Pharmacological Prevention of Ectopic Erythrophagocytosis by Cilostazol Mitigates Ferroptosis in NASH

Abstract

Hepatic iron overload (HIO) is a hallmark of nonalcoholic fatty liver disease (NAFLD) with a poor prognosis. Recently, the role of hepatic erythrophagocytosis in NAFLD is emerging as a cause of HIO. We undertook various assays using human NAFLD patient pathology samples and an in vivo nonalcoholic steatohepatitis (NASH) mouse model named STAMTM. To make the in vitro conditions comparable to those of the in vivo NASH model, red blood cells (RBCs) and platelets were suspended and subjected to metabolic and inflammatory stresses. An insert-coculture system, in which activated THP-1 cells and RBCs are separated from HepG2 cells by a porous membrane, was also employed. Through various analyses in this study, the effect of cilostazol was examined. The NAFLD activity score, including steatosis, ballooning degeneration, inflammation, and fibrosis, was increased in STAMTM mice. Importantly, hemolysis occurred in the serum of STAMTM mice. Although cilostazol did not improve lipid or glucose profiles, it ameliorated hepatic steatosis and inflammation in STAMTM mice. Platelets (PLTs) played an important role in increasing erythrophagocytosis in the NASH liver. Upregulated erythrophagocytosis drives cells into ferroptosis, resulting in liver cell death. Cilostazol inhibited the augmentation of PLT and RBC accumulation. Cilostazol prevented the PLT-induced increase in ectopic erythrophagocytosis in in vivo and in vitro NASH models. Cilostazol attenuated ferroptosis of hepatocytes and phagocytosis of RBCs by THP-1 cells. Augmentation of hepatic erythrophagocytosis by activated platelets in NASH exacerbates HIO. Cilostazol prevents ectopic erythrophagocytosis, mitigating HIO-mediated ferroptosis in NASH models.

Erythrocyte TLR9 is upregulated in metabolic associated fatty liver disease, and is linked to an inflammatory immunometabolic signature

“…MAFLD constitutes a metabolic hepatic disease, where metabolic perturbations precede inflammatory mechanisms. Otogawa et al 18 and Park et al 19 , have reported that hepatic erythrophagocytosis results in inflammation during MAFLD. We have also reported extended erythrocyte changes during MAFLD 20–22,39 .…”

“…Our hypothesis of TLR9-induced pro-inflammatory erythrophagocytosis seems to be contradictory to previous studies with regard to MAFLD. In particular, previous studies claimed that solely iron is responsible for hepatic erythrophagocytosis-induced inflammation 18,19 . However, iron alone is not sufficient for triggering ferroptosis and inflammation.…”

“…Apart from gas exchangers, erythrocytes also possess central role in the immunometabolic regulation 16,17 . Accordingly, erythrocytes exhibit extended lipidome rearrangement, removal signals and pro-inflammatory phagocytosis during MAFLD [18][19][20][21][22] .…”

Ferroptosis in organ fibrosis: From mechanisms to therapeutic medicines

“…[ 149 ] Cilostazol, an antiplatelet drug, was also found to alleviate liver fibrosis through inhibiting hepatocyte ferroptosis via the prevention of ectopic erythrophagocytosis. [ 150 ] However, studies focused on the drugs targeting hepatocyte ferroptosis are still very limited.…”

Design, Synthesis, and Evaluation of Dihydropyrimidine Derivatives as Selective PDE1 Inhibitors for the Treatment of Liver Fibrosis

“…Elevated levels of cAMP and/or cGMP have been found to have antifibrotic effects by inhibiting the myofibroblast-driven ECM production. − Thus, several PDE inhibitors have been used to investigate the antifibrotic effects in different liver fibrosis animal models. For example, the PDE5 inhibitor tadalafil could improve portal hypertension and reduce liver fibrosis induced by bile duct ligation (BDL) in rats. , The PDE3 inhibitor cilostazol could protect rats from alcohol-induced liver fibrosis by suppressing the TGF-β/CTGF activation and the cAMP/EPAC1 signal. , Our group developed a PDE9 inhibitor 4a , which showed significant antifibrotic effects in a BDL-induced rat model …”