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 experimental efficacy of Qushi Huayu Decoction (祛湿化瘀方,QHD) on protein and gene expression of cathepsin B (ctsb) in HepG2 cells induced by free fatty acids (FFAs).Methods: The model of HepG2 steatosis and tumor necrosis factor-α (TNF-α) secretion was induced by long-chain FFAs.HepG2 cells were divided into 4 groups: control group (group C),model group (group M),low-dose QHD group (group L) and high-dose QHD group (group H ).Long-chain FFAs were added to groups M,L and H.The 10% blank-control serum was added to group C and M,while 5% and 10% QHD-containing sera were added to group L and H,respectively.The levels of serum TNF-α and cellular triglyceride (TG) were detected.Cellular p-IκB and ctsb expression were detected using Western blot and PCR.The expression and distribution of ctsb were observed by immunofluorescence.Results: After incubating with FFA for 24 h,TG deposition in HepG2,TNF-α content in cell supernatant,the protein expression of cellular ctsb and P-IκB,as well as mRNA expression of ctsb increased markedly in group M compared with group C (P<0.05,P<0.01).Compared with group M,TG deposition,the expression of cellular ctsb,P-IκB and ctsb mRNA in groups L and H,as well as TNF-α content in group H,decreased significantly (P<0.05).Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA.In this study,these above-mentioned changes were inhibited markedly in groups L and H.Conclusion: QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.
