| Obesity is excessive accumulation of body fat state caused by environmental and genetic factors,which is easy to diabetes,coronary heart disease and respiratory distress syndrome.With the development of social economy and the change of life style,the obesity has become serious public health problem.Depression is one kind of emotional disorder which is characterized by slow thinking,reduced speech lack of pleasure and cognitive impairment.WHO predicts that depression will be the second biggest world's major non fatal diseases in 2020.Depression is a heavy burden to the family and society which prone to combine with diabetes and other metabolic related diseases,endanger the physical and mental health of patients,living and working ability.Epidemiological survey found that the risk of depression in obese patientsincreased significantly,while the prevalence of obesity in patients with depression was higher than that of the general population.Other studies have shown that the same candidate genes for depression and obesity.Comorbidity of depression and obesity seriously affects the health of patients,resulting in adverse outcomes.Leptin is a secreted by adipose tissue protein hormones,inhibit appetite,reduce energy intake,increase energy consumption,inhibit fat synthesis,its physiological function is mainly mediated through the receptor B(LepRb).The increase of peripheral leptin level and the decrease of leptin in central nervous system are resulted from the decrease of central/plasma leptin ratio,which is called leptin resistance.In recent years,some scholars have found that patients with depression have different levels of leptin in normal weight change,such as depression and plasma leptin level in patients with elevated leptin interaction and depressive symptoms is regulated by the degree of abdominal obesity.The absence of LepRb in hippocampus can cause depressive like behavior and the effect of leptin injection in brain has antidepressant effect.These studies suggest that leptin may play an important role in the regulation of depression.However,the mechanism of leptin in obesity and depression is not clear.Obesity leads to chronic inflammation in the body for a long time,and immune mediated inflammation is one of the important reasons leading to obesity.The cytokines in the brain are closely related to the integrity of neurons,and play an important role in the synaptic remodeling.The study found that elevated inflammatory cytokines lead to depression,anorexia,fever,sleep changes and social decline and other depressive behavior.The persistent elevation of inflammatory cytokines may affect the function of neurotransmitters,leading to neurological disorders such as depression.Local injection of cytokines can induce the occurrence of neuropsychiatric symptoms and behavioral changes in patients with depression.More recently,scholars have pointed out that the free fatty acids can increase the level of inflammatory cytokines.IL-1?,IL-6 and TNF-a are considered to be the key factors mediating the depression like behavior and cognitive impairment in inflammatory cytokines.NF-?B is a key regulator of the TLR pathway and is also an important regulator of stress disorder and depression.However,there is no systematic study on the role of inflammatory cytokines in the comorbidity of obesity and depression.High energy food intake is an important factor leading to obesity,so that high-fat diet(HFD)feeding is commonly used to establish the animal model of obesity.At the same time,chronic unpredictable mild stress(CUMS)model is an effective method to establish the animal model of depression,which leads to the loss of appetite and the ability to explore the new environment,such as depression and anxiety symptoms.The purpose of this study was to study obesity induced by a high-fat diet combined with chronic unpredictable mild stress animal model of diet and high-fat establishment of obesity and depression,the concept of validity by behavioral test and evaluation of animal model,and plasma Leptin,LepRb,IL-1?,IL-6,TNF-a and NF-??B protein level and mRNA level.To elucidate the role of Leptin/LepRb and inflammatory factors in comorbid obesity and depression induced by HFD and CUMS.1.Objectives(1)The apparent validity of an animal model of comorbid obesity and depression induced by high-fat diet and chronic unpredictable mild stress.(2)The role of Leptin/LepRb in depression and anxiety like behavior and cognitive impairment in rats withobesity and depression.(3)The role of IL-1,IL-6,TNF-a and NF-?B in hypothalamus and prefrontal cortex in depression and anxiety like behavior and cognitive impairment in rats with obesity and depression.2.Methods(1)Animals68 Wistar rats in experimental group were fed for 7 days.The rats were randomly divided into 2 groups according to the weight of the rats in the experiment group(n ?68).The control group(Regulate diet,RD)was 24,and the high fat diet group was 44.At the end of the eighth week,the weight of rats in high fat diet group was compared with that of control group,of which 26 rats in high fat diet group were treated with diet induced obesity DIO).At the beginning of ninth week,the rats in the RD group and 26 DIO rats were randomly divided into 2 groups,and the rats in the 1 groups were treated with chronic unpredictable mild stress for 21 days.Therefore,there were four groups in the study:control group(Ctr,n=12),CUMS group(CUMS,n=12),obesity group(Ob,n=13),combined obesity and CUMS group(Co,n=13).(2)Chronic unpredictable mild stressExperimental rats were received chronic unpredictable mild stress(CUMS).Stimulation methods include:fasting 8h,water deprivation 8h,45 degree oblique cage,group feeding,circadian rhythm reversed,wet squirrel cage,shaking the 20min level.Every day,a method was used to stimulate rat,the process lasted 3 weeks.(3)The behavioral testsSucrose preference test,open field test,the elevated plus maze and Morris water maze test were used to evaluated depressive and anxiety like behaviors and memorydamage.(4)Weight and fat/body ratioWeight of all rats was measured once a week at a fixed time.Fat/body weight percentage(%)=([retroperitoneal fat(g)+ omental fat(g)+ epididymal fat(g)]/[weight](g))×100%.(5)Serum levels of total cholesterol,triglyceride,high-density lipoprotein and low-density lipoprotein were measured by LX-20 automatic biochemical analyzer.(6)Elisa assay was used to determine the concentration of plasma leptin in rats,and the expression of inflammatory factors in hippocampus and prefrontal cortex was determined by IL-1?,IL-6,TNF-a and NF-?B Elisa kit.(7)ImmunohistochemistryImmunohistochemistry to detect the expression of LepRb in paraffin embedded brain tissue into coronary direction of thickness of 5 mu m slice expression by immunohistochemistry staining detection of LepRb region of hippocampus and hypothalamus,randomly selected 5 high magnification view of cell counting and cells showed brown granules.(8)Western BlotThe expression of LepRb was detected by Western blot respectively ice hippocampus and hypothalamus 30mg homogenate after centrifugation,the supernatant protein concentration was measured in each sample,the same amount of 40ug protein,isolated by SDS-PAGE 12%and transferred to PVDF membrane,skim milk buffer for 1 hours,and then adding a resistance 4 degrees overnight.The next day after washing three times,add the resistance of the room temperature for 1 hours,then add to enhance the development of light emitting liquid.The optical density of the strip was recorded and analyzed using c-digit.(9)Real time quantitative PCRReal time quantitative PCR detection of LepRb in hypothalamus and hippocampus,hippocampus,prefrontal IL-1?,IL-6,TNF-a and NF-?B expression to extract total RNA from tissues using Trizol,reverse transcription and quantitative real-time PCR using standard procedures and reagents.Using 96 hole plate L reaction system of 95?/5min,the following conditions run for 40 cycles:95?/30s,60?/30s,72?/30s,terminated at 72?/5min.All PCR data analysis using 2-Delta CT method.3.Results(1)11 weeks of high-fat feeding resulted in increased body weight,body fat ratio and blood lipid,3 weeks of chronic unpredictable mild stress decreased rat body weight and body fat ratio,the body weight and body fat ratio of comorbidity rats is higher than the control group and the depression group and lower than the obese group.(2)The combination of high fat diet and chronic unpredictable mild stress induced anxiety and depression like behavior of rats in the preference test,open field test and elevated plus maze test,and cognitive impairment of rats in the Morris water maze test.(3)The Leptin level was elevated in the plasma of rats and the expression of LepRb in hippocampus and hypothalamus decreased with the combination of high fat diet and chronic unpredictable mild stress.Sucrose preference,open field test cross grid number,the elevated plus maze of open arm time percentage and the percentage of Morris water maze and target quadrant time in hypothalamus and hippocampus regions of the LepRb protein level was negatively correlated.(4)The combination of high fat diet and chronic unpredictable mild stress caused increase of IL-1?,IL-6,TNF-a and NF-?B levels in hypothalamus and prefrontal cortex of rats.Sucrose preference,open field test cross grid number,the elevated plus maze of open arm time percentage and the percentage of Morris water maze and target quadrant time in hippocampus and prefrontal regions IL-1?,IL-6,TNF-a and NF-?B protein level was negatively correlated.4.Conclusion(1)Chronic unpredictable mild stress may reverse the obesity induced by high fat diet.(2)The combination of high fat diet and chronic unpredictable mild stress can lead to depression anxiety and cognitive impairment in rats.(3)The abnormal expression Leptin in the peripheral blood and LepRb in the hypothalamus and hippocampus may play an important role in the anxiety,depression and cognitive dysfunction in obesity and depression comorbidity.(4)The abnormal expression of IL-1?,IL-6,TNF-and NF-in the hippocampus and prefrontal cortex may play an important role in the anxiety,depression and cognitive dysfunction in obesity and depression comorbidity. |
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