Abstract
•Bile acids, ethanol and fatty acids affect pancreatic ductal fluid and bicarbonate secretion via mitochondrial damage, ATP depletion and calcium overload. •Pancreatitis-inducing factors open the membrane transition pore (mPTP) channel via cyclophilin D activation in acinar cells, causing calcium overload and cell death; genetic or pharmacological inhibition of mPTP improves the outcome of acute pancreatitis in animal models. •Here we show that genetic and pharmacological inhibition of mPTP protects mitochondrial homeostasis and cell function evoked by pancreatitis-inducing factors in pancreatic ductal cells. •The results also show that the novel cyclosporin A derivative NIM811 protects mitochondrial function in acinar and ductal cells, and it preserves bicarbonate transport mechanisms in pancreatic ductal cells. •We found that NIM811 is highly effective in different experimental pancreatitis models and has no side-effects. NIM811 is a highly suitable compound to be tested in clinical trials. Mitochondrial dysfunction plays a crucial role in the development of acute pancreatitis (AP); however, no compound is currently available with clinically acceptable effectiveness and safety. In this study, we investigated the effects of a novel mitochondrial transition pore inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), in AP. Pancreatic ductal and acinar cells were isolated by enzymatic digestion from Bl/6 mice. In vitro measurements were performed by confocal microscopy and microfluorometry. Preventative effects of pharmacological [cylosporin A (2µm), NIM811 (2µm)] or genetic (Ppif-/- /Cyp D KO) inhibition of the mitochondrial transition pore (mPTP) during the administration of either bile acids (BA) or ethanol+fatty acids (EtOH+FA) were examined. Toxicity of mPTP inhibition was investigated by detecting apoptosis and necrosis. In vivo effects of the most promising compound, NIM811 (5 or 10mgkg-1 per os), were checked in three different AP models induced by either caerulein (10×50µgkg-1 ), EtOH+FA (1.75gkg-1 ethanol and 750mgkg-1 palmitic acid) or 4% taurocholic acid (2mlkg-1 ). Both genetic and pharmacological inhibition of Cyp D significantly prevented the toxic effects of BA and EtOH+FA by restoring mitochondrial membrane potential (Δψ) and preventing the loss of mitochondrial mass. In vivo experiments revealed that per os administration of NIM811 has a protective effect in AP by reducing oedema, necrosis, leukocyte infiltration and serum amylase level in AP models. Administration of NIM811 had no toxic effects. The novel mitochondrial transition pore inhibitor NIM811 thus seems to be an exceptionally good candidate compound for clinical trials in AP.
Highlights
Acute pancreatitis (AP) is among the most common gastrointestinal disorders requiring hospitalization in the United States (Fagenholz et al 2007a; Peery et al 2012)
Experiments with TMRM and TOM20 revealed that genetic inhibition of mPTP decreased both the loss of ψ (Fig. 1A) and mitochondrial mass (Fig. 1B) caused by 500 μM chenodeoxycholic acid (CDC; bile acids (BAs)) or co-administration of 100 mM ethanol and 200 μM palmitoleic acid (EtOH + FA)
Co-staining the pancreatic ducts with CytoCalcein Violet, Apopxin Deep Red and Nuclear Green showed that genetic inhibition of mPTP decreased the extent of necrosis and apoptosis during the administration of BA or EtOH + FA (Fig. 1C), suggesting that genetic inhibition of cyclophilin D (Cyp D) has a protective effect on pancreatic ductal epithelial cells (PDECs)
Summary
Acute pancreatitis (AP) is among the most common gastrointestinal disorders requiring hospitalization in the United States (Fagenholz et al 2007a; Peery et al 2012). Among the mechanisms noted above, one of the earliest events in AP is mitochondrial dysfunction (Sah & Saluja, 2011; Maleth et al 2013; Abu-El-Haija et al 2018; Biczo et al 2018) It has been shown in acinar cells that bile acids (BAs) and ethanol and fatty acids (EtOH+FA) open the membrane transition pore (mPTP) channel via cyclophilin D (Cyp D) activation, keeping the channel continuously opened and resulting in mitochondrial depolarization, lower ATP synthesis and cell necrosis (Shalbueva et al 2013; Mukherjee et al 2016; Abu-El-Haija et al 2018). It remains unknown how the pancreatitis-inducing factors noted above modify mPTP channel activity in pancreatic ductal epithelial cells (PDECs), it still seems to be one of the most promising drug targets and calls for further investigation
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