摘要
目的探讨葛根芩连汤(GQD)含药血清对缺氧诱导HepG2肝癌细胞糖代谢的影响及机制。方法(1)在缺氧条件(94%N_(2)、5%CO_(2)、1%O_(2))下,通过考察5个缺氧时间点(6、12、18、24、48 h)对HepG2细胞葡萄糖消耗量和细胞活力的影响,确定最佳缺氧时间。(2)设置正常组(常氧条件,15%空白血清)、模型组(15%空白血清)、二甲双胍组(15%空白血清+2 mmol·L^(-1)二甲双胍)、葛根芩连汤含药血清低剂量组(5%GQD含药血清+10%空白血清)、葛根芩连汤含药血清中剂量组(10%GQD含药血清+5%空白血清)、葛根芩连汤含药血清高剂量组(15%GQD含药血清),检测缺氧18 h后各组细胞的葡萄糖消耗量及细胞活力。(3)采用超高效液相色谱-四级杆-飞行时间质谱(UPLC-Q-TOF-MS)联用技术进行细胞样品的分离和数据采集;采用正交偏最小二乘法判别分析(OPLS-DA)筛选差异变量;通过HMDB、KEGG在线数据库中进行代谢物鉴定,以确定葛根芩连汤含药血清干预缺氧诱导HepG2肝癌细胞的潜在生物标志物;最后,通过Metabo Analyst 5.0网站进行潜在生物标志物代谢通路分析。结果(1)与正常组比较,模型组HepG2细胞缺氧12、18 h的葡萄糖消耗量显著降低(P<0.01),缺氧24、48 h的葡萄糖消耗量显著增多(P<0.01),缺氧6、12、18、24 h的细胞活力无明显变化(P>0.05),缺氧48 h的细胞活力显著升高(P<0.01)。故在1%O2环境下,确定缺氧18 h作为HepG2细胞的模型复制时间。(2)与正常组比较,模型组HepG2细胞的葡萄糖消耗量显著降低(P<0.01);与模型组比较,葛根芩连汤含药血清低、中、高剂量组HepG2细胞的葡萄糖消耗量明显升高(P<0.05,P<0.01)。缺氧18 h,各组间的HepG2细胞活力无明显变化(P>0.05)。(3)共鉴定出缺氧诱导HepG2肝癌细胞的潜在生物标志物12个,其中1个生物标志物含量显著上升,11个生物标志物含量显著下降,而GQD含药血清干预可使上述生物标志物明显回调。12个潜在生物标志物涉及甘油磷脂代谢、亚油酸代谢、α-亚麻酸代谢、花生四烯酸代谢、鞘脂代谢、半胱氨酸和蛋氨酸代谢、嘧啶代谢、嘌呤代谢以及甘氨酸、丝氨酸和苏氨酸代谢等9条代谢通路。结论GQD含药血清可能通过调节脂质代谢、氨基酸代谢、嘌呤及嘧啶代谢来改善缺氧引起的HepG2细胞糖代谢异常,提高葡萄糖消耗量。
Objective To investigate the effect of Gegen Qinlian Decoction(GQD)-containing serum on glucose metabolism in hypoxia-induced HepG2 hepatoma cells and its metabolic mechanism.Methods(1)Under hypoxic conditions(94%N_(2),5%CO_(2),1%O_(2)),the optimal hypoxic time was determined by examining the effects of 5 hypoxic time points(6,12,18,24 and 48 hours)on the glucose consumption and cell viability of HepG2 cells.(2)The normal group(normal oxygen environment,15%blank serum),model group(15%blank serum),Metformin group(15%blank serum+2 mmol·L^(-1)Metformin),low-dose of GQD-containing serum group(5%GQD-containing serum+10%blank serum),medium-dose of GQD-containing serum group(10%GQDcontaining serum+5%blank serum),and high-dose of GQD-containing serum group(15%GQD-containing serum).The glucose consumption and cell viability of the cells in each group were measured after 18 hours of hypoxia.(3)ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-QTOF-MS)was used for the separation of cell samples and data acquisition;orthogonal partial least squares discriminant analysis(OPLS-DA)was used to screen the differential variables;metabolite identification was performed in the HMDB and KEGG online databases to determine the potential biomarkers of GQD-containing serum intervention on hypoxia-induced HepG2 hepatoma cells;finally,the metabolic pathway analysis of potential biomarkers was performed through Metabo Analyst 5.0 website.Results(1)Compared with the normal group,the glucose consumption of HepG2 cells in the model group was significantly decreased for 12 and 18 hours of hypoxia(P<0.01),significantly increased for 24 and 48 hours of hypoxia(P<0.01),with no significant change in cell viability for 6,12,18 and 24 hours of hypoxia(P>0.05),and significantly increased for 48 hours of hypoxia(P<0.01).Therefore,18 hours of hypoxia was determined as the model replication time for HepG2 cells under 1%O2 environment.(2)Compared with the normal group,the glucose consumption of HepG2 cells in the model group was significantly decreased(P<0.01);compared with the model group,the glucose consumption of HepG2 cells in the low-,medium-and high-dose groups of GQD-containing serum was significantly increased(P<0.05,P<0.01).There was no significant change in HepG2 cell viability among the groups after 18 hours of hypoxia(P>0.05).(3)A total of 12 potential biomarkers of hypoxia-induced HepG2 hepatoma cells were identified,of which 1 biomarker was significantly increased and 11 biomarkers were significantly decreased,while the GQD-containing serum could intervene to significantly regress the above biomarkers.The 12 potential biomarkers relate to 9 metabolic pathways including glycerophospholipid metabolism,linoleic acid metabolism,α-linolenic acid metabolism,arachidonic acid metabolism,sphingolipid metabolism,cysteine and methionine metabolism,pyrimidine metabolism,purine metabolism,and acid metabolisms of glycine,serine and threonine.Conclusion GQD-containing serum may improve hypoxia-induced abnormalities in glucose metabolism of HepG2 cell and increase glucose consumption by regulating lipid metabolism,amino acid metabolism,purine and pyrimidine metabolism.
作者
许清松
彭程程
李冰涛
张启云
姜丽
李佳
李洋
曾治君
徐国良
XU Qingsong;PENG Chengcheng;LI Bingtao;ZHANG Qiyun;JIANG Li;LI Jia;LI Yang;ZENGZhijun;XU Guoliang(Research Center for Differentiation and Development of Traditional Chinese Medicine Basic Theory,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Jiangxi Provincial Key Laboratory of Chinese Medicine Etiopathogenesis,Nanchang 330004 Jiangxi,China;Jiangxi Provincial Key Laboratory of Pharmacology of Chinese Medicine,Nanchang 330004 Jiangxi,China;School of Pharmacy,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China;Qihuang State Medical College,Jiangxi University of Chinese Medicine,Nanchang 330004 Jiangxi,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2023年第5期636-645,共10页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家自然科学基金项目(82060826)
江西省卫生和计划生育委员会科技计划项目(202211417)
江西中医药大学校级科技创新团队发展计划项目(CXTD22007)
江西中医药大学校级研究生创新专项资金项目(JZYC22S79)。
