慢性乙肝合并非酒精性脂肪肝细胞模型的建立及火绒草素干预研究

Establishment of the Cellular Model of Chronic Hepatitis B Complicated with Non-alcoholic Fatty Liver Disease and the Intervention of Leontopodic Acid A

关键词:  慢性乙型病毒性肝炎并发脂肪性肝病  细胞模型  乙型肝炎病毒转基因细胞  火绒草素  药物干预

Key words:  chronic hepatitis B complicated with non-alcoholic fatty liver disease  cellular model  HepG2.2.15 cells  leontopodic acid A  drug intervention

摘要:

目的 建立慢性乙型病毒性肝炎(chronic hepatitis B，CHB)合并非酒精性脂肪性肝病(non-alcoholic fatty liver disease，NAFLD)细胞模型，并探讨CHB与NAFLD间的相互关系以及火绒草素的干预作用。方法 采用转染乙型肝炎病毒(hepatitis B virus，HBV)基因组的HepG2.2.15细胞，以脂肪乳和油酸/棕榈酸(2∶1)混合液为诱导剂，以甘油三酯(triglyceride，TG)和油红O染色为评价指标，建立分阶段诱导CHB合并NAFLD细胞模型。运用该模型比较HepG2.2.15细胞脂肪变性前后血红素加氧酶-1(heme oxygenase-1，HO-1)、肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白介素-1β(interleukin-1β，IL-1β)和HBV DNA表达差异，以探讨CHB与NAFLD间的相互关系。通过分析对CHB合并NAFLD模型细胞活力、TG含量和HBV DNA复制的影响，以评价火绒草素的干预作用。结果 经1%脂肪乳诱导12 h后，再用0.5 mmol·L^–1油酸/棕榈酸混合液诱导24 h为最佳CHB合并NAFLD建模条件。HepG2.2.15细胞诱导脂肪变性后与脂肪变性前相比，其HO-1、IL-1β和HBV DNA水平显著下降，而TNF-α表达显著升高。火绒草素在10，50和100 μg·mL^–1时对CHB合并NAFLD模型细胞活力有明显改善作用，同时显著减少TG含量和HBV复制水平。结论 脂肪变性的诱导可能干扰了HBV DNA复制，而相关细胞因子表达的变化可能与脂肪变性产生的炎症有关，初步证明火绒草素具有显著抗CHB合并NAFLD作用。

Abstract:

OBJECTIVE To establish the cellular model of chronic hepatitis B(CHB) complicated with non-alcoholic fatty liver disease(NAFLD), and to explore the correlations of CHB and NAFLD and the intervention of leontopodic acid A. METHODS Hepatitis B virus(HBV)‑transfected HepG2.2.15 hepatoma cells were induced by fat emulsion and oleic acid/palmitic acid(2︰1) mixture in stages to establish a model of CHB complicated with steatohepatitis. Triglyceride(TG) content and oil red O staining were employed to evaluate the degree of steatosis of HepG2.2.15 cells. The above model was employed to study the correlations of CHB and NAFLD by comparison of the levels of heme oxygenase-1(HO-1), tumor necrosis factor-α (TNF-α) and interleukin-1β(IL-1β) and HBV DNA in HepG2.2.15 cells before and after steatosis. The effect of leontopodic acid A on CHB complicated with NAFLD were evaluated by analyzing its effects on the viability, TG content and HBV DNA levels of HepG2.2.15 cells with steatosis. RESULTS The steatohepatitis in HepG2.2.15 cells was induced by 1% fat emulsion for 12 h and then by 0.5 mmol·L^-1 oleic acid/palmitic acid mixture for 24 h as the optimal modeling conditions for CHB complicated with NAFLD. Compared with before steatosis, the levels of HO-1, IL-1β and HBV DNA in HepG2.2.15 cells with steatosis were significantly decreased. Meanwhile, the expression of TNF-α was markedly increased. Additionally, at 10, 50 and 100 μg·mL^-1 of leontopodic acid A improved the viability against CHB complicated with NAFLD and decreased TG contents and HBV DNA levels significantly. CONCLUSION The induced fatty degeneration can interfere with the replication of HBV DNA. The expression changes of the related cytokines can be relevant to the inflammation that exists in the cells with fatty degeneration. This study preliminarily proves that leontopodic acid A has significant viability against CHB complicated with NAFLD.