To establish and validate human liver cell L-02 Ischemia reperfusion injury (IRI) model.

Human liver cell line L-02 IRI model was constructed using liquid paraffin, cobalt chloride (CoCl₂) and hypoxia incubator, respectively. Cell viability was detected by CCK-8 method. The levels of Superoxide Dismutase (SOD) and Malondialdehyde (MDA) were detected by oxidative stress kit, and apoptosis was detected by Annexin-V fluorescein isothiocyanate (FITC)/propidiumiodide (PI) apoptosis Detection Kit. Apoptosis-related aspartic acid specific cysteine proteinase-3 (Caspase-3) and B-cell lymphoma-2-associated X protein (bax) were determined by Western blotting. The above methods were used to verify and statistically analyze the effects of constructing hepatocyte IRI in three ways. T test was used for comparison between the two groups, one-way ANOVA was used for comparison between multiple groups, and P<0.05 was considered statistically significant.

Compared with the control group, the activity of hepatocyte IRI model constructed by liquid paraffin, CoCl₂ and hypoxia incubator was significantly decreased, and the absorbance at 450 nm was 0.698±0.021, 0.622±0.025 and 0.725±0.025 compared with 1.086±0.123, respectively. The difference was statistically significant (F=30.02, P<0.01). The cell activity of liquid paraffin hypoxia and incubator group were higher than that of CoCl₂ group (t=3.660, 4.605, P<0.05). SOD levels in all groups were lower than control group [(117.678±9.797), (100.095±5.588), (105.614±4.880) U/mg protein vs. (172.378±6.208) U/mg protein, F=92.56, P<0.01]. SOD level in liquid paraffin group was higher than that in CoCl₂ group (t=3.118, P<0.05). MDA levels in each group were significantly higher than control group [(62.684±4.584), (70.931±1.849), (63.472±2.316) vs. (16.163±2.106) nmol/mg protein, F=219.90, P<0.01). MDA in CoCl₂ group was higher than that in liquid paraffin group and hypoxia incubator group (t=2.889, 4.347, P<0.05). Compared with the control group, the apoptosis rate was increased [(26.087±4.107)%, (36.797±3.453)%, (31.945±2.859)% vs. (1.840±0.489)%, F=103.50, P<0.01), and the apoptosis rate of CoCl₂ group was higher than that of liquid paraffin group (t=3.992, P<0.01). The relative expression level of P-Caspase-3 in each group was higher than that in the control group (2.460±0.102, 2.629±0.059, 2.574±0.107 vs. 1.000, F=291.23, P<0.01). The relative expression level of bax was also increased compared with the control group (1.506±0.012, 1.618±0.055, 1.631±0.080 vs. 1.000, F=90.80, P<0.01). There was no significant difference in the relative expression level of apoptosis protein among the three groups of liquid paraffin, CoCl₂ and hypoxia incubator (F=4.33, 0.92, P>0.05).

Liquid paraffin, CoCl₂ and hypoxia incubators can induce IRI of hepatocytes successfully. In the absence of hypoxia incubators, liquid paraffin is preferred to construct the IRI model of hepatocytes.

Ischemia-reperfusion injury; L-02 hepatocytes; Cell model; Hypoxia