| With the improvement of living standard and lifestyle changes,the incidence of coronary heart diseases(CHD)is sigificantly increased in recent years by year,and becoming one of the most common diseases which threat people's health and life quality.Dyslipidemia and vascular endothelial injury are considered to be the main pathology of coronary heart diseases.However,with the advance of etiology research,it is proved that chronic inflammation also plays critical roles in the occurrence and development of coronary heart diseases,which can not be ignored.The formation and aggregation of foam cells in intima of myometrial arteries,which formate the atherosclerotic plaques,are the key steps in theprogression of atherosclerosis(AS).Statins,as first-line medication of anti-AS,can effectively reduce the blood lipid levels in patients.Atorvastatin calcium(ATRO)is a potential fat-soluble statin,and is widely used for the treatment of atherosclerosis.However,the toxic and side effects of statins limit their application,such as liver injury,muscle dissolution.Among the toxic and side effects of statins,the elevations of blood bilirubin and transaminases are very common.Bilirubin is usually regarded as an indicator of liver injury,but more and more studies have proved that bilirubin is one of the endogenous antioxidants in eliminating the oxygen free radicals,inhibiting oxidative stress and inflammation,and its functions should be extensively studies.Recently,several studies confirmed that bilirubin could alleviate the development of AS,but its detail roles and mechanismremain to be further studied.Therefore,the aims of this study are to explore the clinical relationship betweenblood bilirubin and the severity,and tother relevant factors of CHD.The in vitro experiments were proposed to detemined the funcitonal roles of biliruin in alleviating the oxidative injury of vascular endothelial cells,and inbiting the formation of foam cells,which is helpful for us to understand the roles and mechanism of bilirubin in preventing the development of atherosclerosis.AIMS:1.To investigate the significance of the blood bilirubin with the severeity of coronary heart diseases(CHD),and the relationship between the blood bilirubin and the risk factors of CHD.Moreover,to analyze the relationship between the elevation of blood bilirubin induced by atorvastatin calcium(ATRO)and the risk factors of CHD.2.To investigate the roles of biliruin in the fatty acid-induced oxidative stress,inflmmation and cell death of vascular endothelial cells,and to determine the protective effects of bilirubin on the vascular endothelial injury.3.To investigate the intervation of bilirubin on the lipid accumulation and oxidative stress of THP-1 derived foam cells,and to detemine the roles of bilirubin in the development of foam cells.METHODS:1.After strictly screening,we retrospectively analyzed 276 cases of newly diagnosed patients with coronary heart disease inin our hospital.According to the blood bilirubin levels,those patients were divided into the high bilirubin group(TBIL> 15.9?M,97 cases)and the low bilirubin group(TBIL<15.9?M,179 cases),and Gensini score system ws used to assess the severity of coronary heart disease.The correlation between the blood bilirubin and the severity of CHD,blood CRP and uric acids were analyzed.Furthermore,according to the dose of atorvastatin calcium(ATRO),the patients were divided into 20mg/d group(158 cases)and 40mg/d group(118 cases)and the changes of bilirubin and related risk factors were observed before and after statin treatment.2.The vascular endothelial cells were isolated and cutured,and induced with high concentration(200 ?M)of oleic acid(OA)and palmitic acid(PA).After treatment with various concentration of bilirubin,the cell numbers were counted,and the MDA,4-HNE levels and the SOD activity were measured.These experiments were proposed to determine the protective roles of bilirubin in the fatty acid-induced oxidative injury and cell death of vascular endothelial cells..3.The THP-1 derived foam cells were induced using ox-LDL and LPS,the cellular triglyceride(TG)were determined after treated with bilirubin.Moreover,the Oil Red O and Bodipy 493/503 stainings were performed to observe the size and number of lipid droplets(LDs)in foam cells.The total proteins of foam cells were extracted,and the expression of LD-associated proteins were detected by Western blot.These experiments could determine the roles of bilirubin in foam cell formation.RESULTS:1.The levels of serum bilirubin in CHD patients were lower compared to normal control population.The levels of serum total,indirect and direct bilirubin in the CHD patients were lower than those in the control group(P <0.01).Moreover,the levels of C-reactive protein and serum uric acid in coronary heart disease group were significantly higher than those in the control group(P <0.01).2.The levels of serum bilirubin were negatively correlated with the severity of coronary heart diseases.The severity of coronary heart diseases was assessed using Gensini score system,and the correlation of serum bilirubin and the severity of CHD.The results showed that the severity of CHD was negatively correlated with the levels of serum bilirubin.In addition,the results of Pearson correlation analysis indicated that the levels of serum bilirubin were also negatively correlated with the f serum CRP and uric acid levels.3.ATRO elevated the serum direct bilirubin.The results of blood chemistry analysis were collected and analyzed from the patients before and 1 month after ATRO treatment.The results indicated that the levels of alanine aminotransferase,aspartate aminotransferase,total bilirubin and direct bilirubin were significantly increased after treatment with 20 mg/d and 40 mg/d ATRO(P <0.05).Meanwhile,the levels of CRP and serum uric acid in coronary heart disease were significantly lower than those before ATRO treatment.Tthere was no significant difference in bilirubin,CRP,serum uric acid,blood pressure and BMI between patients different dose groups(P> 0.05).However,the elevation of high-density lipoprotein(HDL)and the reduction of low-density lipoprotein(LDL)in patients with 40 mg/d ATRO were more significant than that in patients with 20 mg/d ATRO(P <0.05).4.Bilirubin alleviated the oxidative injury of vascular endothelial cells induced by fatty acids.The mouse vascular endothelial cells were isolated and cultured,and the results indicated that 0.5 ?.5 /L bilirubin could alleviate the cell death of vascular endothelial cells induced by high concentration(200?M)oleic acid(OA)and palmitic acid(PA).The results of oxidative stress analysis showed that 0.5 ?M bilirubin could significantly reduce the elevation of 4-Hydroxy-2-nonenal(4HNE)and malondialdehyde(MDA),but increase superoxide dismutase(SOD)activity in fatty acid-induced vascular endothelial cellsd.These results indicated that bilirubin could inhibit the fatty acid-induced oxidative injury of vascular endothelial cells.5.Bilirubin inhibited the foam cell formation.THP-1 cells were induced with oxidized low-density lipoprotein(ox-LDL)and lipopolysaccharide(LPS),and the results of triglyceride(TG)determination using commercial kits showed that bilirubin could significantly decrease the TG contents(P<0.05),which indicated that bilirubin could reduce the lipid accumulation in foam cells.The results of Oil Red O and Bodipy 493/503 staining showed that bilirubin could decrease the number and sizes of lipid droplets(LDs)in foam cells.Moreover,the analysis of oxidative stress showed that bilirubin could significantly reduce the elevation of 4HNE and MDA levels,but increase SOD activity in foam cells induced by ox-LDL and LPS.These results indicated that bilirubin could inhibit the foam cell formation.Conclusion:In this study,we found that the levels of serum bilirubin l in CHD patients were significantly lower than those of normal control population,and the levels of serum bilirubin were correlated with the severity of CHD.Furthermore,atorvastatin calcium could elevate the serum bilirubin,which were consistent with several CHD risk factors,such as CRP and serum uric acids.The in vitro experiments confirmed that bilirubin could protect the vascular endothelial cells from fatty acid-induced oxidative injury,improve cell viability.Moreover,bilirubin could also inhibit the macrophage-derived foam cell formation.This study will help us to further understand the role of bilirubin in cardiovascular protection,and provides a new clue for the therapy of atherosclerosisn. |
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