TAF blocks phosphorylation of AKT in MPs
Immunoblotting was performed to determine the signalling pathway blocked by TAF in in vivo STZ, HFHC diet-fed NASH mouse model treated with mock or TAF. The p-AKT, AKT, p-mTOR, mTOR, and GAPDH protein levels were detected in the NASH mice livers. p-AKT and p-mTOR levels were decreased in the livers of the STZ-injected TAF-treated HFHC diet-fed mice compared to those of the STZ-injected mock-treated HFHC diet-fed mice (Fig 5A).
We investigated TAF’s effects on human monocytes, as monocytes are recruited to damaged livers and differentiate into MPs. We sorted CD14+ monocytes in the peripheral blood mononuclear cells. Sorted human CD14+ monocytes were pre-treated with TAF for 6 h and then stimulated with or without LPS for 24 h. The CD14+ monocyte population was determined using flow cytometry. PD-L1+ and human leukocyte antigen-DR isotype (HLA-DR)+ populations decreased with increasing TAF concentrations compared to those in the mock group (Fig 5B). The HLA-DR+/PD-L1+ cell number increased after LPS treatment but decreased in a concentration-dependent manner in the TAF-stimulated group (Fig 5C). Moreover, TAF treatment decreased HLA-DR and PD-L1 MFI in activated CD14+monocytes compared to that in the mock group (Fig 5D). Representative AKT and p-AKT level plots in activated human CD14+monocytes treated with mock or TAF were analysed using flow cytometry. The phosphorylated AKT population was significantly reduced following TAF addition to activated human CD14+ monocytes based on p-AKT MFI values; AKT phosphorylation was blocked by TAF treatment (Fig 5E). A schematic representation of the effect of TAF on NASH liver is presented in Fig 5F.