latency,total swimming distance,swimming speed and the number of crossing platforms were recorded.5.Histopathology:Frozen sections of the brains of each group were prepared and the morphology of brain neurons and microglia were observed by immunohistochemistry.6.Western blot detection:We detected the relative protein expression of IRS1,PI3K,AKT,p-AKT,GLUT1,GLUT3,GLUT4,Iba-1,GFAP,Aβ,BACE,PS1,IDE in cortex and hippocampus.7.Isolation and identification of exosomes:The exosomes of brains were separated by ultracentrifugation,and identified by the morphology under transmission electron microscopy,the particle size,as well the marker proteins such as.Flotillin-1,Flotillin-2,Alix and IL-1β.Results1.The combined effect of HFD and 1-BP on the body weight of rats:Compared with the Con group and the 1-BP group,the body weight of the HFD group and the HFD+1-BP group increased significantly(P<0.05).2.The combined effect of HFD and 1-BP on glucose metabolism in rats:2.1 GTT and ITT of 6th Week:Compared with two chow diet groups(Con group and 1-BP group),glucose tolerance curves of two high-fat diet groups(HFD group and HFD+1-BP group)were significantly increased(P<0.01).It is suggested that a high-fat diet would disturb glucose metabolism in rats.2.2 GTT and ITT of 15th Week:In the GTT experiment,compared with the Con group,the AUC of the 1-BP group,HFD group and HFD+1-BP group all increased,of which the AUC of HFD group and HFD+1-BP group were significantly higher than that of the Con group(P<0.01,P<0.05);in the ITT experiment,the AUC of the HFD and HFD+1-BP groups were significantly higher than that of the Con and 1-BP groups(P<0.01).Comparing this result with the experimental results of the results in 6th week,we found that the glucose tolerance curve and the insulin tolerance curve of the HFD+1-BP group changed significantly.The two curves of the HFD+1-BP group had an increasing trend compared to the HFD group.Compared with Con group,the two curves of 1-BP group also have the same trend.These results suggest that 1-BP exposure can aggravate the glucose metabolism disorder and insulin resistance caused by HFD.3.The combined effect of HFD and 1-BP on fasting blood glucose and fasting insulin levels in rats:Compared with Con group and 1-BP group,fasting blood glucose level in HFD group and HFD+1-BP group increased significantly(P<0.01);compared with Con group,fasting insulin levels in 1-BP group,HFD group and HFD+1-BP group showed an upward trend,and fasting insulin levels in HFD+1-BP group increased significantly(P<0.05).It is suggested that HFD and 1-BP caused insulin resistance.4.The combined effect of HFD and 1-BP on rat pancreas:There were no obvious pathological changes in HE staining of pancreas.5.The combined effect of HFD and 1-BP on the classical insulin signaling pathway in the rat brain-insulin/IRS 1 PI3K/AKT:Compared with the Con group,the expression level of IRS 1,PI3K and p-AKT in the HFD+1-BP group were significantly reduced(P<0.01,P<0.05).It is suggested combined effect of HFD and 1-BP impaired insulin signaling pathway in the brain.6.The combined effect of HFD and 1-BP on glucose transporter in rats brain:Compared with Con group,the expression levels of GLUT1 and GLUT3 in cortex and hippocampus of HFD+1-BP group were significantly reduced(P<0.01,P<0.05);compared with Con group,the expression level of GLUT4 in hippocampus of HFD+1-BP group was significantly reduced(P<0.05),but there was no significant difference in expression level of GLUT4 in cortex(P>0.05),It suggests that there was a glucose transport disorder in the brain.7.The combined effect of HFD and 1-BP on neuroethology in rats:7.1 Sucrose prefer test:SPT of 1St week,compared with Con group,the behavior of rats in 1-BP exposure group,HFD group and HFD+1-BP group had no significant change(P>0.05);SPT of 16th week,compared with Con group,the behavior of rats in 1-BP group and HFD group showed abnormal behavior but no statistically significant difference compared with Con group.However,the behavior of HFD+1-BP group was significantly abnormal(P<0.05).It is suggested that the combined action of HFD and 1-BP can disturb behavior and preference of rats.7.2 Water maze test:Compared with Con group,the escape latency and total swimming distance of rats in 1-BP group,HFD group,and HFD+1-BP group were prolonged.Compared with Con group,the escape latency and swimming distance in HFD+1-BP group were significantly increased(P<0.05);in the space exploration experiment,compared with the Con group,the number of crossing platforms of rats in the 1-BP group,HFD group,and HFD+1-BP group were decreased,and the difference between the HFD+1-BP group and the Con group was statistically significant(P<0.05).It is suggested that the combined effect of HFD and 1-BP impaired learning and memory ability of rats.8.The combined effect of HFD and 1-BP leads to the loss of neurons and the reduction of dendritic spines in the rat brain:Compared with the Con group,the number of neurons in the cerebral cortex and hippocampus CA1 and CA3 of the HFD+1-BP group is obviously reduced(P<0.05).Compared with the Con group,the expression level of NeuN in HFD+1-BP group was significantly reduced(P<0.01,P<0.05),and dendritic spines on a single neuron decreased.It is suggested that the combined effect of HFD and 1-BP led to neuronal damage.9.The combined effect of HFD and 1-BP leads to the deposition of Aβ protein in the rat brain:Compared with the Con group,the expression of Aβ,APP,BACE and PS 1 protein expression of HFD+1-BP group significantly increased in the cerebral cortex and hippocampus(P<0.05);and IDE expression decreased(P<0.05).It is suggested that the combined effect of HFD and 1-BP cause Aβ protein deposition in the rat brain.10.The effect of the combined effect of HFD and 1-BP on rat glial cells:Compared with the Con group,the microglia number of the HFD+1-BP group was significantly increased(P<0.01).Compared with Con group,the expression of Iba-1 in the cortex and hippocampus of the HFD+1-BP group increased significantly(P<0.01).Compared with the Con group,the expression of GFAP of the HFD+1-BP group in cortex and hippocampus increased significantly(P<0.01,P<0.05).These results suggested that both microglia and astrocytes in the rat brain were activated.11.The combined effect of HFD and 1-BP led to the release of a large amount of exosomes containing inflammatory factors in rat brain:Compared with the Con group,the expression level of IL-1β in the exosomes extracted from the brain tissue of the HFD+1-BP group significantly increased(P<0,05).It is suggested that the combined effect of HFD and 1-BP aggravate the neuroinflammatory reaction.Conclusion1.The combined effect of obesity and 1-BP can aggravate the disorder of glucose and lipid metabolism in rats,and lead to insulin resistance in rat brain by changing the insulin/IRS/PI3K/AKT signaling pathway.2.The combined effect of obesity and 1-BP can increase the production of Aβ in rat brain,and enhanced inflammatory response and neuron loss.3.The combined effect of obesity and 1-BP can can enhance the neurotoxicity of low-dose 1-BP,leading to neurobehavioral changes. |
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