The Inflammatory Mechanism Of High Serum Cortisol Concentration Of Chronic Psychological Stress Contributing To Atherosclerosis

PaperⅠThe Inflammatory Mechanism Of High Serum Cortisol Concentration Of Chronic Psychological Stress Contributing To AtherosclerosisBackgroundCardiovascular diseases are common and multi-onset diseases,which have serious effects on the health of human beings.It has been reported that cardiovascular diseases induced by many risks,such as smoking,fatness,diabetes mellitus,hyperlipoidemia,hypertension,and so on.But epidemiological studies suggested that about 40%atherosclerosis(AS) patients had nothing to do with above risks,they concerned with chronic psychological stress.These psychological stress mainly include negative-emotiom,anxiety,work pressure and a lot of psychosocial stress.Thus,it is so significant to study the correlation between psychological stress and AS,which has profound meanings in preservation and treatment of AS.There are three theories about the pathogenesis of AS at present:lipids infiltration,thrombosis and endothelium injury.So far many studies about the relation between the three theories of AS and psychological stress had been done,but which could not elucidate chronic psychological stress how to effect the development and progression of AS.So,this complicated problem will be interpreted by new ideas.Recently,inflammatory theory has been well elucidate the pathogenesis of AS,this theory suggests AS is a chronic inflammatory diseases occurred in aorta wall,and the various stages of AS represent the various stages of inflammatory reactivity.Coincidentally,prolonged chronic psychological stress contributed to the increasing of serum cortisol concentration,and the high cortisol concentration not only has not anti-inflammation effect,but also contributed to inflammatory factors releasing,which promoted the development of AS.If it is true,which inflammatory factors induced by high cortisol concentration contribute the development and progression of AS? And how do these inflammatory factor change in various stages of AS induced by chronic psychological stress?So,we need to establish animal model,which symptoms are conformed to psychological stress patients,and investigate whether some inflammatory factors in correlation with AS,such as monocyte chemoattractant protein 1(MCP-1),macrophage migration inhibitory factor(MIF), tumor necrosis factor-alpha(TNF-α),high sensitivity C-reactive protein(hs-CRP),and so on, relate to high serum cortisol level or not,and the change tendency of these inflammatory markers in various stages of AS induced by chronic psychological stress.Objective(1)To establish a psychological stress animal model which symptoms are conformed to psychological stress patients and convenient for dynamic investigation of the process of AS induced by chronic psychological stress.(2) To compare the change tendency of these inflammatory markers in various stages of AS induced by chronic psychological stress and high cholesterol diet.(3) To observe the inflammatory markers which relate to not only AS but also chronic psychological stress.Methods1.Experiment groups160 healthy male New Zealand white rabbits were randomly divided into 4 groups:normal group(A),chronic psychological stress group(B),high cholesterol group(C) and chronic psychological stress plus high cholesterol group(D),40 in each group.Rabbits in A and C groups were fed standard diet,and.Rabbits in B and D groups were fed an atherogenic diet containing 1%cholesterol.The experiment last for 16 weeks.2.Establishing the animal model80 rabbits in C and D were given chronic and prolonged psychological stress:the first day, rabbits were given noise stimulus for 3h.The second day,rabbits were given rotation stimulus (80r/min) for 60 minutes.The third day,rabbits were given terror stimulus by dog for 60 minutes (a big cage was divided into two rooms by bars,and a dog stays in a room,rabbits stay in another.).The fourth day,rabbits were put into net-bag and hung in cage for 4h.The fifth day, rabbits were given sole-stroke stimulus(110v,1vic/min) for 60 minutes.The sixth day,rabbits were inhibited drinking for 24h.The seventh day,rabbits were inhibited eating for 24h.Seven days make up a cycle,the next cycle increase 10 minutes based the former cycle.The experiment last for 16 weeks. 3.Evaluating the animal model(1) To detect and record the appetites,weight,blood pressure,heart rates,serum cortisol and inflammatory factors levels at the beginning of experiments and at the end of 4w,8w,12w and 16w of experiments.(2) To detect and record the frequency of self-clearing at the beginning and end of experiments.4.Lipids measurementBlood samples were collected from all rabbits at the baseline,the end of the 4^th,8^th,12^th and 16^th week.Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were measured by enzymic method.5.Serum cortisol and inflammatory markers measurementThe blood samples were collected from all rabbits at the baseline,the end of the 4^th,8^th,12^th and 16^th week.Serum corticosterone(CORT),high sensitivity C-reactive protein(hsCRP),and tumor necrosis factor alpha(TNF-α) were assayed using highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.6.High frequency ultrasoundAt the baseline,end of the 8^th,12^th and 16^th week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system.The intima-media thickness(IMT),the end-diastolic luminal diameters(Dd),the end-systolic luminal diameter(Ds),the mean velocity (Vm),peak velocity(Vp),velocity integral(VTI) and the average image intensity(AⅡ) of the abdominal aorta were measured.7.Histopathological stainingThe abdominal aorta was processed and examined by hematoxylin and eosin,and oil red O staining.8.Immunohistochemical stainingThe expression of TNF-α,MIF,ET-1,CRP,MCP-1,eNOS and IL-10 were determined.9.Real-time PCRThe mRNA expressions of TNF-α,ET-1,CRP,MCP-1,eNOS and IL-10 in the abdominal aorta tissue were analyzed.14.Western blot The protein expressions of TNF-α,MIF,ET-1,CRP,MCP-1,eNOS and IL-10 in the abdominal aorta tissue were analyzed.15.Statistical analysisData are expressed as mean±SD for continuous variables and by frequency count and percentage for qualitative variables.The data were tested for normality and homogeneity of variance.Paired Student's t-test was used to assess the significance of any change in various stages,while unpaired Student's t-test(equal or unequal variance assumed accordingly) was used to assess the significance of any change between groups.Photoshop 7.0 was used to analyze image positive staining area.The SPSS was version 13.0.P＜0.05 was considered statistically significant.Results1.General state of the experimental animals40 rabbits in the normal group,37 rabbits in chronic psychological stress group,40 rabbits in high cholesterol group and 36 rabbits in chronic psychological stress plus high cholesterol group completed the study.2.Evaluating of animal model2.1 The change of appetiteCompared with normal group,the appetites of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously reduced(P＜0.05).Compared with the high cholesterol group,the appetites of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously reduced(P＜0.05).The appetites of high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The appetites of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.2 The change of weightCompared with normal group,the weight of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously reduced(P＜0.05).Compared with the high cholesterol group,the weight of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously reduced(P＜0.05). The weight of high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The weight of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.3 The change of blood pressureCompared with normal group,the blood pressure of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,at the end of 8^th,12^th,and 16^th,the blood pressure of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).The blood pressure of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.4 The change of heart ratesCompared with normal group,the heart rates of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.01).The heart rates of high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The heart rates of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.5 The change of self-clearing frequencyCompared with normal group,the self-clearing frequency of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).The self-clearing frequency of high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The self-clearing frequency of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.6 The change of serum lipids levelCompared with normal group,the serum lipids level of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,the serum lipids level of chronic psychological stress group significantly decreased(P＜0.05),but the serum lipids level of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the chronic psychological stress group,the serum lipids level of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).2.7 The change of serum cortisol levelAt the end of 4^th week,compared with normal group,the serum cortisol level of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,the serum cortisol level of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the chronic psychological stress group,the serum cortisol level of chronic psychological stress plus high cholesterol group obviously increased (P＜0.05).The serum cortisol level of chronic psychological stress group in comparison with high cholesterol group didn't obviously change(P＞0.05).At the end of 8^th,12^th,and 16^th week,compared with normal group,the serum cortisol level of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,the serum cortisol level of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).The serum cortisol level of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).2.8 The change of serum hs-CRP levelCompared with normal group,the serum hs-CRP level of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,the serum hs-CRP level of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased (P＜0.05,P＜0.01).Compared with the chronic psychological stress group,the serum hs-CRP level of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).2.9 The change of serum TNF-αlevelAt the end of 4^th week,compared with normal group,the serum TNF-αlevel of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the high cholesterol group,the serum TNF-αlevel of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the chronic psychological stress group,the serum TNF-αlevel of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05). The serum TNF-αlevel of chronic psychological stress group in comparison with high cholesterol group didn't obviously change(P＞0.05).At the end of 8^th,12^th,and 16^th week,compared with normal group,the serum TNF-αlevel of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the high cholesterol group,the serum TNF-αlevel of chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).The serum TNF-αlevel of high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The serum TNF-αlevel of chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).3.High frequency ultrasound measurementCompared with normal group,the IMT and AⅡ%of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).Compared with the high cholesterol group,the IMT and AⅡ%of chronic psychological stress group significantly decreased(P＜0.05).The IMT and AⅡ%of chronic psychological stress group in comparison with high cholesterol group didn't obviously change(P＞0.05).Compared with the chronic psychological stress group,the IMT and AⅡ%of chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).4.Pathologic stainingHE staining showed atherosclerosis plaque formed between 12^th week and 16^th week. Compared with normal group,the plaque of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05, P＜0.01).Compared with the high cholesterol group,the plaque of chronic psychological stress group significantly decreased(P＜0.05).The plaque of chronic psychological stress plus high cholesterol group in comparison with high cholesterol group didn't obviously change(P＞0.05).Red-oil O showed,lipids collection occurred at the end of 4^th week,fatty streak occurred at the end of 8^th week,lipid droplet in plaque occurred at the end of 12^th week,lipid pool in plaque formed at the end of 16^th week.5.Immunohistochemical stainingCompared with normal group,the positive expression of eNOS of high cholesterol group, chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously decreased(P＜0.05).At the end of 4^th week,the positive expression of IL-10,MIF, ET-1 and TNF-αof high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).At the end of 12^th and 16^th week,the positive expression of CRP and MCP-1 of high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the high cholesterol group,at the end of 8^th,12^th and 16^th week,the positive expression of TNF-αof chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).The positive expression of TNF-αof high cholesterol group in comparison with normal group didn't obviously change(P＞0.05). The positive expression of TNF-αof chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).6.Real-time PCRCompared with normal group,the mRNA expression level of eNOS of high cholesterol group, chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously decreased(P＜0.05).At the end of 4^th week,the mRNA expression levels of IL-10,MIF. ET-1 and TNF-αof high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).At the end of 12^th and 16^th week,the mRNA expression levels of CRP and MCP-1 of high cholesterol group, chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the high cholesterol group,at the end of 8^th,12^th and 16^th week,the mRNA expression levels of TNF-αof chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).The mRNA expression levels of TNF-αof high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The mRNA expression levels of TNF-αof chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).7.Western blotCompared with normal group,the protein expression level of eNOS of high cholesterol group, chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously decreased(P＜0.05,P＜0.01).At the end of 4^th week,the protein expression levels of IL-10,MIF,ET-1 and TNF-αof high cholesterol group,chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).At the end of 12^th and 16^th week,the protein expression levels of CRP and MCP-1 of high cholesterol group, chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05).Compared with the high cholesterol group,at the end of 8^th,12^th and 16^th week,the protein expression levels of TNF-αof chronic psychological stress group and chronic psychological stress plus high cholesterol group obviously increased(P＜0.05,P＜0.01).The protein expression levels of TNF-αof high cholesterol group in comparison with normal group didn't obviously change(P＞0.05).The protein expression levels of TNF-αof chronic psychological stress group in comparison with chronic psychological stress plus high cholesterol group didn't obviously change(P＞0.05).Conclusions(1) The rabbit psychological stress model,which is mimic to human diseases and suitable for the observation of chronic psychological stress effecting on AS.(2) Inflammatory factors MCP-1,CRP and TNF-αall trended to rise in the process of AS induced by chronic psychological stress.Inflammatory factors MCP-land CRP trended to rise in the process of AS induced by high cholesterol diet,TNF-αsignificantly increased at the end of 4^th week,but it began to decline at the end of 8^th week,and recover to normal at the end of 12^th and 16^th week.(3) TNF-αnot only related to AS but also related to chronic psychological stress. PaperⅡThe Effects for Macrophages Producing Tumor Necrosis Factor-alpha Protein Induced by Hydrocortisone in Different ConcentrationsBackgroundEpidemiological studies suggested that chronic psychological stress is a key risk that aggravated the development and progression of atherosclerosis(AS),but the mechanism of which is unclear at present.In contrast to other stress hormones,the serum concentrations of cortisol could reflect the character of moods better.Clinic studies reported the serum concentrations of cortisol and inflammatory markers in depress patients were significantly higher than which in the healthy.Tumor necrosis factor-alpha(TNF-α) is a glycoprotein produced by activated macrophage, a cytokine produced in the process of stress response,and which could inspire cascade response of cytokines.The cascade response induced TNF-α,interleukin-1(IL-1),interleukin-6(IL-6) and other cytokines collecting and releasing.Studies indicated that chronic psychological stress could obviously increase the serum level of TNF-α,and which could extend the original inflammatory signal,and then contributed more and more macrophages collecting and infiltrating,and contributed to the development of AS.In paperⅠ,it was identified that the change tendency of TNF-αin coincidence with the change tendency of cortisol in vivo.TNF-αis mainly produced by macrophage.However,in vitro, whether different concentrations of cortisol could affect TNF-αlevel produced by macrophages or not? What about the tendency?Objective(1) To investigate the effects for macrophage producing TNF-αinduced by cortisol in different concentrations;(2) To investigate the effects for macrophage producing TNF-αinduced by cortisol at different time.MethodsTo investigate the effects for macrophage producing TNF-αinduced by cortisol in different concentrations and at different time,our experiment put cortisol in different concentrations(0 ng/mL,675 ng/mL,1250 ng/mL,2500 ng/mL,5000 ng/mL,10000 ng/mL) into medium of macrophage,and incubated for 2h,4h,8h,12h,24h and were observed under inverted microscope.The effectes of cortisol in different concentrations and at different time on TNF-αprotein expression were investigated by western blot.ResultsInverted microscope showed that the normal macrophage cells were at good condition, adherence stable,fusiform or round,tight cell-cell junction,more cleavage phase,less suspension cells,cell shrinkage,intercellular space broaden and more suspension cells in cortisol damage group.675-1250 ng/mL cortisol TNF-αprotein expression significantly increased(P＜0.01);＞1250 ng/mL cortisol TNF-αprotein expression significantly decreased(P＜0.05).Incubated for 24 h is the optimized stimulus for the present study.Conclusion(1) 675-1250ng/mL cortisol TNF-αprotein expression significantly increased,it is the optimized stimulus concentrations.(2)With the incubated time prolonging,TNF-αprotein expression showed a rise tendency,and 24 h is the optimized stimulus for the present study.