去甲斑蝥素对LPS 诱导的体外培养的肝细胞损伤的保护作用

目的:通过体外观察去甲斑蝥素(NCTD) 对LPS 所致肝细胞损伤和TNF-α 表达的影响,探讨NCTD 的作用及其机制。方法:胶原酶Ⅳ灌流分离培养大鼠肝细胞,将细胞分为对照组、LPS 组、NCTD 组。对照组用无血清DMEM 培养, LPS 组用LPS (40 mg/L) 诱导,NCTD 组用不同浓度NCTD(0.5,1.0,2.5 μg/mL) 与LPS (40 mg/L) 共同作用,各组作用时间均为24 h。MTT 法检测肝细胞的增殖情况、测定培养上清液乳酸脱氢酶(LDH) 含量,ELISA 法检测TNF-α 和IL-6 表达。结果:LPS (40 mg/L) 作用于原代培养大鼠肝细胞24 h 后,与对照组相比,细胞生长抑制率达27%,培养上清液LDH 含量增加20 倍,TNF-α 和IL-6 的表达以及NF-κB DNA 结合活性均明显增加(P<0.05)。给予不同浓度NCTD 后,培养上清液LDH,TNF-α 和IL-6 的含量及NF-κB DNA 结合活性均明显下降,且呈量效关系。结论:NCTD 拮抗LPS 所致的肝细胞损伤,其保护作用的机制可能与其抑制TNF-α 和IL-6 的表达有关。

Effects of norcantharidin on lipopolysaccharide-induced hepatocyte injury in vitro

Objective: To study the e ects of norcantharidin(NCTD) on lipopolysaccharide(LPS)–induced hepatocyte injury and the expression of TNF-α and IL-6 in vitro.Methods: Hepatocytes were isolated from male Sprague-Dawley rats by collagenase perfusion.LPS at concentration of 40 mg/L was used to induce injury to the cultured cells,and NCTD(0.5,1.0,2.5 μg/mL) was added at the same time.After 24 h of incubation,the cell proliferation rates were detected by MTT.LDH,TNF-α and IL-6 were measured by appropriate reagent kits.NF-κB DNA binding activity was measured.Results: 40 mg/L LPS caused a 27% growth inhibition in primary hepatocytes.LDH leakage was 20fold higher in NCTD-treated hepatocytes than in normal ones.TNF-α and IL-6 expression significantly increased.In cells treated with NCTD at doses of 0.5,1.0 and 2.5 μg/mL,LDH leakage,TNF-α and IL-6 expression,and NF-κB DNA binding activity were attenuated in a dose dependent manner.Conclusion: NCTD protects hepatocytes from injury induced by LPS;the protection is associated with suppression of the in ammatory cytokine TNF-α and IL-6.