METTL3 Deficiency Aggravates Hepatic Ischemia/Reperfusion Injury in Mice by Activating the MAPK Signaling Pathway

Abstract

Background: Inflammatory responses, apoptosis, and oxidative stress, are key factors that contribute to hepatic ischemia/reperfusion (I/R) injury, which may lead to the failure of liver surgeries, such as hepatectomy and liver transplantation. The N6-methyladenosine (m⁶A) modification has been implicated in multiple biological processes, and its specific role and mechanism in hepatic I/R injury require further investigation. Methods: Dot blotting analysis was used to profile m⁶A levels in liver tissues at different reperfusion time points in hepatic I/R mouse models. Hepatocyte-specific METTL3 knockdown (HKD) mice were used to determine the function of METTL3 during hepatic I/R. RNA sequencing and western blotting were performed to assess the potential signaling pathways involved with the deficiency of METTL3. Finally, AAV8-TBG-METTL3 was injected through the tail vein to further elucidate the role of METTL3 in hepatic I/R injury. Results: The m⁶A modification levels and the expression of METTL3 were upregulated in mouse livers during hepatic I/R injury. METTL3 deficiency led to an exacerbated inflammatory response and increased cell death during hepatic I/R, whereas overexpression of METTL3 reduced the extent of liver injury. Bioinformatic analysis revealed that the MAPK pathway was significantly enriched in the livers of METTL3-deficient mice. METTL3 protected the liver from I/R injury, possibly by inhibiting the phosphorylation of JNK and ERK, but not P38. Conclusions: METTL3 deficiency aggravates hepatic I/R injury in mice by activating the MAPK signaling pathway. METTL3 may be a potential therapeutic target in hepatic I/R injury.

Keywords: Hepatic ischemia/reperfusion injury; MAPK.; METTL3; apoptosis; inflammatory response; m6A.

Figure 1

m⁶A levels and METTL3 expression were upregulated during liver I/R. (A-H) Wild-type C57BL/6 mice were subjected to sham or 1 h of ischemia and subsequent reperfusion for 3 h, 6 h, 12 h, and 24 h (n = 5 each time point). Relevant indicators were detected. (A-B) Serum ALT/AST levels. (C) Representative HE staining images of liver sections. (D) Necrotic area proportion and (E) The Suzuki's scores of liver sections. (F) The m⁶A methylation of total RNA was detected by Dot Blot analysis. (G-H) The mRNA level (G) and protein level (H) of METTL3 in the liver tissue harvested from sham and I/R group. (I) Western blot analysis of METTL3 expression in primary hepatocytes or L02 cells after 6 h of hypoxia followed by 6 h of reoxygenation.

Figure 2

METTL3 deficiency exacerbated liver damage after I/R injury. (A) Schematic representation of the generation of METTL3-HKD and METTL3-Flox mice. (B) The expression of METTL3 in major organs of HKD mice. (C-E) Representative HE staining images (C), necrotic area pro-portion (D) and Suzuki' score (E) of liver sections, and serum AST/ALT levels (F-G) of METTL3-HKD mice and METTL3-Flox mice in the sham group and I/R 6 h group (n = 5 per group).

Figure 3

METTL3 deficiency aggravated inflammatory response and cell death during hepatic I/R injury. (A-E) Mice were subjected to sham or I/R 6 h injury (n = 5 per group). (A) Representative TUNEL staining images and number of TUNEL-positive cells from liver sections of METTL3-HKD and METTL3-Flox mice at 6 h after suffering I/R injury. (B-C) The mRNA levels (B) and protein levels (C) of apoptosis-related factors in the liver. (D-E) The mRNA levels of inflammatory factors (D) and protein expression of NF-κB signaling pathway (E) in the liver. (F) L02 cells were transfected with three different shMettl3 plasmids. (G-J) Cell death and inflammation-related factors were detected in L02 cells subjected to 6 h of hypoxia and 6 h of reoxygenation after being transfected with shMettl3 or control plasmids.

Figure 4

AAV-mediated hepatocyte-specific METTL3 overexpression attenuated hepatic I/R injury in mice. (A) Wild type C57BL/6 mice were injected with AAV8-GFP or AAV-TBG-METTL3 through tail vein and then subjected to I/R 6 h injury (n = 5 per group). (B-C) Western blot (C) and qPCR (B) analysis of METTL3 in the liver of mice injected with AAV8-GFP or AAV-TBG-METTL3. (D-G) Representative HE staining images (D), necrotic area proportion and Suzuki' score (E) of liver sections, and serum AST/ALT levels (F-G) of mice injected with AAV8-GFP or AAV-TBG-METTL3 and further subjected to I/R 6 h injury.

Figure 5

Overexpression of METTL3 inhibited inflammation and apoptosis during hepatic I/R injury. (A) Representative TUNEL staining images and numbers of TUNEL-positive cells from liver sections, which were obtained from mice injected with AAV8-GFP or AAV-TBG-METTL3 and further subjected to I/R 6 h injury (n = 5 per group). (B-C) The protein levels (B) and mRNA levels (C) of apoptosis-related factors in the liver of mice injected with AAV8-GFP or AAV-TBG-METTL3 and further subjected to I/R 6 h injury. (D-E) The mRNA levels of inflammatory factors (D) and protein expressions of NF-κB signaling pathway (E) in the liver of mice injected with AAV8-GFP or AAV-TBG-METTL3 and further subjected to I/R 6 h injury.

Figure 6

METTL3 participated in hepatic I/R injury by inhibiting the MAPK pathway. (A-B) GSEA analysis of the biological processes related to apoptosis (A) and inflammatory response (B). (C) Volcano plots showing the significantly differential genes between the METTL3-HKD and METTL3-Flox mice subjected to I/R 6 h injury. MAPK signaling pathway-related genes are high-lighted in green. (D) Major biological pathways determined by KEGG enrichment analysis of RNA-seq of liver tissues from METTL3-HKD and METTL3-Flox mice subjected to I/R 6 h injury. (E-F) Heatmap generated based on the expression of apoptosis-related genes (E) and inflammation-related genes (F) in the livers from METTL3-HKD and METTL3-Flox mice detected by RNA-seq analyses. (G-I) Western blot analyses of the total and phosphorylated protein levels of ERK, JNK, and P38 in liver and L02 cells after I/R or H/R insult.