Hu Xiao-Na, Wang Jiao-Feng, Huang Yi-Qin, Wang Zheng, Dong Fang-Yuan, Ma Hai-Fen, Bao Zhi-Jun
Department of Gastroenterology, Huadong Hospital Affiliated to Fudan University, Shanghai, China.
Shanghai Key Laboratory of Clinical Geriatric Medicine, Shanghai, China.
PeerJ. 2018 Jun 26;6:e5145. doi: 10.7717/peerj.5145. eCollection 2018.
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.
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 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 nuclear factor (NF-κB) and IκBα were detected by immunofluorescence.
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 (TNF-α and IL-6) and oxidants Malonaldehyde (MDA), 4-hydroxynonenal (HNE) and reactive oxygen species (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 expression of IκBα increased, while the expression of NF-κB decreased.
Fatty liver cells showed an obvious senescence and apoptosis phenomenon. Huperzine A suppressed hepatocyte senescence, and it might exert its anti-aging effect via the NF-κB pathway.
本研究旨在检测游离脂肪酸（FFA）是否诱导L-02细胞发生肝细胞衰老，以及石杉碱甲对脂肪肝细胞是否具有抗衰老作用。
用不同浓度的FFA混合物（油酸/棕榈酸，比例为3:0、2:1、1:1、1:2和0:3）处理L-02细胞。分别通过细胞计数试剂盒8和尼罗红染色评估细胞活力和脂肪积累率。选择细胞活力和脂肪积累率最高的混合物继续进行以下实验。将L-02细胞分为五组，包括对照组、FFA组、FFA + 0.1 μmol/L石杉碱甲（LH）组、FFA + 1.0 μmol/L石杉碱甲（MH）组和FFA + 10 μmol/L石杉碱甲（HH）组，并培养24小时。用SA-β-半乳糖苷染色试剂盒检测衰老相关β-半乳糖苷酶（SA-β-gal）的表达。通过qRT-PCR测量衰老基因的表达水平。用蛋白质免疫印迹法检测凋亡蛋白的表达水平。用ELISA试剂盒检测炎症因子和氧化应激产物。通过免疫荧光检测核因子（NF-κB）和IκBα的表达。
0.5 mmol/L的FFA混合物（油酸/棕榈酸，比例为2:1）具有最高的细胞活力和脂肪积累率，这对于建立体外脂肪肝模型是优选的。L-02脂肪肝细胞中炎症因子（TNF-α和IL-6）以及氧化剂丙二醛（MDA）、4-羟基壬烯醛（HNE）和活性氧（ROS）的表达也增加。衰老标志物和衰老基因如SA-β-gal、p16、p21、p53和pRb在L-02脂肪肝细胞中的表达水平比在L-02细胞中增加得更多。凋亡相关蛋白Bcl2、Bax、Bax/Bcl-2、CyCt和裂解的半胱天冬酶9的总水平在L-02脂肪肝细胞中也上调。在石杉碱甲与FFA联合处理组中，上述所有基因和蛋白均下调。在L-02脂肪肝细胞中，IκBα的表达降低，而NF-κB的表达增加。石杉碱甲与FFA联合处理后，IκBα的表达增加，而NF-κB的表达降低。
脂肪肝细胞表现出明显的衰老和凋亡现象。石杉碱甲抑制肝细胞衰老，其可能通过NF-κB途径发挥抗衰老作用。
