[Establishment and evaluation of hepatocyte injury model induced by LPS/D-galactosamine in vitro]

Objective To establish a novel hepatocyte injury model induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in vitro. Methods Freshly isolated mouse primary hepatocytes were cultured in vitro and treated with different doses of tumor necrosis factor-α (TNF-α) and 5 mg/mL of D-GalN. The supernatants from hepatocyte culture were detected for alanine aminotransferase (ALT) activity by chemiluminescence assay. Bone marrow-derived macrophages (BMDMs) were stimulated with 1 μg/mL of LPS and the level of TNF-α in supernatants were detected by ELISA. Primary hepatocytes were treated with the BMDM supernatants combined with 5 mg/mL D-GalN or 50 ng/mL actinomycin D (ActD) for 24 hours. The level of ALT from hepatocyte supernatant was detected and morphology of hepatocytes was observed with microscopy. BMDMs and hepatocytes were co-cultured and treated with 1 μg/mL of LPS combined with D-GalN or ActD for 24 hours. Hepatocyte injury was reflected by the ALT activity and hepatocyte morphology. Results The ALT activity was significantly increased in the supernatants of hepatocytes treated with TNF-α and D-GalN, indicating the obvious hepatocyte injury. Co-treatment with LPS-primed BMDM supernatants and D-GalN or ActD could cause hepatocyte injury, as reflected by markedly increased ALT activity and the deformed and cracked hepatocytes. In the context of co-culture of BMDM and hepatocytes, treatment with LPS and D-GalN led to obvious hepatocyte injury as expected. LPS combined with ActD could not cause hepatocyte injury, since the BMDMs started to die earlier than they could secret TNF-α to destruct hepatocytes. Hepatocytes with normal morphology and deformed BMDMs were observed. Conclusion LPS/D-GalN can be used to induce hepatocyte injury in vitro. D-GalN, rather than ActD, should be used as a transcriptional inhibitor when the TNF-α -induced hepatocyte injury is evaluated in a co-culture system of BMDMs and hepatocytes.