罗汉果皂苷Ⅵ对LPS诱发肝细胞损伤的保护作用及机制研究

Study on the Protective Effect and Mechanism of MogrosideⅥon LPS Induced Hepatocyte Injury

目的:探讨罗汉果皂苷Ⅵ(MⅥ)对脂多糖(LPS)致体外肝细胞损伤的保护作用及其机制。方法:构建LPS诱导的L02细胞损伤模型,采用不同浓度(7.5、15、30μmol/L)MⅥ或30μmol/L MⅥ联合4 mmol/L PGC-1α抑制剂SR-18292处理24 h,MTT检测细胞增殖活性;比色法检测细胞培养上清中丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平,以及细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;流式细胞术检测细胞凋亡率;Mito-Tracker Green染色法观察细胞线粒体分裂;qRT-PCR检测线粒体DNA(mtDNA)的拷贝数;qRT-PCR和Western blotting检测细胞中PGC-1α、NRF-1、TFAM的表达水平。结果:与对照组比较,LPS组L02细胞增殖活性及胞内GSH-Px和SOD活性均明显降低(P<0.05);细胞凋亡率,上清液中ALT、AST水平及胞内MDA含量均明显升高(P<0.05);线粒体分裂水平、mtDNA拷贝数及PGC-1α、NRF-1、TFAM的mRNA和蛋白表达水平均明显降低(P<0.05)。与LPS组比较,不同剂量MⅥ处理后,细胞增殖活性及胞内GSHPx和SOD活性均明显升高(P<0.05);细胞凋亡率,上清液中ALT、AST水平及胞内MDA含量均明显降低(P<0.05);线粒体分裂水平、mtDNA拷贝数及PGC-1α、NRF-1、TFAM的mRNA和蛋白表达水平均明显升高(P<0.05)。然而,SR-18292处理可明显抑制MⅥ对细胞损伤的改善效果。结论:MⅥ能有效改善LPS致体外肝细胞损伤,其潜在的分子机制可能是通过增强PGC-1α/NRF-1/TFAM信号通路,进而促进肝细胞线粒体生物合成,降低细胞内氧化应激水平,抑制细胞凋亡。

Objective:To study the effect of mogrosideⅥ(MⅥ)on lipopolysaccharide(LPS)induced liver injury and its possible mechanisms in vitro.Methods:The L02 cell injury model induced by LPS was constructed with different concentrations(7.5,15,30μmol/L)MⅥor 30μmol/L MⅥcombined with 4 mmol/L PGC-1αinhibitor SR-18292 was treated for 24 hours,and the cell proliferation activity was detected by MTT.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH PX)and the content of malondialdehyde(MDA)in cell culture supernatant were detected by colorimetry.The cellular apoptosis rate was detected by flow cytometry.Mito-Tracker Green staining was used to observe the division of mitochondria.The copy number of mitochondrial DNA(mtDNA)was detected by qRT PCR.The expression levels of PGC-1α,NRF-1 and TFAM were detected by qRT PCR and Western blotting.Results:Compared with the control group,the activities of GSH-Px and SOD in L02cells of the LPS group were significantly reduced(P<0.05).The apoptosis rate,the levels of ALT and AST in culture supernatant,and the concentration of MDA in L02 cells were significantly increased(P<0.05).The mitochondrial division level,the copy number of mtDNA,and the expression levels of PGC-1α,NRF-1,and TFAM in L02 cells were all significantly decreased(P<0.05).Compared with the LPS group,after treatment with different dose of M VI,the cell proliferation viability and the activities of GSH-Px and SOD were significantly increased(P<0.05).The apoptosis rate,the levels of ALT and AST in supernatant and the content of intracellular MDA were significantly decreased(P<0.05).The level of mitochondrial division,the copy number of mtDNA and the m RNA and protein expression levels of PGC-1α,NRF-1 and TFAM were significantly increased(P<0.05).However,SR-18292 treatment could significantly inhibit the improvement effect of MⅥon cell injury.Conclusion:MⅥcould effectively improve hepatocyte injury induced by LPS in vitro.Its potential molecular mechanism may be to enhance PGC-1α/NRF-1/TFAM signal pathway,promote hepatocyte mitochondrial biosynthesis,reduce the level of intracellular oxidative stress and inhibit apoptosis.