基于SREBPs通路的益肾通络方改善脂质代谢异常作用机制研究

Study on the mechanism of Yishen tongluo formula improving abnormal lipid metabolism based on SREBPs pathway

目的基于固醇调节元件结合蛋白(SREBPs)通路探究益肾通络方改善脂质代谢异常的作用机制。方法以C57BLKS/J(db/db)小鼠为模型动物、C57BLKS/J(db/m)小鼠为正常对照,通过测定小鼠肝脏系数及血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)含量,观察小鼠肝组织脂肪变性和脂质蓄积情况,测定小鼠肝组织中SREBP-1、SREBP-2蛋白和Srebp-1c、Srebp-2及其下游脂质代谢相关靶基因(Fasn、Acc1、Scd5、Fads1、Hmgcr、Dhcr24、Insig-1、Fdps)的mRNA转录水平,来考察1、2.5、5 g/kg益肾通络方改善db/db小鼠脂质代谢异常的作用机制。以低脂培养的人肝癌细胞HepG2为体外脂质代谢异常细胞模型,以25-HC(SREBPs抑制剂,10μmol/L)为抑制剂对照,考察125、250、500μg/mL益肾通络方干预24 h后对细胞中SREBP-1、SREBP-2蛋白和SREBP-1c、SREBP-2其下游脂质代谢相关靶基因mRNA转录水平的影响,进行体外机制验证。结果1、2.5、5 g/kg益肾通络方均可显著降低模型小鼠TC、TG、LDL水平,显著降低肝组织脂滴阳性面积百分比、肝脏系数,显著下调肝组织中Pre-SREBP-1、n-SREBP-1、Pre-SREBP-2、n-SREBP-2蛋白以及Srebp-1c、Srebp-2及其下游靶基因的mRNA转录水平,并可显著升高HDL水平,差异均有统计学意义(P<0.05或P<0.01)。体外细胞实验中,125、250、500μg/mL益肾通络方作用24 h后细胞中上述蛋白及基因的表达的变化与动物实验一致,且抑制剂对照与250、500μg/mL益肾通络方给药后细胞中上述蛋白和基因表达水平相比差异均无统计学意义(P>0.05)。结论益肾通络方可能通过调控转录因子SREBPs表达,进而抑制脂肪酸及胆固醇合成相关基因的高表达,促进TC、TG的降解,改善脂质代谢异常,抑制脂质蓄积,从而发挥降脂作用。

OBJECTIVE To explore the mechanism of Yishen tongluo formula(YSTLF)in improving abnormal lipid metabolism based on the sterol regulatory element binding proteins(SREBPs)pathway.METHODS Using C57BLKS/J(db/db)mice as model and C57BLKS/J(db/m)mice as normal control,the mechanism of 1,2.5 and 5 g/kg YSTLF improving abnormal lipid metabolism of db/db mice was investigated by determining the liver coefficient,the contents of serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL),observing steatosis and lipid accumulation in liver tissue of mice,detecting the protein expressions of SREBP-1 and SREBP-2 as well as mRNA transcription levels of Srebp-1c,Srebp-2 and their downstream lipid metabolism-related target genes(Fasn,Acc1,Scd5,Fads1,Hmgcr,Dhcr24,Insig-1,Fdps)in liver tissue of mice.Using low-fat cultured human liver cancer cell HepG2 as an in vitro cell model for abnormal lipid metabolism,and 25-HC(SREBPs inhibitor,10μmol/L)as the control,the effects of 125,250 and 500μg/mL YSTLF on protein expressions of SREBP-1 and SREBP-2 as well as mRNA transcription of SREBP-1c,SREBP-2 and their downstream lipid metabolism-related target genes were investigated to verify the mechanism in vitro.RESULTS 1,2.5,5 g/kg YSTLF significantly reduced the levels of TC,TG and LDL,the percentage of lipid droplet-positive region in liver tissue and liver coefficient,significantly down-regulated protein expressions of Pre-SREBP-1,n-SREBP-1,Pre-SREBP-2 and n-SREBP-2,and mRNA transcription of Srebp-1c,Srebp-2 and their downstream target genes in liver tissue,while significantly increased HDL level,with statistical significance(P<0.05 or P<0.01).In the cell experiment in vitro,the expressions of the above-mentioned proteins and genes in the cells treated with YSTLF at 125,250 and 500μg/mL for 24 hours were consistent with those in the animal experiment;there was no significant difference in the expressions of the above-mentioned proteins and genes between inhibitor control group and 250,500μg/mL YSTLF groups(P>0.05).CONCLUSIONS YSTLF can regulate the expression of transcription factor SREBPs,so as to inhibit the high expression of fatty acid and cholesterol synthesis-related genes,promote the degradation of TC and TG,improve the abnormality of lipid metabolism and inhibit lipid accumulation,thus playing the role of lipid-lowering.