黄素单加氧酶3及其代谢产物水平的变化对高原地区胆囊胆固醇结石形成影响的实验研究

Experimental study of changes in flavin monooxygenase 3 and its metabolite levels in formation of gallbladder cholesterol stone in plateau areas

背景与目的:西藏等高原地区胆囊胆固醇结石(GCS)的发病率为平原地区的数倍,给高原地区居民带来严重困扰。既往研究表明脂质代谢紊乱是GCS形成的始发因素,黄素单加氧酶3(FMO3)及其代谢产物三甲胺-N-氧化物(TMAO)共同参与了GCS的形成(平原地区)。然而,FMO3和TMAO在高原GCS发病中的作用尚不明确。本研究在高原地区建立的小鼠GCS模型上观察FMO3和TMAO水平的变化并分析其潜在的临床意义。方法:将成年雄性C57BL/6J小鼠饲养在西藏那曲(海拔4500 m),随机均分为GCS模型组与对照组。模型组采用高脂饮食(基础日粮+15%脂肪+1%胆固醇+0.5%胆酸)以诱发GCS,对照组采用正常饲料进行饲喂。连续8周后处死小鼠收集标本(模型组以胆囊内出现胆固醇结石为造模成功标准,两组各取20只用于实验)。分别用全自动生化分析仪测定血脂和胆汁生化指标,高效液相色谱法(HPLC)测定血浆TMAO水平,qRT-PCR检测肝脏FMO3 mRNA表达水平,Spearman相关系数分析观测指标间的相关性,多因素Logistic回归分析危险因素。结果:模型组血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、胆汁TC、胆汁酸、磷脂和胆固醇饱和指数(CSI)均明显高于对照组,高密度脂蛋白(HDL)明显低于对照组(均P<0.05)。模型组血浆TMAO和肝脏FMO3 mRNA水平均明显高于对照组(均P<0.05)。Spearman相关系数分析显示,FMO3 mRNA、TMAO与血浆TC、TG、LDL、胆汁TC、胆汁酸、磷脂和CSI均呈明显正相关,与HDL呈明显负相关(均P<0.05)。多因素Logistic回归显示,FMO3 mRNA和TMAO升高是影响高原GCS的危险因素(均P<0.05)。结论:FMO3和TMAO升高可能与高原GCS形成密切相关,机制可能与两者升高导致的脂代谢紊乱有关,这为高原地区GCS的防治提供新的策略及方向。

Background and Aims:The incidence of gallbladder cholesterol stone(GCS)in Tibet and other plateau areas is several times higher than that in plain regions,which brings serious harm to the residents in the plateau areas.Previous studies have shown that the disorder of lipid metabolism is the initial factor for the formation of GCS.Flavin monooxygenase 3(FMO3)and its metabolite trimethylamine-N-oxide(TMAO)are involved in the formation of GCS(plain region).However,the roles of FMO3 and TMAO in the pathogenesis of GCS in plateau areas is not clear.This study was conducted to observe the changes in FMO3 and TMAO levels in mice GCS model created in a plateau region and then analyze the potential clinical significance.Methods:Adult male C57BL/6J mice were reared in Naqu,Tibet(4500 m above sea level)and randomly divided into GCS model group and control group.Mice in model group were fed high-fat diet(basic diet+15%fat+1%cholesterol+0.5%cholic acid)to induce GCS,and those in control group were given normal diet.Eight weeks later,the mice were sacrificed,and the samples were harvested(the presence of cholesterol stones in the gallbladder in model group was a standard for successful model creation,and 20 mice were used in each group).Blood lipids and bile biochemical indexes were measured by automatic biochemical analyzer,plasma TMAO level was measured by high performance liquid chromatography(HPLC),the expression of FMO3 mRNA in liver was detected by qRT-PCR.Spearman correlation coefficient was used to analyze the correlation among the observed variables,and multivariate Logistic regression analysis was used to analyze the risk factors.Results:The serum total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),bile TC,bile acid,phospholipid and cholesterol saturation index(CSI)in model group were significantly higher than those in the control group,while the high-density lipoprotein(HDL)in model group was significantly lower than that in the control group(all P<0.05).The plasma level of TMAO and FMO3 mRNA expression level in the liver in model group were significantly higher than those in control group(both P<0.05).Spearman correlation coefficient analysis showed that FMO3 mRNA and TMAO were positively correlated with plasma TC,TG,LDL,bile TC,bile acid,phospholipid and CSI,and negatively correlated with HDL(all P<0.05).Multivariate Logistic regression showed that the increase of FMO3 mRNA and TMAO were the risk factors for GCS in plateau areas.Conclusion:The high FMO3 and TMAO levels are closely related to the occurrence GCS in plateau areas,and the mechanism may be probably associated with the lipid metabolism disorders caused by their increase.This provides a new strategy and direction for the prevention and treatment of GCS in plateau areas.