Protective effects and mechanisms of bilirubin nanomedicine against acute pancreatitis

Highlights

• Bilirubin nanomedicine holds potent therapeutic potentials aginst acute pancreatitis.

• Silk firboin based nanoparticle release the bilirubin in an enzyme resposnive manner.

• Bilirubin nanomedicine selectively accumulate at the inflammatory pancreas.

• Bilirubin nanomedicine suppressed oxidative stress by activating Nrf2/HO-1 pathway.

• Bilirubin nanomedicine reduced inflammation by inhibiting NF-κB pathway.

Abstract

Acute pancreatitis (AP) is a sudden inflammatory reaction, caused by the activation of pancreatic enzymes in the pancreas, and in severe cases can lead to systemic inflammation and multiple organ failure. Oxidative stress contributed to the further deterioration of inflammation and played an important role in AP development. Bilirubin has been found to exert antioxidative, anti-inflammatory, and anti-apoptotic effects in a series of diseases accompanied by a high level of oxidative stress. However, the therapeutic effects of bilirubin for AP management have not yet been demonstrated. Additionally, the poor solubility and potential toxicity of bilirubin also limit its application. Thus, we developed bilirubin encapsulated silk fibrin nanoparticles (BRSNPs) to study the protective effects and mechanisms of bilirubin nanomedicine for the treatment of AP. BRSNPs could selectively delivery to the inflammatory lesion of the pancreas and release bilirubin in an enzyme-responsive manner. In the model of AP caused by L-Arginine hyperstimulation, BRSNPs exerted strong therapeutic effects against AP by the reduction of oxidative stress, decreased expression of pro-inflammatory cytokines, and impaired recruitment of macrophages and neutrophils. The mechanism study indicated that BRSNPs protected acinar cells against extensive oxidative damage and inflammation through inhibiting NF-κB pathway and activating the Nrf2/HO-1 pathway. Collectively, for the first time, this study demonstrated that bilirubin nanomedicine, BRSNPs, are effective in alleviating experimental acute pancreatitis, and the mechanisms are associated with its inhibition of NF-κB regulated pro-inflammatory signaling and activation of Nrf2-regulated cytoprotective protein expression.

Introduction

Acute pancreatitis (AP) is a sudden inflammatory reaction, begins with hyperstimulation of acinar cell caused by premature activation of pancreatic enzymes in the pancreas, and could lead to systemic inflammation and multiple organ failure in severe condition. The mortality in severe AP patients is surprisingly up to 20% due to the massive amplification of inflammation [1]. Acute pancreatitis can result from various etiologies such as alcohol abuse, drugs, biliary abnormalities, and autoimmune conditions. The pathogenesis of AP is not completely elucidated for now, but it is believed that oxidative stress plays a critical role in AP progression [1]. In the early stage of AP, excessive reactive oxidative species (ROS) cause oxidative damage to acinar cells and modulate the transcription and transduction of redox-related pathways to enlarge the inflammation. For example, nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical redox sensitive transcription factor that protect cells against oxidative stress induced damages. Studies showed that upon oxidative stress, Nrf2 binds to the antioxidant response element (ARE) and trigger antioxidant defense genes to defends against redox homeostatic failure [2], which could be applied to protect the acinar cells in AP condition. Sanfey et al. demonstrated that pre-treatments of antioxidants, such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), was beneficial in the AP treatment, indicating the involvement of oxidative stress in AP progress [3]. Among the pre-mentioned ARE, HO-1 can integrate NADPH and degrade heme into biliverdin to exert endogenous protective effects on the injured cells against excessive oxidative stress and inflammatory reactions. The upregulation of HO-1 was found to play a protective role in acute pancreatitis by decreasing leukocyte infiltration and pro-inflammatory cytokines level [4]. Nuclear factor-κB (NF-κB) is an important nuclear transcription factor in cells. Studies had shown that the NF-κB was involved in the inflammatory mediator's expression in acute pancreatitis [5], such as pro-inflammatory cytokines and adhesion molecules. Current clinical AP treatment often only involved in the inhibition of trypsin secretion and promoting the microcirculation to restrain the systematic inflammation. Considering the mechanism of AP development, directly mediating inflammation and redox signaling, e.g., NF-κB and Nrf2, might provide a new therapeutic alternation for the intervention of acute pancreatitis.

Bilirubin is a demonstrated endogenous antioxidant compound derived from heme catabolism. A low level of bilirubin in tissues could sufficiently scavenge ROS and attenuate the intracellular oxidative stress. Recent studies have shown that bilirubin nanomedicine [[6], [7], [8], [9], [10], [11]] can play an anti-inflammatory and immunomodulatory effect in a series of inflammatory models, such as asthma, hepatic ischemia-reperfusion injury, and improve islet transplantation microenvironment in the diabetics, even selectively target the tumors sites with high ROS content. Our previous study also demonstrated that bilirubin could protect the pancreatic islets during hypothermic preservation as well as islet transplantation [12,13]. Given the utility of bilirubin as a prophylactic agent in many acute inflammatory models, we hypothesized that bilirubin might offer a therapeutic benefit in experimental pancreatitis.

It is of clinical interest to explore the use of bilirubin for the AP treatment and associated mechanisms. However, the therapeutic application of bilirubin is limited with low aqueous solubility and hyperbilirubinemia induced potential toxicity. Nanotechnology-based formulation strategy could address the solubility issues of bilirubin and facilitate the precise delivery to inflammatory lesions by ROS responsiveness of bilirubin [14,15]. However, current bilirubin nanomedicine often developed by self-assembly from biomaterials conjugations, which might result in the incomplete release of bilirubin and compromised drug actions in the targeted site. As repeatedly mentioned, AP is characterized by over released pancreatic enzymes. Thereby, an enzyme-responsive nanomedicine that could rapidly release bilirubin with the presence of excessive enzymes is of impotence for the bilirubin application for AP treatment. It has been known that the silk fibrin (SF) protein can be degraded by many proteolytic enzymes, and is thus a natural biological material for the design of enzyme-responsive nanoparticles [16].

Encouraged by the intrinsic antioxidant and anti-inflammatory effects of bilirubin and the limitation of current bilirubin formulation, we prepared bilirubin encapsulated silk fibrin nanoparticles (BRSNPs) for AP treatment (Scheme. 1). The designed BRSNPs not only improved the water-solubility that avoided the abundance of free bilirubin induced jaundice but also retained antioxidative and anti-inflammatory effects. We hypothesized that BRSNPs could selectively travel into the pancreatic lesions and release bilirubin in an enzyme-responsive manner, and more importantly, provide efficient protection on the damaged acinar cells and attenuate the severity of AP. The protective effects and associated mechanisms of BRSNPs were assessed in oxidative injured acinar cells and hyper L-arginine induced AP rat model. Here we report the first application of BRSNPs as bilirubin nanomedicine for the treatment of acute pancreatitis and explore the therapeutic mechanisms.