CMPK2 accelerates liver ischemia/reperfusion injury via the NLRP3 signaling pathway

Abstract

Cytidine monophosphate kinase 2 (CMPK2) is a mitochondrial nucleotide monophosphate kinase which is important for the substrates of mitochondrial DNA synthesis and has been reported to participate in macrophage activation and the inflammatory response. The purpose of the present research was to determine the potential role of CMPK2 in hepatic ischemia/reperfusion (I/R) injury and to elucidate the underlying molecular mechanisms. The present study investigated the role of CMPK2 in regulating the NLRP3 pathway and liver dysfunction induced by hepatic I/R both in vivo and in vitro. It was revealed that hypoxia/reoxygenation (H/R) treatment enhanced the mRNA expression levels of CMPK2, NLRP3, IL-18, IL-1β and TNF-α in RAW 264.7 cells. The protein expression levels of IL-18, IL-1β and cleaved-caspase-1 were decreased following CMPK2 knockdown. Furthermore, the inhibition of AIM2 downregulated the expression level of IL-1β, IL-18 and cleaved-caspase-1 in the CMPK2 knockdown group followed by H/R treatment, while the inhibition of NLRP3 did not. CMPK2 deficiency also decreased alanine aminotransferase and aspartate aminotransferase expression in mice serum, as well as the pathological changes in the liver. Similarly, the release of IL-18 and IL-1β in mouse serum was also restrained with the decline of CMPK2. In conclusion, the results of the present study demonstrate that CMPK2 is indispensable for NLRP3 inflammasome activation, making CMPK2 an effective target to relieve the liver from I/R injury. In addition, the function of CMPK2 is closely associated with NLRP3 inflammasome activation, instead of AIM2.

Keywords: AIM2; CMPK2; NLRP3 inflammasome; hepatic I/R injury; inflammation.

Figure 1

mRNA expression levels of CMPK2, NLRP3 and inflammatory factors in H/R model of RAW 264.7 cells. (A) The mRNA levels of CMPK2 were measured at different time points after reoxygenation. The mRNA expression levels of (B) NLRP3, (C) TNF-α, (D) IL-1β and (E) IL-18 were detected following H/R treatment. ^*P<0.05, ^**P<0.01 vs. control groups. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; H/R, hypoxia/reoxygenation.

Figure 2

Knockdown of CMPK2 decreased the NLRP3-associated inflammation factor. (A) Protein detection of CMPK2 after H/R treatment in scramble siRNA or CMPK2 siRNA-transfected RAW 264.7 cells. (B) Western blotting and subsequent quantification of (C) CMPK2, (D) NLRP3, (E) AIM2, (F) pro-IL-1β, (G) cleaved-caspase-1 (H) IL-1β and (I) IL-18 protein levels. (J) IL-18 and IL-1β levels in the supernatant were detected by ELISA. ^**P<0.01, ^***P<0.001 vs. control group, ^#P<0.05, ^&&P<0.01 vs. H/R group. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; H/R, hypoxia/reoxygenation; siRNA, small interfering RNA; AIM2, absent in melanoma 2.

Figure 3

Knockdown of CMPK2 decreases IL-18 and IL-1β by inhibiting the NLRP3 inflammasome, and not AIM2. Protein detection of (A) NLRP3 or (B) AIM2 following H/R treatment in scramble siRNA or specific siRNA-transfected RAW 264.7 cells. (C) Protein detection and respective quantification of (D) cleaved-caspase-1, (E) IL-18 and (F) IL-1β in the H/R + CMPK2 siRNA, H/R + CMPK2 siRNA + NLRP3 siRNA, and H/R + CMPK2 siRNA + AIM2 siRNA groups. ^##P<0.01 vs. H/R +CMPK2 siRNA group. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; H/R, hypoxia/reoxygenation; AIM2, absent in melanoma 2; siRNA, small interfering RNA.

Figure 4

Suppression of CMPK2 alleviates I/R-induced deterioration of hepatic injury. (A) Protein detection of CMPK2 in liver tissue. Secretion of (B) IL-18 and (C) IL-1β was measured by ELISA. Hepatocellular function of experimental mice was evaluated by detecting the release of (D) AST and (E) ALT. (F and G) Histological changes of the liver in the sham, I/R, I/R + scramble siRNA and I/R + CMPK2 siRNA groups. Original magnification, x200. ^**P<0.01, ^***P<0.001 vs. sham group; ^#P<0.05, ^##P<0.01 vs. I/R group. CMPK2, cytidine monophosphate kinase 2; NLRP3, NLR family pyrin domain containing 3; I/R, ischemia/reperfusion; siRNA, small interfering RNA; ALT, alanine aminotransferase; AST, aspartate aminotransferase.