Nrf2 is a leucine zipper protein that is covalently repressed by its regulator Keap1 in the cytoplasm through ubiquitination [
20,
21,
22]. Stress induces the separation of these two molecules via modification of the Keap1 cysteine residues, allowing Nrf2 to enter the nucleus and heterodimerize with small Maf Proteins (MafP) (
Figure 1). The Nrf2/MafP complex binds to genes which promote the transcription of multiple antioxidant enzymes [
23,
24,
25,
26,
27]. Yagishita et al. have demonstrated that the Nrf2/Keap1 system plays a critical role in the protection of pancreatic β-cells from oxidative damage through repressed apoptosis and enhanced proliferation [
28]. Nrf2/Keap1 thus promises a potential way of reducing the oxidative damage that occurs in ICT.
Dh404, dimethyl fumarate (DMF), and epigallocatechin gallate (EGCG) are the Nrf2 activators that have been most studied in context of pancreatic inflammation and islet cell transplantation.
Nrf2 Activators
Dh404, formally CDDO-9,11-dihydro-trifluoroethyl amide (CDDO-dhTFEA) [
29] is a synthetic oleanane triterpenoid (SO) plant derivative used in oriental medicine [
30] for anti-inflammatory and anti-tumorigenic purposes [
31,
32]. Since Yates et al.’s first report of dh404 as a protective agent against aflatoxin-induced tumorigenesis [
33], dh404 has been studied in oncology, chronic renal disease, and recently in diseases of the pancreas. As a group, SOs are unique because they are one of the most potent inducers of the Nrf2 pathway with selective induction of phase II detoxifying and antioxidant enzymes [
34].
Dh404 has proven to be protective in the pathogenesis of acute pancreatitis. Dh404 (1 mg/kg)-treated rats 24 h before L-arginine (600 mg/100 g)-induced pancreatitis showed reductions in inflammatory cells, acinar structural damage, edema, necrosis (
p < 0.001), and rates of apoptosis (
p < 0.05) [
35]. Malondialdehyde (MDA), which is an indicator of lipid peroxidation, was also reduced (
p < 0.05) [
35]. MDA was further shown to be lower in dh404-cultured cells compared to controls when in 200 μM H
2O
2 for a 24-h period [
35]. Lastly, the effect of dh404 was shown to be temporally dependent, as cells that were incubated with 500 nM dh404 for 1 h had nearly twice the intranuclear Nrf2 concentration as cells incubated for 30 min. When dh404 treatment was prolonged for 24 h, the presence of anti-oxidant enzymes such as Heme Oxygenase-1 (HO-1), superoxide dismutase (SOD), and catalase (CAT) was recorded [
35].
The mechanism of dh404-mediated Nrf2 activation is not yet clear. Ichikawa et al. showed that dh404 is involved in a unique interaction with Cys-151 of Keap1, which under physiological conditions binds Cul3/Rbx1 E3 ligase complex to target Nrf2 ubiquitination and subsequent digestion [
36]. On another hand, Li et al. have shown that dh404-mediated Nrf2-activated pathway involves the autophagy of toxic ubiquitinated proteins driven directly by Nrf2 induction, and not by ROS [
37]. Because ROS were previously shown [
38] to endogenously drive the autophagy process as a defense mechanism to inflammation, these findings suggest that dh404 activates Nrf2 to simultaneously carry out two actions that are not mutually exclusive. Whether this response is entirely due to the Nrf2 or supplemented by an additional pathway activated by dh404 necessitates further investigation.
Dimethyl fumarate, otherwise known as BG-12 or Tecfidera, is a fumarate ester that started out as a recognized anti-carcinogen [
39], in the 1990s it was licensed in Germany for treatment of psoriasis, and more recently in 2013 has received approval by the US Food and Drug Administration (FDA) for the treatment of relapsing-remitting multiple sclerosis [
40]. Our lab examined the role of DMF as a Nrf2 activator in the setting of pancreatitis [
41,
42]. Pancreata of rats treated with DMF (25 mg/kg) 24 h prior to L-arginine (3 g/kg)-induced acute pancreatitis showed reductions in the severity of inflammatory cell infiltration, acinar damage, perilobar edema, and cell necrosis (
p < 0.001) [
41]. Similarly, rats that were orally fed DMF (25 mg/kg) prior to and after L-arginine-induced-chronic pancreatitis resulted in improved glucose tolerance, better-preserved tissue architecture (less atrophy, edema, and fatty infiltration) (
p < 0.05), significantly lower levels of inflammatory markers (myeloperoxidase (MPO) and MDA), and significantly higher expression of antioxidants (i.e., HO-1) [
42]. Zhang et al. corroborated similar findings and also demonstrated that animals transplanted with DMF-treated-cells had lower blood glucose (
p < 0.01) and preserved β-cell function [
43].
Interestingly, and conveniently, DMF has demonstrated to be most efficacious under stressful conditions. In a study performed by Schultheis et al., islet cells from adult mice were cultured for 12-16 h in DMF, and then for 2 or 48 h under control or glucolipotoxic conditions (25 mmol/L glucose and 100 µmol/L palmitate) [
44]. Compared to controls, cells in the glucolipotoxic medium had a decrease in oxidized status, superior insulin secretion, and a higher mitochondrial membrane potential (50 vs. 10 µmol/L) at 48 h [
44]. While the benefits of DMF in the treatment of inflammatory conditions have been shown to be due to a sundry of anti-inflammatory responses [
45,
46,
47,
48], the specific mechanism behind Nrf2-activation necessitates further investigation.
Epigallocatechin gallate is a main ingredient of green tea and has been described since the 1990s to have anticarcinogenic, antioxidant, antiangiogenic, antiviral properties, and more recently antidiabetic properties [
49,
50,
51,
52]. It has been shown to act as a neutralizing agent for ROS, and to have anti-inflammatory effects that have reduced liver fibrosis [
53] and even contribute to hepatic regeneration [
54].
EGCG has been shown to suppress cytokine-induced pancreatic β-cell damage in vitro. Pretreatment of RINm5f cells with EGCG (0–200 µg/mL) in presence of proinflammatory cytokines resulted in no cell apoptosis compared to the 55% that became apoptotic in the absence of EGCG [
55]. In fact, the response was noted to be concentration-dependent, with 200 µg/mL EGCG nearly fully blocking the cell death response, abrogating the three-fold increase in NO
2 seen in control, and completely inhibiting the production of inducible NO synthase (iNOS) [
55]. To simulate the inflammatory environment of T1DM in vivo, the authors induced autoimmune diabetes with a 250 mg/kg streptozotocin (STZ) dose in C57BL/KsJ mice for five consecutive days. In the experimental group, EGCG (100 mg/kg) was administered daily with STZ, and alone for the five days thereafter [
56]. Relative to the control group, the EGCG-treated mice had a significantly reduced STZ-induced hyperglycemia, and markedly suppressed iNOS mRNA expression [
56].
Despite current evidence that EGCG is an activator of the Nrf2/Keap1 pathway [
57], the exact mechanism of how this occurs has yet to be elucidated. There is evidence that the activation involves the mitogen-activated protein kinase (MAPK) cascade [
58], electrophilic interactions with the cysteine residues in Keap1 [
59], as well as ROS-derived auto-oxidation of EGCG facilitating the release of Nrf2 from its complex [
60].
Having introduced the activators with most promise in the setting of pancreatic islet cell transplantation, next we discuss how the Nrf2/Keap1 pathway is a potential target at each step of the transplantation process to protect islet cells from oxidative damage.