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Influence of Baical in on the Expression of Resist in and CD36Induced by Iipopolysaccharide

Author: SunDongMei
Tutor: WanXing
School: Guangzhou University of Traditional Chinese Medicine
Course: Chinese and Western medicine combined with the basis
Keywords: Baicalin Inflammation Atherosclerosis Resistin CD36
CLC: R285.5
Type: Master's thesis
Year: 2012
Downloads: 83
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Abstract


ObjectiveInflammation is a defense response to damage factor of what happens to living tissue of vascular system, which exists in some diseases especially chronic diseases. At present atherosclerosis (AS) is regarded as a chronic inflammatory response in the pathological aspect and its development has always been associated with inflammation, chronic inflammation mediated the occurrence and development of the whole process. As a pathological foundation of heart and head vessel diseases, strengthening the research of AS etiology, mechanisms and the prevention and control can delay and reverse the progress, and reduce the incidence and mortality. With the development of research, we found resistin had been associated with AS as an inflammation factor, and promoted macrophages to absorb lipid because of the increase of scavenger receptor CD36and had an important role in obesity and insulin resistance and type2diabetes. Resistin may be a new therapeutic target. And we have confirmed the effect of baicalin on anti-inflammatory and reducing the plaque in the previous studies, but the mechanism has not been clear entirely. The study aims to explore:the effect of baicalin on the expressions of resistin and CD36induced by lipopolysaccharide (LPS);the role of baicalin in the inflammation and lipid disorders.Methods1. Mice macrophage (RAW264.7cells) were cultured in vitroCells were placed in the25c m2cultural flask, with DMEM culture medium including10%fetal bovine serum(FBS),100U/ml penicillin and100μg/ml streptomycin, under the conditions of5%CO2and37℃. When80%had fused, detached them with0.25%trypsin-EDTA solution and passaged them. 2. The inhibition rate of baicalin to RAW264.7cellCells were incubated with culture medium including baicalin of different concentrations (0、1、5,10,50,100mol/L)for24h, then applyed the MTT to detect the inhibition rate of baicalin to the cells.3. The effect of baicalin to macrophage phagocytosing lipid3.1Oil red0dyeingFive groups were divided. The control grouprthe cells were incubated with DMEM culture medium;the model grouprthe cells were incubated with culture medium including LPS0.1μg/ml;the baicalin pretreatment groups:firstly to add different concentrations of baicalin (10,50,100μ mol/L respectively) for1h, then added LPS0.1μg/ml to continue the cultivation for16h. Then removed the old culture medium, changed it and added oxLDL50mg/L for24h, then collected the cells of climbing, dyed with oil red O, collected images and analysis.3.2The content of the intracellular lipidThe experimental groups were as follow:the control group,the model group, the high、middle and low dose of baicalin groups. Among them, the baicalin groups were to add different concentrations of baicalin (10,50,100μ mol/L respectively) for1h firstly, then joined LPS (0.1μ g/ml) for24h. After the end of incubation, used the enzymes method to determine the content of TC, TG.4. The effect of baicalin on the content of resistin in cell supernatantThe experimental groups were similar to3.2, but the concentration of LPS was5u g/ml. After the end of incubation, used elisa to detect.5. The expression of CD36induced byLPS of different concentrations or recombination resistin and the intervention of baicalinLPS of different concentrations (0,0.01,0.1,1,10μ g/ml) stimulated cells for16h;recombination resistin of different concentrations (0,20,50,100ng/ml) for24h. Detected the cell surface protein CD36by FCM. According to the results, determined the best stimulating concentration. Then groups were divided:the control group, the model group and the baicalin intervention groups. Finally detected the CD36expression.6. The effect of baicalin on the mRNA expressions of resisin and CD36CD36:LPS or recombination resistin stimulated cells for0,4,8,12,24h and detected the mRNA expression. According to the result,the best stimulation time was determined, further pretreated with baicalin;Resistin:LPS stimulated cells for0,4,8,12,24h and detected the mRNA expression. According to the result the best stimulation time was determined, further pretreated with baicalin to detect.Results1. The mice macrophages (RAW264.7cells) were cultured in vitroThe RAW264.7cells, round or irregular, had a "cobblestones" style.2. The inhibition rate of baicalin to the proliferation fo RAW264.7cellWith the increasing concentration of baicalin, the inhibition rate increased, in a dose-dependent manner. The IC50of baicalin was4.44×104μ mol/L. In the following experiment the concentrations of baicalin used (maximum100μ mol/L) were in this range, which could avoid the effect of baicalin itself to the cells.3. The effect of baicalin on macrophage phagocytosing lipid3.1Oil red0dyeingIt had been found that compared with the normal group, the intracellular lipid droplets increasd in the model group. Pretreated with the high, medium and low concentrations of baicalin, the intracellular lipid droplets significantly reduced compared with the model group, and showed a dose-dependent trend.3.2The quantity of the intracellular lipidIt showed that the quantity of TC and TG increased in the model group compared with the control group (P<0.05, P<0.01). Pretreated with baicalin, the quantity of TC and TG in high and medium dose groups descended compared with the model group (P<0.05).4. Effect of baicalin on the content of resistin in cell supernatant.LPS5μ g/ml could make the cells secrete more resistin than the control group (P<0.05). Baicalin of high and midle concentrations could restrain the expression of the resistin induced by LPS (P<0.05)5. The expression of CD36induced byLPS of defferent concentrations or recombination resistin and the intervention of baicalinThe expression of CD36declined with the increased concentrations of LPS, and was in a rising trend with the increased concentrations of recombination resistin, LPS0.1μg/ml or resistin100ng/ml was the most obvious increase(P<0.01). Baicalin in the high, medium and low dose group could significantly reduce the rise of CD36induced by LPS (P<0.01).6. The influence of baicalin on the mRNA expression of resistin and CD36It showed that the resistin and CD36mRNA expressions were extremely low in normal circumstances. After LPS stimulation, resistin and CD36expressions raised, and the stimulation for24h were the most obvious increase compared with the control (P<0.01). Pretreated with baicalin, the low dose group had no difference with the LPS group(P>0.05), the high and middle groups could reduce the resistin and CD36mRNA expressions (P<0.05). In addition, the stimulation of recombination resistin for24h was the obvious increase compared with the control(P<0.05).Baicalin of high and medium groups could reduce the mRNA expression of CD36induced by recombination resistin (P<0.05),but the low group had no effect.Conelus ionsThis study adopted the method of vitro experiments to discusse the mechanism of baicalin inhibiting AS. The results showed that LPS could make intracellular lipid gather together, resistin and CD36of macrophage increase, and recombination resistin could promote the protein and mRNA expression of CD36. Baicalin intervention reduced intracellular lipid, and the protein and mRNA expressions of resistin and CD36indused by LPS, and inhibitde the mRNA expression of CD36stimulated by recombination resistin.So we conclude that may be LPS promote macrophages phagocytose lipid especially oxLDL to form the foam cells and make lipid metabolism disorder by increasing the expression of resistin, and then CD36rises up.CD36as the receptor of oxLDL, their binding causes intracellular lipid accumulation. Baicalin can restrain the expressions of resistin and CD36induced by LPS, and inhibit the expression of CD36stimulated by recombination resistin. That is to say baicalin can restrain inflammatory reaction and the formation of foam cells which could be one of the mechanisms of baicalin restraining AS.

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