The Impact of Acute Matriptase Inhibition in Hepatic Inflammatory Models

Abstract

Purpose. Dysfunction of matriptase-2 can be involved in iron regulatory disorder via downregulation of hepcidin expression. In the present study, we investigated the effects of 3-amidinophenylalanine-derived matriptase inhibitors on porcine hepatic inflammatory cell models. Methods. Hepatocyte-Kupffer cell cocultures (ratio of 2 : 1 and 6 : 1) were treated with four structurally related matriptase inhibitors at 50 μM. Cell cytotoxicity and relative expressions of IL-6 and IL-8 and the levels of hepcidin were determined by MTS and porcine-specific ELISA. The extracellular H2O2 contents were analyzed by Amplex Red method. Results. Matriptase inhibitors at 50 µM for 24 h did not increase cell death rate. The elevated ROS production observed after short-term application of inhibitor MI-441 could be correlated with lowered hepcidin expression. MI-460 could significantly enhance hepcidin levels in the supernatants of cocultures (by 62.21 ± 26.8% in hepatocyte-Kupffer cell, 2 : 1, and by 42.6 ± 14.3% in hepatocyte-Kupffer cell, 6 : 1, cocultures, resp.). No significant changes were found in IL-6 and IL-8 levels in cocultures exposed to matriptase inhibitors. Conclusions. Based on in vitro findings, administration of MI-460 via modulation of hepcidin expression without cytotoxic and oxidative stress inducing properties might be a reliable alternative to treat iron overload in human and veterinary clinical practice.

3-Amidinophenylalanine-derived matriptase inhibitors can modulate hepcidin production in vitro

“…The solutions of the inhibitors in phenol red free Williams' Medium E at 50 μM (in the case of MI-461, 1, 10, 30, and 50 μM) were prepared freshly prior to each experiment from the stock solutions. It was found that the selected inhibitors can be used at 50 μM concentration safely for 24 h (cell death rate was less than 5% in controls and in treated samples) in Hep mono-and HepK6 co-cultures (Pomothy et al 2016) using MTS assay and this concentration could be applied in further experiments to characterize the effects of MT-1/MT-2 modulation in our hepatic models for 24 h. After incubation with the inhibitors for 24 h, the mono-and co-cultures were washed twice with plain medium before being subjected to the subsequent procedures.…”

“…An inhibition of MT-1 and MT-2 is also achieved by sulfonylated 3-amidinophenylalanine derivatives (Hammami et al 2012). The most potent analogues of this inhibitor-type contain an N-terminal dichloro-or dimethoxy-biphenyl-3-sulphonyl group and inhibit MT-1 in the low one-digit nanomolar range, whereas the best inhibition constants for MT-2 are close to 100 nM (Hammami et al 2012;Pomothy et al 2016). However, the effects of these combined MT-1/2 inhibitors on the compensation for iron homeostatic imbalances caused by dysregulated hepcidin production have not been elucidated yet.…”

Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect

“…ROS and cell viability were also measured by establishing the effects of 3,3 0 -diindolylmethane (DIM) antioxidant agent on HIEC-6 51 . Using MI-1900 and MI-1907, the extracellular hydrogen-peroxide production and release of pro-inflammatory cytokines such as IL-6 and IL-8 were monitored and it was confirmed that both compounds at 50 mM did not induce excessive oxidative or inflammatory responses in HIEC-6 similarly to other Phe(3-Am)-derived inhibitors such as MI-460 and 461 in hepatocytes-based cell cultures 52,53 .…”

“…The impact of several Phe(3-Am)-derived inhibitors was investigated on porcine intestinal epithelial cell line, IPEC-J2, hepatocyte mono-and hepatocyte-Kupffer cell co-cultures. It was found that 50 mM MI-432, 441, 460, and 461 did not exert any detrimental effects on cell viability of hepatocyte-Kupffer cell co-cultures 52 . In addition, a significant increase in hepcidin levels was detected in hepatocyte mono-and hepatocyte-Kupffer cell co-cultures after acute treatments 53 .…”

Potential Therapeutic Agents and Associated Bioassay Data for COVID-19 and Related Human Coronavirus Infections

“… 120 Finally, compounds MI460 and CAS RN 944925–37–5 may also inhibit proinflammatory cytokines and block blood coagulation-related factors, respectively. 121 , 122 …”