Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Dec;305(12):G950-63.
doi: 10.1152/ajpgi.00235.2013. Epub 2013 Oct 24.

Purinergic Receptor X7 Is a Key Modulator of Metabolic Oxidative Stress-Mediated Autophagy and Inflammation in Experimental Nonalcoholic Steatohepatitis

Affiliations
Free PMC article

Purinergic Receptor X7 Is a Key Modulator of Metabolic Oxidative Stress-Mediated Autophagy and Inflammation in Experimental Nonalcoholic Steatohepatitis

Suvarthi Das et al. Am J Physiol Gastrointest Liver Physiol. .
Free PMC article

Abstract

Recent studies indicate that metabolic oxidative stress, autophagy, and inflammation are hallmarks of nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanisms that link these important events in NASH remain unclear. In this study, we investigated the mechanistic role of purinergic receptor X7 (P2X7) in modulating autophagy and resultant inflammation in NASH in response to metabolic oxidative stress. The study uses two rodent models of NASH. In one of them, a CYP2E1 substrate bromodichloromethane is used to induce metabolic oxidative stress and NASH. Methyl choline-deficient diet feeding is used for the other NASH model. CYP2E1 and P2X7 receptor gene-deleted mice are used to establish their roles in regulating metabolic oxidative stress and autophagy. Autophagy gene expression, protein levels, confocal microscopy based-immunolocalization of lysosome-associated membrane protein (LAMP)2A and histopathological analysis were performed. CYP2E1-dependent metabolic oxidative stress induced increases in P2X7 receptor expression and chaperone-mediated autophagy markers LAMP2A and heat shock cognate 70 but caused depletion of light chain 3 isoform B (LC3B) protein levels. P2X7 receptor gene deletion significantly decreased LAMP2A and inflammatory indicators while significantly increasing LC3B protein levels compared with wild-type mice treated with bromodichloromethane. P2X7 receptor-deleted mice were also protected from NASH pathology as evidenced by decreased inflammation and fibrosis. Our studies establish that P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, thereby modulating hepatic inflammation. Furthermore, our findings presented here form a basis for P2X7 receptor as a potential therapeutic target in the treatment for NASH.

Keywords: 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts; CYP2E1; GABA-A receptor-associated protein; cytokines; light chain 3 isoform B; lipid peroxidation; tyrosine nitration.

Figures

Fig. 1.
Fig. 1.
A: 4-Hydroxynonenal (4-HNE), (a marker of lipid peroxidation) immunoreactivity in liver slices from diet-induced obesity (DIO), DIO + bromodichloromethane (BDCM), CYP2E1 knockout (KO), methylcholine-sufficient (MCS)-diet-fed mice, and methylcholine-deficient (MCD)-diet-fed mice (iv). B: nitrotyrosine immunoreactivity as shown by immunohistochemistry in liver slices (iv; DIO, DIO+BDCM, CYP2E1 knockout, MCS, and MCD groups). C: morphometry (percentage) of 4-hydroxynonenal-reactive cells (*P < 0.05). D: morphometry (percentage) of nitrotyrosine (NTyr)-immunoreactive cells (*P < 0.05). E: 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-nitrone adducts, a marker of protein free radicals in toxin model of nonalcoholic steatohepatitis (NASH) as assessed by ELISA with anti-DMPO antibody (*P < 0.05). F and G: CYP2E1 mRNA expression as measured by quantitative real-time PCR (P < 0.05) in toxin-induced and MCD models of NASH. H: Western blot analysis of CYP2E1 protein levels. Lanes 1–5 represent DIO, DIO+BDCM, CYP2E1 Knockout, MCS, and MCD, respectively. I: band quantification of the immunoblots using normalization against β-actin. The Western analysis was done using liver homogenates from the respective groups. Representative immunoblot is shown.
Fig. 2.
Fig. 2.
A and B: mRNA expression of purinergic receptor X7 (P2X7) receptor as assessed by quantitative real-time PCR, normalized against DIO and MCS groups. CYP2E1 KO represents liver mRNA expression from CYP2E1 gene-depleted mice fed with high-fat diet (*P < 0.05). C: immunohistochemistry of P2X7 receptor protein from liver slices of DIO, DIO+BDCM, CYP2E1 KO, MCS, and MCD groups. The localization of the receptor-positive staining is shown by arrows.
Fig. 3.
Fig. 3.
Metabolic oxidative stress influences autophagy protein profiles in NASH. A and B: mRNA expressions of early and late autophagy-related proteins in toxin and diet models of NASH (*P < 0.05) as measured by quantitative real-time PCR. GABARAP: GABA-A receptor-associated protein; Atg-2A, autophagy-related protein 2A; LC3B, light chain 3 isoform B; Hsc 70, heat shock cognate 70; LAMP2A, lysosome-associated membrane protein. Immunoreactivity of GABARAP (C) and LAMP2A (D) as measured by immunohistochemistry is shown. iv: DIO, DIO+BDCM, CYP2E1 KO, MCS, and MCD, respectively. E and F: Western blot image for Hsc 70 and its corresponding band quantification analysis (*P < 0.05). The normalization was carried out against the levels of β-actin in the liver homogenates.
Fig. 4.
Fig. 4.
P2X7 receptor deletion modulates autophagy in NASH. A and B: mRNA expressions of early and late autophagy-related proteins in toxin and diet models of NASH (*P < 0.05) as measured by quantitative real-time PCR in DIO+BDCM and P2X7 receptor gene-deleted mice, coexposed to high-fat diet and toxin, as well as MCD and P2X7 receptor gene-deleted mice fed with MCD diet. C: Western blot analysis of GABARAP and LC3B and their corresponding band quantification analysis (D) (*P < 0.05). E: Western blot analysis of LC3B protein in DIO, DIO+BDCM, MCS, and MCD liver homogenates. Bands are normalized against β-actin. F: immunohistochemistry of LC3B protein in liver slices of DIO, DIO+BDCM, and P2X7 receptor knockout mice coexposed to high-fat diet and BDCM showing perivenular areas.
Fig. 5.
Fig. 5.
A: immunoreactivity of LAMP2A as analyzed by immunohistochemistry. iiv: DIO+BDCM, P2X7 receptor-deleted group coexposed to high-fat diet and BDCM (toxin), as well as MCD and P2X7 receptor knockout group exposed to MCD diet, respectively. B and C: Western blot analysis of Hsc 70 in DIO+BDCM, P2X7 receptor-deleted group coexposed to high-fat diet and BDCM (toxin), as well as MCD and P2X7 receptor-deleted group (MCD) (*P < 0.05). C represents the band quantification analysis.
Fig. 6.
Fig. 6.
A: LAMP2A colocalization with lysosomal membrane (×60 zoom 3). Confocal laser scanning image of LAMP2A and membrane lipid (Bodipy stain) from liver sections of DIO, DIO+BDCM, and P2X7 receptor-deleted group coexposed to high-fat diet and BDCM. B: LAMP2A colocalization with lysosomal membrane (×60). Confocal laser scanning image of LAMP2A and membrane lipid (Bodipy stain) from liver sections of MCS, MCD, and P2X7 receptor-deleted group fed with MCD diet. Gray arrows show LAMP2A localizations, whereas white arrows depict colocalizations of LAMP2A with lysosomal membrane depicted by bodipy staining. Number of colocalizing events as shown by overlay of images were counted to reveal the extent of LAMP2A attachment to the lysosomal membrane.
Fig. 7.
Fig. 7.
P2X7 receptor modulates inflammation in NASH. A and B: mRNA expression of proinflammatory mediators, as measured by quantitative real-time PCR in DIO, DIO+BDCM, P2X7 receptor gene-deleted mice, coexposed to high-fat diet and BDCM (toxin), MCS, MCD, and P2X7 receptor gene-deleted mice fed with MCD diet. The expression levels were normalized against DIO in toxin model and MCS in diet model. C: Western blot analysis of cleaved caspase-1 (active caspase-1) and its corresponding band quantification analysis (D) (*P < 0.05). Individual lanes representing DIO+BDCM and P2X7 receptor gene-deleted groups were separated from the parent blot to ensure better visibility. E and F: immunoreactivity of IL-1β and IFN-γ as analyzed by immunohistochemistry. ivi: DIO, DIO+BDCM, P2X7 receptor-deleted group coexposed to high-fat diet and BDCM (toxin), MCS, MCD, and P2X7 receptor-deleted group exposed to MCD diet, respectively.
Fig. 8.
Fig. 8.
A: Western blot analysis of high-motility group box protein (HMGB)-1 from liver homogenates of DIO+BDCM, P2X7 receptor-deleted group coexposed to high-fat diet and BDCM (toxin), MCD, and P2X7 receptor-deleted group exposed to MCD diet, respectively. B: band quantification analysis following normalization against β-actin (*P < 0.05). C: quantitative real-time PCR analysis of the mRNA expression profiles of fibrosis markers in DIO+BDCM, P2X7 receptor-deleted group coexposed to high-fat diet and BDCM (toxin), MCD, and P2X7 receptor-deleted group exposed to MCD diet, respectively (*P < 0.05). SMA, smooth muscle actin. D: hematoxylin and eosin staining of liver sections from DIO+BDCM group and P2X7 gene-deleted group coexposed to BDCM and high-fat diet. Areas of necrosis are shown in demarcated gray dashed lines and arrow heads. E: Picro-Sirius red staining for fibrosis in liver sections from DIO+BDCM group and P2X7 gene-deleted group coexposed to BDCM and high-fat diet.

Similar articles

See all similar articles

Cited by 23 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback