P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice

Abstract

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl4-treated hepatocytes and generating redoxmediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and post-translational nitration, primarily in Kupffer cells, at 24 h post-CCl4 administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase- and P2X7 receptor-dependent, correlated well with the release of TNF- α and MCP-2 from Kupffer cells. The Kupffer cells in CCl4-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

Purinergic receptor X7 is a key modulator of metabolic oxidative stress-mediated autophagy and inflammation in experimental nonalcoholic steatohepatitis

“…We and others (5,31,32) have shown that toxininduced steatohepatitis models produce metabolic oxidative stress. The toxin-and diet-induced steatohepatitic injury models that produce metabolic oxidative stress are relevant to clinical outcomes because patients with NASH show oxidative stress and have been hypothesized to augment disease progression (27).…”

“…Metabolic oxidative stress in obesity-induced steatohepatitic injury is caused following increased CYP2E1 activity, mitochondrial uncoupling of the electron transport chain, or toxin metabolism following administration of hepatotoxic drugs (1,5,11,22). We and others (5,31,32) have shown that toxininduced steatohepatitis models produce metabolic oxidative stress.…”

“…Metabolic oxidative stress leads to cellular stress and induces cell death mechanisms, which modulate inflammatory microenvironment in NASH (5,24).…”

“…One of the key components of the inflammatory microenvironment in various disease pathologies is the inotropic purinergic receptors (P2 receptors) that respond to the damage-associated molecular patterns (3,28). There is an increased focus on a subclass of these receptors (P2X7 receptor), which bind ATP with high affinity and are known to regulate inflammation in early steatohepatitic injury in mice (5). Apart from the binding to ATP, purinergic receptor X7 (P2X7) receptor (P2X7r) has been shown to increase NADPH oxidase activity, increase Kupffer cell-major histocompatibility complex (MHC) class II expression, and regulate inflammation (5).…”

“…There is an increased focus on a subclass of these receptors (P2X7 receptor), which bind ATP with high affinity and are known to regulate inflammation in early steatohepatitic injury in mice (5). Apart from the binding to ATP, purinergic receptor X7 (P2X7) receptor (P2X7r) has been shown to increase NADPH oxidase activity, increase Kupffer cell-major histocompatibility complex (MHC) class II expression, and regulate inflammation (5). With increased focus on purinergic receptors, inflammasome activation, and autophagy in NASH, we explored the possibility of P2X7r as a key regulator of autophagy and the resultant inflammation in NASH.…”