Abstract：
BACKGROUND: Nonalcoholic steatohepatitis (NASH) is one of the most frequent liver diseases at present, and there is no radical treatment. The consequences of a variety of ginsenoside compounds on this situation have before been reported, however, the specific effect on the monomeric ginsenoside Rg1 (Rg1) and its associated underlying molecular mechanism stay unknown.
METHODS: In vitro, the cell models were constructed by exposing free fatty acids (FFAs) to HepG2 cells. A methionine and choline deficiency (MCD)-induced NASH mouse model was also established over 5-6 weeks of treatment. Rg1 is a traditional Chinese medicine monomer. These NASH models were treated with Rg1 and analyzed by qRT-PCR, Western Blot, sequencing, Oil red O staining, immunofluorescence, enzyme activity, HE staining, ELISA, double luciferase reporter assay, and immunohistochemistry.
RESULTS: Overexpression of ATG2B, an autophagy-related protein, attenuated lipid droplet accumulation and reduces ALT, AST, inflammatory cytokines, hydrogen peroxide, and pyroptosis in established mouse and cellular models of NASH and increased levels of ATP and autophagy. The binding sites of miR-375-3p and ATG2B were verified by bioinformatic prediction and a dual-luciferase reporter gene. Knockdown of miR-375-3p promoted autophagy and inhibited pyroptosis. ATG2B knockdown substantially attenuated the impact of miR-375-3p on NASH. Rg1 appears to regulate the occurrence and development of NASH inflammation through miR-375-3p and ATG2B in vitro and in vivo, and is regulated by PTEN-AKT pathway.
CONCLUSIONS: This study showed that Rg1 participates in autophagy and pyroptosis through the miR-375-3p/ATG2B/PTEN-AKT pathway, thereby alleviating the occurrence and development of NASH, for that reason revealing Rg1 as a candidate drug for NASH.
METHODS: In vitro, the cell models were constructed by exposing free fatty acids (FFAs) to HepG2 cells. A methionine and choline deficiency (MCD)-induced NASH mouse model was also established over 5-6 weeks of treatment. Rg1 is a traditional Chinese medicine monomer. These NASH models were treated with Rg1 and analyzed by qRT-PCR, Western Blot, sequencing, Oil red O staining, immunofluorescence, enzyme activity, HE staining, ELISA, double luciferase reporter assay, and immunohistochemistry.
RESULTS: Overexpression of ATG2B, an autophagy-related protein, attenuated lipid droplet accumulation and reduces ALT, AST, inflammatory cytokines, hydrogen peroxide, and pyroptosis in established mouse and cellular models of NASH and increased levels of ATP and autophagy. The binding sites of miR-375-3p and ATG2B were verified by bioinformatic prediction and a dual-luciferase reporter gene. Knockdown of miR-375-3p promoted autophagy and inhibited pyroptosis. ATG2B knockdown substantially attenuated the impact of miR-375-3p on NASH. Rg1 appears to regulate the occurrence and development of NASH inflammation through miR-375-3p and ATG2B in vitro and in vivo, and is regulated by PTEN-AKT pathway.
CONCLUSIONS: This study showed that Rg1 participates in autophagy and pyroptosis through the miR-375-3p/ATG2B/PTEN-AKT pathway, thereby alleviating the occurrence and development of NASH, for that reason revealing Rg1 as a candidate drug for NASH.
摘要：
背景：非酒精性脂肪性肝炎（NASH）是目前最常见的肝脏疾病之一，没有激进的治疗方法。多种人参皂苷化合物对这一状况的影响以前已有报导，然而,对单体人参皂苷Rg1（Rg1）的具体作用及其相关的潜在分子机制尚不清楚。
方法：体外，通过将游离脂肪酸(FFA)暴露于HepG2细胞来构建细胞模型。还在5-6周的治疗期间建立了甲硫氨酸和胆碱缺乏(MCD)诱导的NASH小鼠模型。Rg1是一种中药单体。这些NASH模型用Rg1处理，并通过qRT-PCR分析，西方印迹，测序,测序油红O染色，免疫荧光，酶活性，HE染色,ELISA,双荧光素酶报告基因测定，和免疫组织化学。
结果：ATG2B过表达，自噬相关蛋白，减少脂滴积累和减少ALT，AST,炎性细胞因子，过氧化氢,在已建立的NASH小鼠和细胞模型中以及ATP和自噬水平升高。通过生物信息学预测和双荧光素酶报告基因验证miR-375-3p与ATG2B的结合位点。miR-375-3p的敲低促进自噬并抑制焦凋亡。ATG2B敲低显著减弱miR-375-3p对NASH的影响。Rg1似乎通过miR-375-3p和ATG2B在体外和体内调节NASH炎症的发生和发展，受PTEN-AKT通路调控。
结论：本研究显示Rg1通过miR-375-3p/ATG2B/PTEN-AKT通路参与自噬和焦亡，从而缓解NASH的发生和发展，因此，Rg1是NASH的候选药物。
方法：体外，通过将游离脂肪酸(FFA)暴露于HepG2细胞来构建细胞模型。还在5-6周的治疗期间建立了甲硫氨酸和胆碱缺乏(MCD)诱导的NASH小鼠模型。Rg1是一种中药单体。这些NASH模型用Rg1处理，并通过qRT-PCR分析，西方印迹，测序,测序油红O染色，免疫荧光，酶活性，HE染色,ELISA,双荧光素酶报告基因测定，和免疫组织化学。
结果：ATG2B过表达，自噬相关蛋白，减少脂滴积累和减少ALT，AST,炎性细胞因子，过氧化氢,在已建立的NASH小鼠和细胞模型中以及ATP和自噬水平升高。通过生物信息学预测和双荧光素酶报告基因验证miR-375-3p与ATG2B的结合位点。miR-375-3p的敲低促进自噬并抑制焦凋亡。ATG2B敲低显著减弱miR-375-3p对NASH的影响。Rg1似乎通过miR-375-3p和ATG2B在体外和体内调节NASH炎症的发生和发展，受PTEN-AKT通路调控。
结论：本研究显示Rg1通过miR-375-3p/ATG2B/PTEN-AKT通路参与自噬和焦亡，从而缓解NASH的发生和发展，因此，Rg1是NASH的候选药物。
