目的 探究豆甾醇对油酸联合低密度脂蛋白胆固醇（LDL-C）诱导脂肪性肝炎细胞模型炎症损伤的保护作用及可能机制。方法 将LO2肝细胞分为4组：对照组（CON）,普通培养液培养;模型组（MOD）,普通培养液添加200 μmol/L的油酸和50 mg/L的LDL-C孵育36 h诱导建立脂肪性肝炎细胞模型;豆甾醇干预组（STG）,在MOD组细胞培养液额外添加50 μmol/L的豆甾醇;转染组（si-CYP7B1）,使用50 nmol/L的氧甾醇7α羟化酶（CYP7B1）si-RNA预处理STG组细胞24 h。结果 STG组的胞内总胆固醇和游离胆固醇含量均明显低于MOD组（P<0.05）,STG组培养液中的谷丙转氨酶和谷草转氨酶活性和细胞NLRP3炎性小体、半胱氨酸天冬酶-1和白介素-1β基因表达水平较MOD组均明显降低（P<0.05）;相较于MOD组,STG组肝细胞CYP7B1和胆盐输出泵的mRNA相对表达量明显升高（P<0.05）;si-RNA特异性沉默CYP7B1基因表达后,豆甾醇干预对细胞胆固醇沉积和炎症损伤影响消失。结论 豆甾醇通过提高肝细胞中CYP7B1表达,减少由油酸联合LDL-C处理引起的肝细胞胆固醇沉积,并抑制NLRP3炎性小体信号通路活化,从而减轻细胞炎症损伤。
Abstract
Objective To investigate the protective effect and the underlying mechanism of stigmasterol on inflammatory injury in steatotic hepatocytes induced by oleic acid and low-density lipoprotein cholesterol (LDL-C). Methods LO2 cells were divided into 4 groups. The control group (CON) was cultured in the DMEM high-glucose medium, and the model group (MOD) was cultured in the DMEM high-glucose medium supplemented with 200 μmol/L oleic acid and 50 mg/L LDL-C for 36 h to induce the steatohepatitis cell model. In the stigmasterol intervention group (STG), 50 μmol/L of stigmasterol was added on the basis of the culture medium of the MOD group. In the transfection group (si-CYP7B1), 50 nmol/L of si-RNA was used to silence oxysterol 7α-hydroxylase (CYP7B1) gene expression in the STG-treated cells for 24 h. Results The contents of total cholesterol and free cholesterol in the STG group were significantly lower than those in the MOD group (P<0.05). The activities of alanine transaminase, aspartate aminotransferase and gene expression levels of NLRP3, caspase-1 and interleukin-1β in the STG group were significantly decreased compared with those in the MOD group (P<0.05). Compared with the MOD group, the relative mRNA expression levels of CYP7B1 and bile salt export pump in hepatocytes of the STG group were significantly increased (P<0.05). After CYP7B1 gene expression was specifically silenced by the si-RNA for CYP7B1, stigmasterol intervention no longer had significant effects on cellular cholesterol deposition and inflammatory injury. Conclusions tigmasterol alleviates cholesterol deposition in steatotic hepatocytes induced by oleic acid and LDL-C through increasing CYP7B1 expression and inhibiting NLRP3 inflammasome signaling pathway activation, thereby protecting against cellular inflammatory injury.
关键词
豆甾醇 /
脂肪性肝炎 /
胆固醇代谢 /
L-O2肝细胞
Key words
stigmasterol /
steatohepatitis /
LO2 cells
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基金
国家自然科学基金（No. 82273622）; 广东省基础与应用基础研究基金（No. 2021B1515140057）
