OSBPL3在代谢相关脂肪性肝病中的作用及机制研究

The Role and Mechanism of OSBPL3 in Metabolism-related Fatty Liver Disease

目的:探究氧化固醇结合蛋白类似物3(Oxysterol Binding Protein-like 3,OSBPL3)在代谢相关脂肪性肝病中的作用及可能机制。方法:建立肝脏特异性沉默OSBPL3小鼠模型和空载体对照组,分别予以普食和高脂喂养12周。分为正常对照组、OSBPL3沉默组、肥胖对照组、肥胖OSBPL3沉默组。观察小鼠一般情况,Real-time PCR检测脂质合成基因及脂质分解基因mRNA水平,western blot检测Akt/mTOR通路关键蛋白的表达。人HepG2细胞株给予不同浓度油酸(oleic acid,OA)处理,观察油红O染色的变化,western blot检测OSBPL3表达水平。结果:正常对照组与OSBPL3沉默组小鼠各项指标相比无统计学差异(P>0.05);与对照组相比,肥胖对照组及肥胖OSBPL3沉默组体质量、内脏脂肪及内脏脂肪指数较高(P<0.05);与肥胖对照组相比,肥胖OSBPL3沉默组体质量、内脏脂肪及内脏脂肪指数较低(P<0.05)。与对照组相比,肥胖对照组及肥胖OSBPL3沉默组总胆固醇(Total cholesterol,TC)、甘油三酯(triglycerides,TG)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)较高(P<0.05);与肥胖对照组相比,肥胖OSBPL3沉默组TC、TG、LDL-C及HDL-C较低(P<0.05)。与正常对照组与OSBPL3沉默组小鼠SREBP-1C、FAS及PPARα表达水平相比无统计学差异(P>0.05);与对照组相比,肥胖对照组及肥胖OSBPL3沉默组SREBP-1C、FAS较高,PPARα表达水平较低(P<0.05);与肥胖对照组相比,肥胖OSBPL3沉默组SREBP-1C、FAS表达水平较低,PPARα表达水平较高(P<0.05)。与对照组相比,肥胖对照组Akt及mTOR磷酸化表达水平较高(P<0.05);与肥胖对照组相比,肥胖OSBPL3沉默组Akt及mTOR磷酸化表达水平较低(P<0.05)。随着OA作用浓度的升高,油红O染色逐渐加深。与0μmol/L油酸相比,油酸以剂量依赖性方式增加HepG2细胞OSBPL3 mRNA水平(P<0.05)。结论:OSBPL3能够调控脂质代谢的表达,可能通过调控Akt/mTOR信号通路发挥生物学功能,有望为研究NAFLD疾病发生发展及治疗提供参考依据。

Objective:To explore the role of OSBPL3 in mediating metabolism-related fatty liver disease and its possible mechanism.Methods:Study on the role and mechanism of OSBPL3 in metabolism related fatty liver disease.Hepatic specific silencing OSBPL3 mouse model and empty vector control mouse model were established.They were fed normal diet and high fat diet for 12 weeks respectively.They were divided into normal control group,OSBPL3 silencing group,obese control group and obese OSBPL3 silencing group.To observe the general situation of mice,the lipid synthesis genes and lipid decomposition gene mRNA were detected by Real time PCR,and the key proteins of Akt/mTOR pathway were detected.Human HepG2 cell lines were treated with OA at different concentrations,and the staining changes of O were observed after treatment with different oleic acid concentrations.OSBPL3 expression levels were detected by resting western blot assay.Result:There were no significant differences in all indexes between normal control group and OSBPL3 silencing group(P>0.05).Compared with the control group,the body mass,visceral fat and visceral fat index of the obese control group and the obese OSBPL3 silent group were higher(P<0.05).Compared with the obese control group,the body mass,visceral fat and visceral fat index in the obese OSBPL3 silencing group were lower(P<0.05).Compared with the control group,the TC,TG,LDL-C and HDL-C of the obese control group and the obese OSBPL3 silent group were higher(P<0.05).Compared with the obese control group,the TC,TG,LDL-C and HDL-C in the obese OSBPL3 silenced group were lower(P<0.05).There were no significant differences in the expression levels of SREBP-1C,FAS and PPARα compared with normal control group and OSBPL3 silencing group(P>0.05).Compared with the control group,the levels of SREBP-1C,FAS and PPARα were higher in the obese control group and the obese OSBPL3 silenced group(P<0.05).Compared with the obese control group,the expression levels of SREBP-1C and FAS in the obese OSBPL3 silenced group were lower,and the expression levels of PPARα were higher(P<0.05).Compared with the control group,the expression levels of Akt and mTOR phosphorylation in the obese control group were higher(P<0.05).Compared with the obese control group,the expression levels of Akt and mTOR phosphorylation in the obese OSBPL3 silencing group were lower(P<0.05).Compared with 0μmol/L oleic acid,oil red O staining was gradually deepened with the increase of OA concentration.Compared with 0μmol/L oleic acid and 0μmol/L oleic acid,oil red O staining was gradually deepened with the increase of OA concentration.Compared with 0μmol/L oleic acid,OSBPL3 mRNA levels of 50μmol/L,100μmol/L and 400μmol/L oleic acid were increased successively,with statistical difference(P<0.05).The expression level of OSBPL3 increased with the increase of lipid accumulation.Conclusion:OSBPL3 can regulate the expression of lipid metabolism,and play a biological role by regulating Akt/mTOR signal pathway,which is expected to provide references to study the occurrence,development and treatment of MAFLD.