Objective: To explore the mechanism underlying the hepatoprotective effect of dihydromyricetin (DMY) against lipid accumulation in light of the lipophagy pathway and the inhibitory effect of DMY on HepG2 cell proliferation.
Methods: LO2 cells were cultured in the presence of 10% FBS for 24 h and treated with 100 μg/mL DMY, or exposed to 50% FBS for 24 h followed by treatment with 50, 100, or 200 μg/mL DMY; the cells in recovery group were cultured in 50% FBS for 24 h and then in 10% FBS for another 24 h. Oil red O staining was used to observe the accumulation of lipid droplets in the cells, and the levels of TC, TG, and LDL and activities of AST, ALT and LDH were measured. The expression of LC3 protein was detected using Western blotting. AO staining and transmission electron microscopy were used to determine the numbers of autophagolysosomes and autophagosomes, respectively. The formation of autophagosomes was observed with MDC staining, and the mRNA expression levels of LC3, ATG7, AMPK, mTOR, p62 and Beclin1 were determined with q-PCR. Flow cytometry was performed to analyze the effect of 50, 100, and 200 μg/mL DMY on cell cycle and apoptosis of HepG2 cells; DNA integrity in the treated cells was examined with cell DNA fragmentation test.
Results: DMY treatment and pretreatment obviously inhibited lipid accumulation and reduced the levels of TC, TG, LDL and enzyme activities of AST, ALT and LDH in LO2 cells (P < 0.05). In routinely cultured LO2 cells, DMY significantly promoted the formation of autophagosomes and autophagolysosomes and upregulated the expression of LC3 protein. DMY obviously attenuated high FBS-induced inhibition of autophagosome formation in LO2 cells, up- regulated the mRNA levels of LC3, ATG7, Beclin1 and AMPK, and downregulated p62 and mTOR mRNA levels (P < 0.05 or 0.01). In HepG2 cells, DMY caused obvious cell cycle arrest, inhibited cell proliferation, and induced late apoptosis and DNA fragmentation.
Conclusion: DMY reduces lipid accumulation in LO2 cells by regulating the AMPK/ mTOR-mediated lipophagy pathway and inhibits the proliferation of HepG2 by causing cell cycle arrest and promoting apoptosis.
目的: 基于脂噬途径研究二氢杨梅素（DMY）对LO2细胞脂质蓄积的防治效果及其机制，并探索其对HepG2细胞的增殖抑制。
方法: FBS诱导LO2细胞建立脂肪变性模型，研究分别设置对照组：10%FBS-DMEM正常培养；模型组：50%FBS-DMEM；阳性对照组：100 μg/mL DMY+10%FBS-DMEM；处理组（L-DMY组：50 μg/mL DMY+50%FBS-DMEM；M-DMY组：100 μg/mL DMY+50%FBS-DMEM；H-DMY组：200 μg/mL DMY+50%FBS-DMEM）及恢复对照组：先50% FBS-DMEM培养，后10% FBS-DMEM培养。油红O染色测定脂滴累积。试剂盒测定TC、TG、LDL水平和AST、ALT和LDH酶活性。AO染色观察自噬溶酶体数量，透射电镜观察自噬体数量。Western blot检测自噬蛋白LC3表达量。MDC染色观察自噬体形成情况，q-PCR测定LC3、ATG7、AMPK、mTOR、p62和Beclin1的mRNA表达水平。细胞周期阻滞实验分析HepG2细胞周期比例，流式细胞术检测HepG2细胞凋亡情况，细胞DNA断裂实验检测HepG2细胞DNA的完整情况。
结果: DMY干预和预处理可抑制LO2细胞的脂质蓄积，并且降低TC、TG、LDL水平和AST、ALT和LDH酶活性，差异具有统计学意义（P＜0.05）；DMY可促进LO2细胞的自噬体和自噬溶酶体的形成，并促进自噬蛋白LC3的表达；DMY可减轻高FBS诱导的LO2细胞自噬体形成抑制，并上调LC3（P＜0.01）、ATG7（P＜0.01）、Beclin1（P＜0.05）和AMPK（P＜0.001）的mRNA水平，下调p62（P＜0.001）和mTOR（P＜0.001）的mRNA水平。DMY可阻滞HepG2细胞的有丝分裂和细胞增殖（53.90% vs 14.62%; 32.31% vs 70.17%），诱导HepG2细胞的晚期凋亡（4.74% vs 57.86%）和DNA断裂。
结论: DMY通过调节AMPK/mTOR介导的脂噬途径减少LO2细胞的脂质累积，通过阻滞细胞周期和促进凋亡抑制HepG2增殖，发挥体外护肝作用。
Keywords: AMPK/mTOR; dihydromyricetin; lipophagy; nonalcoholic fatty liver disease; proliferation inhibition.