Peer Review #1 of "Huperzine A attenuates nonalcoholic fatty liver disease by regulating hepatocyte senescence and apoptosis: an in vitro study (v0.1)"

Abstract

Objective: This study was undertaken to detect if free fatty acids (FFA) induce hepatocyte senescence in L-02 cells and if huperzine A has an anti-aging effect in fatty liver cells.Methods: L-02 cells were treated with a FFA mixture (oleate/palmitate, at 3:0, 2:1, 1:1, 1:2 and 0:3 ratios) at different concentrations. Cell viability and fat accumulation rate were assessed by a Cell Counting Kit 8 (CCK8) kit and Nile Red staining, respectively. The mixture with the highest cell viability and fat accumulation rate was selected to continue with the following experiment. The L-02 cells were divided into five groups, including the control group, FFA group, FFA+0.1 μmol/L huperzine A (LH) group, FFA+1.0 μmol/L huperzine A (MH) group and FFA+10 μmol/L huperzine A (HH) group, and were cultured for 24 h. The expression of senescence-associated β-galactosidase (SA-β-gal) was detected by an SA-β-gal staining kit. The expression levels of aging genes were measured by qRT-PCR.The expression levels of apoptosis proteins were detected by a Western blot. ELISA kits were used to detect inflammatory factors and oxidative stress products. The expression of NF-κB and IκBα were detected by immunofluorescence. Results: The FFA mixture (oleate/palmitate, at a 2:1 ratio) of 0.5 mmol/L had the highest cell viability and fat accumulation rate, which was preferable for establishing an in vitro fatty liver model. The expression of inflammatory factors (TNFα, and oxidants (MDA, HNE and ROS) also increased in the L-02 fatty liver cells. The expression levels of aging markers and aging genes, such as SA-β-gal, p16, p21, p53 and pRb, increased more in the L-02 fatty liver cells than in the L-02 cells. The total levels of the apoptosis-associated proteins Bcl2, Bax, Bax/Bcl-2, CyCt, and cleaved caspase 9 were also upregulated in the L-02 fatty liver cells. All of the above genes and proteins were downregulated in the huperzine A and FFA co-treatment group. In the L-02 fatty liver cells, the expression of IκBα decreased, while the expression of NF-κB increased. After the huperzine A and FFA co-treatment, the Manuscript to be reviewedHuperzine A attenuates nonalcoholic fatty liver disease by regulating hepatocyte senescence and apoptosis: an in vitro study Xiao- Manuscript to be reviewed [Abstract] Objective: This study was undertaken to detect if free fatty acids (FFA) induce hepatocyte senescence in L-02 cells and if huperzine A has an anti-aging effect in fatty liver cells.Methods: L-02 cells were treated with a FFA mixture (oleate/palmitate, at 3:0, 2:1, 1:1, 1:2 and 0:3 ratios) at different concentrations. Cell viability and fat accumulation rate were assessed by a Cell Counting Kit 8 (CCK8) and Nile Red staining, respectively. The mixture with the highest cell viability and fat accumulation rate was selected to continue with the following experiment. The L-02 cells were divided into five groups, including the control group, FFA group, FFA+0.1 μmol/L huperzine A (LH) group, FFA+1.0 μmol/L huperzine A (MH) group and FFA+10 μmol/L huperzine...