| IntroductionAtherosclerosis (AS) is an important disease threatened the health of people , and coronary atherosclerotic heart disease is one of its features, which was called coronary heart disease( CHD) in short. In Europe and America, 1/3 - 1/ 2 individuals died of CHD. In our country, its morbidity is increasing. Insulin resistance (IR) is considered as one of independent risk factors of AS. The individual with IR is often accompaned by hyperinsulinemia, high plasma glucose and dyslipidemia, which are associated with high risk of CHD. Studies have shown that the patient with poor plasma glucose control has a distinct dyslipidemia. Hyperinsulinemia may be a contributive factor to promote the development of AS directly. However, the mechanism and physiopathologic changes of AS are not clear completely.Macrophages play an important role in the development of AS, which uptake lipid through scavenger receptors ( SRs) to turn into macrophage - derived foam cells lo deposit under endothelium of artery wall and form atherosclerotic lesion. Most of SRs promote lipid uptake and belong to proathrogenic factors. But scavenger receptor class B type 1 ( SR - BI) , which has recently been found and identified as a high density lipoprotein ( HDL) receptor, is considered as an anti - atherogenic factor. On the one hand, it mediates the selective uptake of cholesteryl esters from HDL. in liver and steroidogenic tissues, on the other hand, it can also stimulate the bi - directional flux of free cholesterol between cells and extracellular lipoprotein particles and plays an important role in elimi-nating the excrescent lipid from the cells in artery wall and the circulation. It has been demonstrated that the rate of cholesterol efflux from various cell types correlates with the expression of SR - BI. In the present study, we observe the change of expression of SR - BI in monocyte/macrophages with different metabolic situations to indicate the probable influence on cholesterol efflux in these conditions.Section A. Changes of Expression of Scavenger Receptor Class B Type I in U937 Monocytes Differentiating to MacrophagesObjectiveIn order to explore the early change of monocyte/macrophages of artery wall and prepare for below experiment, we use phorbol ester (PMA) to differentiate U937 cells to macrophages and detect the changes of expression of SR - BI during this process.Methods1. Cell culture and differentiation U937 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 100U/ml penicillin, lOOjxg/ml streptomycin and transferred every 2-3 days. In order to induce phagocytic differentiation, U937 cells were cultured in the presence of lOOnmol/L PMA for 24, 48 or 72 hours. CD 14 positive cell rate was measured by flow cytometry to identify differentiation efficacy.2. Measurement of expression of SR - BI Expression of SR - BI protein on the cell surface was analyzed by using and - SR - BI polyclonal antibody (Novus Biologicals, Inc) and a labeled streptavidin/biotin kit. Cytomembrane protein was extracted and SR - BI protein expression was detected by Western blotting. Reverse Transcription Polymerase Chain Reaction ( RT - PCR) were used to detect SR - BI mRNA during the PMA - inducing differentiation.Results1. The morphological change and expression of CD14 U937 monocytes were spherical and suspended in the medium. The cells stopped proliferating, attached to the culture dishes, became larger and spindle - shaped after differentiation. CD 14, a kind of differentiation maker of macrophages, was measured during differentiation. Positive cell rate of CD14 significantly increased at 24h after differentiation, which indicated a successful differentiation. ( see table 1)2. Immunocytochemistry was used to determine the expression of SR - BI protein expression and discretional 30 cells in each picture were analyzed by Me-ta Morph Imaging System, the integrated absorptance values ( A value) of SR -BI protein expression in different group were showed in table 1.Table 1Group CD14 positive cell percentage SR - BI protein (A value)control (9.9 ±4.3)% 7.76 ±1.74Differentiating 24h (76.9 ± 10.4)% *' 15.94 ±3. 56"Differentiating 48h (80. 6 ±9. 8) % *' 27. 86 ±4. 39**Differentiating 72h (78.1 ± 13.5) % * *<sub><sub><sub><sub><sub><sub><sub><sub><sub>9.08 ±2.37<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>ii: P <0.01 vs control group3 . Western Blot showed that differentiation resulted ina2.08-(P< 0. 01) , 3.15 - ( P < 0. 01) , and 1. 24 - fold ( P > 0. 05 ) increment in the expression of SR - BI protein at 24, 48 and 72h, compared with U937 undifferen-tiating cells.4. RT - PCR showed that relative SR - BI mRNA expression in different group is 0.112 ±0.006,0.235 ±0. 014,0. 344 ±0. 14 and 0. 138 ±0.010, respectively -24h - and48h - differentiating group significantly increased ( P <0.01).Conclusion1. Most of U937 cells differentiate into macrophages at 24h after treated with PMA.2. SR - BI protein and mRNA was increased after differentiation, reached a peak on 48 hours, and decreased on 72 hours. It is a transcriptional unstable.3. SR - BI expression levels are not associated with CD14 changes. High expression levels of SR - BI in macrophages may be correlated with foam cell formation.Section B. Effects of Glucose and Insulin on Expression of Scavenger Receptor Class B Type I in U937 MacrophagesObjectiveU937 macrophages were treated with different concentration glucose and/or insulin. Measure the changes of expression of SR - BI to explore the influence of hyperglycemia and hyperinsulinemia on monocyte/macrophages of artery wall.Methods1. Cell treatment Three kinds of treatments were used in the experiment. A. glucose treatment: U937 macrophages differentiating for 48 h were incubated with different concentration glucose (5.6, 11.1,16.7 and 33. 3mmol/L) for 24 hours. B. insulin treatment; U937 macrophages were incubated with different concentration insulin (0,10,20,100,200 and 400jxU/ml). C. mixed treatment : U937macrophages were incubated with glucose and/or insulin (5.6mmol/L glucose but no insulin, 5. 6mmol/L glucose + 20n,U/ml insulin, 5. 6mmol/L glucose + 200|xU/ml insulin, 16. 7mmol/L glucose but no insulin, 16. 7mmol/ Lglucose + 20 |xU/ml insulin and 16. 7 mmol/L glucose + 200 |xU/mlinsu-lin).2. Measurement of expression of SR - BI protein by Western blotting.3. Reverse Transcription Polymerase Chain Reaction ( RT - PCR) were used to detect SR — BI mRNA during insulin treatment.Results1. Effect of glucose on SR - BI expression Relative SR - BI protein expression among different group 100% , (97. 5 ± 10. 1) % , (94. 6 ± 9. 3 ) % , (89.7 ±7. 8)% .respectively. There are no statistically significant differences among them (P > 0.05).2. Effect of insulin on SR -BI expression Result showed that 10,20fxU/ ml insulin upregluated SR - BI protein, with relative SR - BI protein 182% and 195% vs control (OpOJ/ml insulin) group. 100, 200 and 400jjiU/ml insulin downregulated SR - BI protein, compared with that treated with basal insulin (P<0.01). However, SR — BI mRNA expression is stable at basal or high insulin levels.3. SR - BI protein was downregulated by high glucose and insulin synerget-ically. At basal insulin level, high glucose had no statistically significant down-regulation effect on SR — BI, while at high insulin level, SR - BI protein expression significantly decreased by high plasma glucose.Conclusion1. The change of glucose concentration itself cant influence SR - BI protein expression.2. Basal physiological insulin levels can promote SR - BI protein expression of macrophages. High levels of insulin significantly downregulates SR - BI.3. Different concentration of insulin had no effect on SR - BI mRNA expression. It is a transcriptional stable change.4. It is one of contributors that high glucose and insulin accelerates atherosclerosis through synergetically downregulating SR - BI protein expression anddecreasing cholesterol efflux of macrophages.Section C. Advanced Glycation End Products Promotes U937 Macrophages Expression of Scavenger Receptor Class B Type IObjectiveDifferent concentration glucose has no significant regulation effect to SR -BI expression, which indicated that acute hyperglycemia in human body could not influence lipid metabolism in artery wall through SR - BI. Whether advanced glycation end products (AGEs) , long - term products of Maillard reaction due to chronic hyperglycemia, can influence macrophages SR - BI expression? In the present study, we learn the effects of AGEs on expression of SR -BI protein and mRNA in U937 macrophages.Methods1. Preparation of AGEs - BSA 2. Og bovine serum albumin ( BSA) was dissolved in 10 ml sodium phosphate buffer with 3. 0 gram D - glucose. Each sample was incubated at 37T! for 90 days, and dialyzed against PBS. Nongly-cated BSA was prepared simultaneously as the same method but no glucose in the sample. AGEs were identified by fluorescence speetrophotometer.2-. Cell treatment U937 macrophages differentiating for 48 h were incubated with different concentration AGEs (100, 200 and 400mg/L) or 400 mg/L BSA as control for 24 hours. Another group of macrophages were incubated with 400mg/L AGEs for different time (0, 6, 12, 24, 48 hours).3. The expression of SR - BI protein and mRNA were detected by Western blot analysis and RT - PCR.
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