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.