4.3. Immortalized Mouse Kupffer Cells (ImKCs) and Hep3B Cell Culture

Immortalized mouse kupffer cells (ImKCs) were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada), 10% FBS (fetal bovine serum) was added to Dulbecco’s Modified Eagle’s Medium (DMEM). Incubation occurred in an environment of 37 °C, 5% CO₂. To evaluate the mtROS inhibitory effect of ZC, ImKCs (5 × 10⁴ cells/mL) were treated with lipopolysaccharide (LPS, 500 ng/mL), ZC (10 μM) and Mito-TEMPO (50 μM) for 6 h. In order to measure the protein level of the related gene, ImKCs (4 × 10⁵ cells/mL) were pre-treated with ZC (10 μM) for 5 h and then simultaneously treated with lipopolysaccharide (LPS, 250 ng/mL) for 1 h. To confirm the anti-inflammatory effect of ZC and to measure mRNA levels, ImKCs (2 × 10⁵ cells/mL) were treated with lipopolysaccharide (LPS, 125 ng/mL) and ZC (10 μM) for 6 h. Lipopolysaccharide (LPS, 250 ng/mL), ZC (10 μM), and Mito-TEMPO (50 μM) were treated in ImKCs (4 × 10⁵ cells/mL) for 6 h to measure the protein level of the related gene.

Hep3B cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. In order to evaluate the mtROS inhibitory efficacy of ZC, Hep3B (3 × 10⁴ cells/mL) cells were treated with tumor necrosis factor (TNF)-α (100 ng/mL), ZC (10 μM) and Mito-TEMPO (50 μM) for 24 h. To measure the anti-lipid effects and mRNA levels of ZC, Hep3B (2 × 10⁵ cells/mL) cells were treated with TNF-α (30 ng/mL), ZC (10 μM) and Mito-TEMPO (50 μM) for 24 h.