黄连生物碱改善小鼠原代肝细胞糖异生与脂质蓄积  

Coptis alkaloids improve gluconeogenesis and lipid accumulation in mouse primary hepatocytes

作　　者：颜家琦 王思齐 王峥 李隽 YAN Jiaqi;WANG Siqi;WANG Zheng;LI Jun(Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院生物化学与分子生物学系,北京100005

出　　处：《基础医学与临床》2023年第6期923-930,共8页Basic and Clinical Medicine

基　　金：国家自然科学基金(81903767);中国医学科学院中央级公益性科研院所基本科研业务费专项资金(2022-JKCS-14)。

摘　　要：目的筛选能改善小鼠原代肝细胞糖异生与脂质蓄积的黄连生物碱单体并探讨其作用机制。方法分离小鼠原代肝细胞,使用不同剂量黄连总生物碱或单体成分处理,CCK-8法检测细胞存活率。使用丙酮酸钠、乳酸钠与胰高血糖素诱导小鼠原代肝细胞糖异生模型,应用试剂盒检测培养基中葡萄糖含量,RT-qPCR检测糖异生相关基因表达,蛋白印迹检测p-AKT水平。应用棕榈酸与油酸诱导脂质蓄积模型,油红O检测细胞内脂质含量,蛋白印迹检测FAS、PLIN1、PPARγ蛋白水平。结果低剂量(0.5、1、2μg/mL)的黄连总生物碱处理24 h后小鼠原代肝细胞存活率在80%以上。糖异生模型组葡萄糖含量与G6PC、PCX的mRNA水平较对照组升高(P<0.05),p-AKT蛋白水平较对照组降低(P<0.05);黄连总生物碱(1、2μg/mL)与小檗碱(2.5、5μmol/L)处理后葡萄糖含量与G6PC、PCX的mRNA水平较模型组降低(P<0.05),p-AKT蛋白水平较模型组升高(P<0.05)。脂质蓄积模型组油红O染色较对照组增加(P<0.01),FAS、PLIN1、PPARγ蛋白水平较对照组升高(P<0.01);黄连总生物碱(1、2μg/mL)、黄连碱(2.5、5μmol/L)与小檗碱(2.5、5μmol/L)组的油红O染色较模型组减少(P<0.05),FAS、PLIN1、PPARγ蛋白水平较模型组降低(P<0.05)。结论小檗碱通过上调p-AKT水平而降低小鼠原代肝细胞糖异生,黄连碱与小檗碱通过降低FAS、PLIN1、PPARγ的表达而减少小鼠原代肝细胞脂质蓄积。Objective To screen the alkaloid monomer of coptis chinensis,which can improve the gluconeogenesis and lipid accumulation of mouse primary hepatocytes,and to explore its mechanism.Methods The mouse primary hepatocytes were isolated and incubated with different doses of coptis alkaloids and coptis chinensis monomer.The cell viability was measured by CCK-8 method.Sodium pyruvate,sodium lactate and glucagon were added to induce the gluconeogenesis model of mouse primary hepatocytes.The content of glucose in the medium was detected by the kit.The expression of gluconeogenesis-related genes was detected by RT-qPCR.The protein p-AKT was detected by Western blot.Palmitic acid and oleic acid were used to induce lipid accumulation model.The lipid content was detected by oil red O staining.The protein level of FAS,PLIN1 and PPARγwas detected by Western blot.Results The cell viability of mouse primary hepatocytes was more than 80%after 24 h treatment with low dose of coptis alkaloids(0.5,1,2μg/mL).Content of glucose and mRNA of G6PC and PCX in gluconeogenesis model group were higher than that in control group(P<0.05),while p-AKT protein level was lower than that in control group(P<0.05).Compared with model group,the glucose content and the mRNA levels of G6PC and PCX were lower in that treated with coptis alkaloids(1,2μg/mL)and berberine(2.5,5μmol/L)(P<0.05),while the protein level of p-AKT was higher(P<0.05).Oil red O staining in the lipid accumulation model group increased compared with that in the control group(P<0.01),and the protein level of FAS,PLIN1,PPARγwas higher than that of the control group(P<0.01).Oil red O staining of groups that treated with coptis alkaloids(1,2μg/mL),coptisine(2.5,5μmol/L)and berberine(2.5,5μmol/L)were less than that of model group(P<0.05),and the protein level of FAS,PLIN1,PPARγwas lower than that of the model group(P<0.05).Conclusions Berberine decreases the gluconeogenesis of mouse primary hepatocytes through p-AKT pathway.Coptisine and berberine reduce the expression of FAS,PLIN

关 键 词：黄连生物碱 原代肝细胞 糖异生 脂质蓄积 

分 类 号：R34[医药卫生—基础医学]