Deletion of p38γ attenuates ethanol consumption- and acetaminophen-induced liver injury in mice through promoting Dlg1

Abstract

Acetaminophen (APAP) is one of the major causes of drug-induced acute liver injury, and ethanol may aggravate APAP-induced liver injury. The problem of ethanol- and APAP-induced liver injury becomes increasingly prominent, but the mechanism of ethanol- and APAP-induced liver injury remains ambiguous. p38γ is one of the four isoforms of P38 mitogen activated protein kinases, that contributes to inflammation in different diseases. In this study we investigated the role of p38γ in ethanol- and APAP-induced liver injury. Liver injury was induced in male C57BL/6 J mice by giving liquid diet containing 5% ethanol (v/v) for 10 days, followed by gavage of ethanol (25% (v/v), 6 g/kg) once or injecting APAP (200 mg/kg, ip), or combined the both treatments. We showed that ethanol significantly aggravated APAP-induced liver injury in C57BL/6 J mice. Moreover, the expression level of p38γ was up-regulated in the liver of ethanol-, APAP- and ethanol+APAP-treated mice. Knockdown of p38γ markedly attenuated liver injury, inflammation, and steatosis in ethanol+APAP-treated mice. Liver sections of p38γ-knockdown mice displayed lower levels of Oil Red O stained dots and small leaky shapes. AML-12 cells were exposed to APAP (5 mM), ethanol (100 mM) or combined treatments. We showed that P38γ was markedly increased in ethanol+APAP-treated AML-12 cells, whereas knockdown of p38γ significantly inhibited inflammation, lipid accumulation and oxidative stress in ethanol+APAP-treated AML-12 cells. Furthermore, we revealed that p38γ could combine with Dlg1, a member of membrane-associated guanylate kinase family. Deletion of p38γ up-regulated the expression level of Dlg1 in ethanol+APAP-treated AML-12 cells. In summary, our results suggest that p38γ functions as an important regulator in ethanol- and APAP-induced liver injury through modulation of Dlg1.

Keywords: Dlg1; P38γ; acetaminophen; ethanol; fatty liver; steatohepatitis.

Fig. 1. EtOH aggravated APAP-induced liver injury in C57BL/6 J mice.

a Liver tissues stained with HE. b, c Serum ALT and AST assay. d–f Levels of MDA, GSH, and SOD in serum; (g-h). Levels of TNF-α and IL-6 in serum. i IHC and quantitative analysis of F4/80. j The expression levels of SREBP-1 and Fasn detected by real-time PCR and Western blotting. k Oil Red O staining in mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Pair group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs APAP or EtOH alone group.

Fig. 2. P38γ was elevated in EtOH- and APAP-induced liver injury in C57BL/6 J mice.

a IHC analysis of p38γ. b The expression level of p38γ and p-p38γ was detected by real-time PCR and Western blotting. c Albumin and p38γ colocalized in the liver by double immunofluorescence. d Immunofluorescence analysis of p38γ. **P < 0.01, ***P < 0.001 vs Pair group. ^##P < 0.01 vs APAP or EtOH alone group.

Fig. 3. EtOH enhanced inflammation secretion, lipid accumulation and oxidative stress in response to APAP.

a–d Real-time PCR and Western blotting detected the levels of PPAR-α, SREBP-1 (the cleaved nuclear (~68 kDa) forms of SREBP-1) and Fasn. e, f Real-time PCR detect the level of TNF-α and IL-6. g Western blotting detected the level of NOX-4. h, i The production of ROS detected by DCF and DHE assay. **P < 0.01, ***P < 0.001 vs Normal group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs APAP or EtOH alone group.

Fig. 4. P38γ was also increased in EtOH+APAP-induced AML-12 cells.

a–c Immunofluorescence, Western blotting and real-time PCR were used to detect the p38γ expression level. d, e. Real-time PCR and Western blotting of the level of p38γ after transfection with pEGFP-C1-p38γ. f, g. Real-time PCR and Western blotting of the level of p38γ after transfection with p38γ-siRNA. h Immunofluorescence analysis of p38γ after transfection with pEGFP-C1-p38γ and p38γ-siRNA. *P < 0.05, **P < 0.01, ***P < 0.001 vs Normal group. ^#P < 0.05, ^##P < 0.01 vs APAP or EtOH alone group.

Fig. 5. P38γ interference mitigated inflammation, lipid accumulation and oxidative stress in EtOH + APAP-induced AML-12 cells.

a–e The expression levels of IL-1β, IL-6, TNF-α, iNOS and NOX4 detected by Western blotting and real-time PCR. f–j The expression levels of PPAR-α, ACOX1, Fasn and SREBP-1 (the cleaved nuclear (~68 kDa) forms of SREBP-1) detected by Western blotting and real-time PCR. k The production of ROS detected by DCF and DHE assay. l-p. The expression levels of IL-1β, IL-6, TNF-α, iNOS, and NOX4 detected by Western blotting and real-time PCR after over-expression of p38γ. q–u The expression levels of PPAR-α, ACOX1, Fasn, and SREBP-1 detected by Western blotting and real-time PCR after over-expression of p38γ. v. The production of ROS detected by DCF and DHE assay after over-expression of p38γ. *P < 0.05, **P < 0.01, ***P < 0.001 vs Normal. ^#P < 0.05, ^##P < 0.01 vs EtOH or APAP alone. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 5. P38γ interference mitigated inflammation, lipid accumulation and oxidative stress in EtOH + APAP-induced AML-12 cells.

a–e The expression levels of IL-1β, IL-6, TNF-α, iNOS and NOX4 detected by Western blotting and real-time PCR. f–j The expression levels of PPAR-α, ACOX1, Fasn and SREBP-1 (the cleaved nuclear (~68 kDa) forms of SREBP-1) detected by Western blotting and real-time PCR. k The production of ROS detected by DCF and DHE assay. l-p. The expression levels of IL-1β, IL-6, TNF-α, iNOS, and NOX4 detected by Western blotting and real-time PCR after over-expression of p38γ. q–u The expression levels of PPAR-α, ACOX1, Fasn, and SREBP-1 detected by Western blotting and real-time PCR after over-expression of p38γ. v. The production of ROS detected by DCF and DHE assay after over-expression of p38γ. *P < 0.05, **P < 0.01, ***P < 0.001 vs Normal. ^#P < 0.05, ^##P < 0.01 vs EtOH or APAP alone. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 6. P38γ interacted with Dlg1 in AML-12 cells.

a–c IHC and Western blotting analysis of Dlg1 in mice. d, e P38γ could interact with Dlg1 in AML-12 cells, as demonstrated by a co-IP assay. with Dlg1. f Double immunofluorescence displayed the typical colocalization of p38γ and Dlg1 in AML-12 cells. g, h Western blotting and real-time PCR of the levels of Dlg1 after transfection with p38γ-siRNA in AML-12 cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs Normal group. ^##P < 0.01, ^###P < 0.001 vs EtOH or APAP alone group. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 7. P38γ interference mitigated inflammation, lipid accumulation and oxidative stress in EtOH + APAP-induced AML-12 cells by interacting with Dlg1.

a–d IF, Western blotting and real-time PCR were used to detect the expression levels of p38γ and Dlg1 in AML-12 cells after transfection with p38γ-siRNA and Dlg1-siRNA. e–g Western blotting and real-time PCR were used to detect TNF-α, Fasn and SREBP-1 (the cleaved nuclear (~68 kDa) form of SREBP-1). h The production of ROS detected by DCF and DHE assays. ***P < 0.001 vs Normal group. ^###P < 0.001 vs APAP or EtOH alone group. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 8. P38γ silencing alleviated liver dysfunction and injury in EtOH- and APAP-treated mice.

a Mouse in vivo imaging analysis showed that AAV9-shRNA-p38γ was specifically located in mouse liver tissue. b–d The mRNA and protein expression levels of p38γ and p-p38γ. e. HE staining in liver tissue. f. Oil Red O staining in liver tissue. g, h. Serum ALT and AST assay. i Western blotting detected the expression levels of p38γ and Dlg1. **P < 0.01, ***P < 0.001 vs Normal group. ^##P < 0.01, ^###P < 0.001 vs APAP or EtOH alone group. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 9. P38γ KD protected against liver inflammation, lipid accumulation and oxidative stress in EtOH- and APAP-induced mice.

a–c IHC analysis of p38γ, Dlg1, and F4/80. d The protein expression levels of IL-6, Fasn, PPAR-α, and SREBP-1 (the cleaved nuclear (~68 kDa) forms of SREBP-1). e–g The expression levels of MDA, SOD, and GSH. *P < 0.05, **P < 0.01, ***P < 0.001 vs Normal group. ^##P < 0.01, ^###P < 0.001 vs APAP or EtOH alone group. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.

Fig. 10. P38γ disruption alleviated inflammation, lipid accumulation and oxidative stress in primary hepatocytes extracted from EtOH- and APAP-induced mice.

a–f The mRNA and protein expression levels of SREBP-1, Fasn, PPAR-α, p38γ, and Dlg1 in the pair group and model primary hepatocytes. g–k The mRNA and protein expression levels of SREBP-1 (the cleaved nuclear (~68 kDa) forms of SREBP-1), Fasn, p38γ and TNF-α in Pair p38γ KD and model p38γ KD primary hepatocytes. l DCF analysis of ROS production. **P < 0.01, ***P < 0.001 vs Normal group. ^##P < 0.01, ^###P < 0.001 vs APAP or EtOH alone group. ^$$P < 0.01, ^$$$P < 0.001 vs EtOH+APAP group.