To investigate the effect of cytokines secreted by activated human peripheral blood mononuclear cell (h-PBMC) on α-1, 3-galactosyltransferase knockout pig hepatocytes (GalT-KO-hep) in xenogenic hepatocytes or liver transplantation.

LPS, poly(I∶C)-activated h-PBMC were co-cultured with immortalized GalT-KO-hep. Albumin, ALT, AST and urea in co-culture supernatant were detected by biochemical analysis. Flow cytometry was applied to detect the GalT-KO-hep apoptosis. Western blot was used to analysis the expression of cleaved caspase-3 and the proteins related to the signal transduction pathways of c-Jun N-terminal kinase (JNK), nuclear factor kappa-B, p38 mitogen-activated protein kinase (p38 MAPK), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT). The mRNA levels of cytokines of activated h-PBMC at different point of time, supernate biochemical indexes of co-culture model at different point of time, expression of cleaved caspase-3 and cell apoptosis of GalT-KO-hep were compared by analysis of variance. Further pairwise comparison was performed with LSD method. P <0.05 was considered statistically significant.

The ALT levels in co-culture supernatant at the time point of 6 hours after cocultivation were higher than 24, 48 hours (P all <0.05). The AST levels in co-culture supernatant at the time point of 48 hours after cocultivation were higher than 6, 24 hours (P all <0.05). The urea levels in co-culture supernatant at the time point of 24, 48 hours after cocultivation were higher than 6 hours (P all <0.05). The expression of cleaved caspase-3 and cell apoptosis in GalT-KO-hep increased 24, 48 hours later after cocultivation; and the expression of p-STAT3, p-JNK, p-IκBα, p-P38 MAPK, p-AKT and p-ERK (1/2) in GalT-KO-hep all increased 0.5, 1, 6, 12 hours later after cocultivation.

The cytokines released by h-PBMC can induce cell injury and apoptosis of GalT-KO-hep and promote the activation of inflammatory gene-related molecular pathways.