Heme Oxygenase-1 Inhibits the NLRP3 Inflammasome to Protect Against Severe Acute Pancreatitis

Research Article

Ann Hematol Onco. 2023; 10(5): 1436.

Heme Oxygenase-1 Inhibits the NLRP3 Inflammasome to Protect Against Severe Acute Pancreatitis

Yuansong Sun1#; Jinwei Qi2,3#; Tingting Yao1; Chunlin Yin1; Min Yang1; Weiwei Ge2,3

¹The Second Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, China

²The First Affiliated Hospital of Anhui Medical University, Anhui Medical University, Hefei, China

³Anhui Public Health Clinical Center, Hefei, China

*Corresponding author: Weiwei Ge, MD The First Affiliated Hospital of Anhui Medical University, Hefei, China. Tel: (+86) 13865990867; (+86) 13721040363 Min Yang, MD The Second Affiliated Hospital of Anhui Medical University, Hefei, China. Email: doctor_vivige@hotmail.com; 512130761@qq.com

#These authors have contributed equally to this article.

Received: July 20, 2023 Accepted: August 19, 2023 Published: August 26, 2023

Abstract

Background: Systemic Inflammatory Response Syndrome (SIRS) and IL-1β and IL-18 maturation and production are encouraged by the NLRP3 inflammasome. Severe Acute Pancreatitis (SAP) may be less severe if certain tactics are used to prevent the NLRP3 inflammasome. The rate-limiting enzyme in the breakdown of heme, Heme Oxygenase-1 (HO-1), has anti-inflammatory, antioxidant, and anti-proliferative properties. HO-1 activity profoundly affects the host’s ability to forbear infection by reducing tissue damage or affecting resistance and increasing the capacity to pathogen load. We postulated that hemin, a strong HO-1 inducer, could decrease NLRP3 inflammasome activation, which would lessen the severity of SAP and acute lung injury caused by pancreatitis.

Methods: By administering intraperitoneal injections of Caerulein (Cae) and Lipopolysaccharide (LPS) to SD rats, a model of SAP was created. Hemin and zinc protoporphyrin IX (Znpp, a HO-1 inhibitor) pretreatments respectively stimulated and inhibited the HO-1 enzyme.

Results: The SAP model rats’ pancreas and lungs suffered considerable pathological damage after Cae and LPS injection, and their serum levels of amylase, lipase, and the cytokines IL-1β and IL-18 also rose. Hemin pretreatment decreased IL-1β and IL-18 release in the serum and prevented pancreatic and pulmonary damage. Hemin dramatically reduced oxidative stress, downregulated the expression of the proteins NLRP3, ASC, and Caspase-1, and elevated the expression of the protein HO-1. On the contrary, there were no discernible changes between the SAP and Znpp groups.

Conclusions: These results show that hemin prevents Cae and LPS-induced lung and pancreatic harm through suppression of the NLRP3 inflammasome. Hemin’s impact on the activity of the NLRP3 inflammasome depends critically on HO-1.

Keywords: Severe acute pancreatitis; NLRP3; HO-1; Inflammation

Abbreviation: SIRS: Systemic Inflammatory Response Syndrome; SAP: Severe Acute Pancreatitis; HO-1: Heme Oxygenase-1; Cae: Caeruelin; LPS: Lipopolysaccharide; Znpp: Zinc Protoporphyrin IX; ALI: Acute Lung Injury; ARDS: Acute Respiratory Distress Syndrome; NLRs: Nod-Like Receptors; SD: Sprague-Dawley; CO: Carbon Monoxide; MDA: Malondialdehyde; SOD: Superoxide Dismutase; ANOVA: One-Way Analysis of Variance

Introduction

A pancreatic inflammation that poses a serious risk to life is known as Severe Acute Pancreatitis (SAP). Inflammatory mediators have an essential effect on the evolution of AP from mild to severe and in the occurrence of MODS in SAP. The causes of SAP death are mostly related to MODS, among which Acute Lung Injury (ALI) and ALI-induced Acute Respiratory Distress Syndrome (ARDS) are the most severe and common. During SAP, premature trypsin activation within pancreatic acinar cells causes pancreatic auto-digestion, leading to a local inflammatory process; these mediators release many relevant pro-inflammatory factors. The increase of capillary permeability and extravascular lung water is a crucial pathophysiological change in ALI/ARDS, and the severe ventilatory blood flow ratio imbalance caused by increases of extravascular lung water is an important reason for refractory hypoxemia and high mortality of ALI/ARDS. The inflammatory cascade brought on by sterile inflammasome activation has been associated with SAP and ALI. Inflammasome NLRP3 is an oligomeric molecular complex activated not only by bacteria and viruses but also by its "danger signals."

The specific intracellular pattern recognition molecules known as Nod-Like Receptors (NLRs) control the host's innate immune response [1]. The aggregation of inflammasomes is facilitated by NLRP3, which functions similarly to other NLR proteins. A NOD-like receptor, adaptor protein ASC [Caspase-1 Activator domain-containing protein (CARD)], and caspase-1 itself make up the macromolecular multi-protein NLRP3 inflammasome. The NLRP3 inflammasome is created when NLRP3 is activated, ASC, and pro-caspase-1 are recruited, and then caspase-1 is activated, which results in the conversion of pro-IL-1β and pro-IL-18 into their activated versions [2].

The inducible isomer of Heme Oxygenase-1 (HO-1) is a rate-limiting enzyme that catalyzes the oxidative breakdown of heme [3]. Its physiological role is to accelerate the production of heme, which releases ferrous iron, biliverdin, and Carbon Monoxide (CO). HO-1 is an essential metabolic enzyme and a vital intermediate between stress response and cellular injury adaptation. In many cellular and preclinical damage models, the transcriptional regulation of HO-1 has been widely related to cytoprotection and the protective inhibition of inflammation [4,5]. HO-1 and heme degradation products may modulate inflammation. CO can stimulate the mitochondrial to produce ROS, promoting the autophagy program, activating HIF-1a, and downregulating the pro-inflammatory transcription factor Egr1. Additionally, recent research suggests that CO can influence how the NLRP3 inflammasome, which controls the production of IL-1β and IL-18, is activated [6].

Two hypotheses were intended to be proved by this investigation. Firstly, by preventing the NLRP3 inflammasome from becoming activated in the Cae and LPS-induced SAP mice, hemin could reduce organ injury and inflammation. Second, it's possible that HO-1 contributes to the NLPR3 inflammasome's activation.

Materials and Methods

Animals

Healthy male Sprague-Dawley rats weighing 260–300g were procured from the Experimental Animal Center of Anhui Medical University. The National Society for Medical Research and Guidelines for Laboratory Animal Care provided the animals with humane treatment. The rats were kept in a temperature-controlled space (25±1°C) with a 12-hour light/dark cycle and unrestricted access to food and drink.

Animal Model of Severe Acute Pancreatitis

Twenty-four male SD rats were divided into four groups at random: control, SAP, Hemin (40g/kg; Sigma Chemical, St. Louis, MO) and Zn-PP (40g/kg; Sigma Chemical, St. Louis, MO) groups. The HO-1 stimulator Hemin was injected intraperitoneally 30 min after SAP induction, and the HO-1 inhibitor Zn-PP was injected intraperitoneally 30 min later. SAP was induced by injecting 50g/kg of cerulein (Sigma-Aldrich) in 0.9% Nacl. LPS (Sigma-Aldrich) was administered intraperitoneally to the animals at a dose of 5 mg/kg following the last injection. 24 hours after the development of SAP, rats were given intraperitoneal sodium pentobarbital (40 g/kg) to induce anesthesia before being put to death. Intracardiac puncture blood samples were centrifuged, and the serum was kept at -80°C for investigation of the levels of amylase, lipase, IL-1β, and IL-18. Western blot and histological analysis of the pancreatic and lung tissues were used to compare the histopathological alterations in the organs between subgroups.

Histological Examination Analysis

Pancreas and lung tissue samples were fixed in 10% formalin solution, embedded in paraffin, and sectioned following hematoxylin and eosin staining for use in light microscopy. For each rat, The sections were evaluated blindly by two observers. Alveolar walls were characterized by diffuse reactions, thickening, and the presence of inflammatory cells (neutrophil and mononuclear) [7]. The scale for the score is 0 to 4; an injury severity scale would seem as follows: Injury levels range from 0 (lack of injury) to 4 (severe or intense injury), where 4 denotes the highest level of injury. The average scores were taken as the final score. Pancreas damage [8] was scored by grading, as shown in Table 1. The average scores were taken as the final score.