茵陈醇提物抗游离脂肪酸对体外培养HepG2细胞脂毒性的作用及机制研究

Research of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity

目的:研究茵陈醇提物对游离脂肪酸刺激HepG2细胞所致肝脂毒性的抑制作用及机制。方法:制备大鼠的正常血清和药物血清,在经毒性试验确定无药物毒性的前提下,分设正常组、模型组和茵陈醇提物组(10%,1%,0.1%3个剂量),以相应浓度的正常血清和药物血清培养HepG2细胞,同时添加长链游离脂肪酸(FFA)刺激HepG2细胞24 h。观察:①细胞上清肿瘤坏死因子(TNF-α)含量(ELISA法);②细胞内甘油三酯(TG)含量、细胞脂肪油红O染色;③细胞内磷酸化κB抑制蛋白(P-IκB)、组织蛋白酶B(ctsb)、凋亡抑制基因相关X蛋白(Bax)蛋白表达(W estern B lotting法);④细胞TNF-,αctsb和Bax基因表达(real-tim e PCR);⑤细胞内ctsb的表达和分布(免疫荧光法)。结果:模型组细胞内TG及上清TNF-α含量显著升高,分别达(590±186)mg.g-1,(77±11)pg.mg-1,细胞内ctsb,P-IκB的蛋白表达以及ctsb,TNF-α的mRNA表达显著增强;而10%茵陈醇提物组细胞内TG和上清TNF-α含量较模型组显著降低,仅为(335±54)mg.g-1,(55±7)pg.mg-1,且显著抑制细胞内ctsb,P-IκB的蛋白表达以及ctsb,TNF-α的mRNA表达。结论:茵陈醇提物对FFA诱导的HepG2细胞脂肪变性,TNF-α分泌有显著的抑制作用,其作用与抑制ctsb等基因和蛋白表达有关。

Objective： To study the efficiency and effect mechanism of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity model in vitro. Method： Preparation rat regular serum and medicine serum. Under the safty of medicine thickness by toxicity testing, normal and model groups were added 10% normal rat serum, Herba Artemisiae Scoporiae group was added 10% medicine serum incubation for 24 h, FFA was added to all the groups but the normal incubation for 24 h. The indices were tested below： the content of serum tumor necrosis factor α （TNF-α） by ELISA, cellular triglyceride content （ TG）, Oil Red Staining ; protein expression of cellular Bcl-2 Assaciated X protein （Bax）, phospho-h（B （P-IκB） and Cathepsin B （ctsb）by Western Blotting; gene expression of cellular TNF-α, Bax and ctsb by real-time PCR ; the expression and distribution of ctsb observed by immunofluorescence. Result： After being incubated with FFA for 24 hours, TG deposition of HepG2 in the model group increased markedly. Compared with normal group, not only the content of serum TNF-α, but also the protein expression of cellular ctsb, P-IκB and mRNA expression of ctsb, TNF-α in- creased significantly. Contrast to model group, TG deposition decreased markedly in the Herba Artemisiae Scoporiae group. The Herba Artemisiae Scoporiae inhibited TNF-α content, the protein expression of cellular etsb, P-IκB and mRNA expression of TNF-α siguificantly. Conclusion： Herba Artemisiae Scoporiae has a direct inhibition on HepG2 steatosis and TNF-α secretion induced by long-chain FFA. The effect mechanism of Herba Artemisiae Scoporiae inhibits the hepatic lipotoxicity has close relationship with inhibition on the protein expression and mRNA expression of ctsb.