Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice - PubMed (original) (raw)

Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice

Ping-Guo Liu et al. World J Gastroenterol. 2008.

Abstract

Aim: To determine the effects of allopurinol, an inhibitor of xanthine oxidase, and apocynin, an inhibitor of NADPH oxidase, on oxidant stress and liver injury caused by hepatic ischemia/reperfusion (I/R) procedure in mice.

Methods: Mice were pretreated with a xanthine oxidase inhibitor, allopurinol, or NADPH oxidase (NOX) inhibitor, apocynin before the hepatic I/R procedure. Then treated or untreated mice underwent the hepatic I/R procedure. The effects on hepatic injury and superoxide anions were determined after starting reperfusion.

Results: A standard warm hepatic I/R procedure led to a marked increase in superoxide anion production as indicated by a superoxide anion tracer, MCLA. At the same time, the procedure caused profound acute liver injury, as indicated by elevated serum alanine aminotransferase and tumor necrosis factor-alpha levels, reduced liver glutathione levels and elevated malondialdehyde contents, as well as a high apoptotic cell count. All these changes were reversed by the use of apocynin or allopurinol prior to the hepatic I/R procedure.

Conclusion: Allopurinol and apocynin exerted protective effects on hepatic ischemia/reperfusion injury. The protection is associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting xanthine oxidase and NADPH oxidase activity.

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Figures

Figure 1

Figure 1

Attenuation of I/R-induced liver injury by apocynin and allopurinol in mice. A-D: Representative micrographs of liver histology (× 100). A: Normal liver; B: I/R-induced liver injury 6 h after starting reperfusion; C and D: Attenuation of liver injury with apocynin (APO) (C) or Allo (allopurinol) (D); E: Serum ALT levels in mice receiving apocynin or allopurinol and subsequently the hepatic I/R procedure. Serum ALT levels were determined 6 h and 24 h after starting reperfusion (n = 6 each group), and expressed as mean ± SEM. a_P_ < 0.01 vs I/R procedure plus normal saline (N.S.) at 6 h or 24 h.

Figure 2

Figure 2

Representative micrographs of in situ TUNEL staining of apoptotic cells in liver tissue after the hepatic ischemia/reperfusion procedure. A-D: The TUNEL staining assay was performed as described in the text, and the positive apoptotic cells were stained in red (× 100). A: Control; B: I/R procedure plus N.S. (6 h after starting reperfusion); C: I/R plus apocynin; D: I/R plus allopurinol. E-H: The corresponding liver sections stained with DAPI to illustrate nuclei as a cell number control.

Figure 3

Figure 3

The hepatic I/R-induced superoxide anion production and the effects of apocynin or allopurinol in mouse liver. The superoxide anion production was determined in mouse liver homogenates with a superoxide anion tracer, MCLA, and the MCLA chemiluminescent emission was recorded in a luminometer and expressed as relative light unit per second per micrograms of protein (mean ± SEM). a_P_ < 0.05, b_P_ < 0.01 vs the sham controls, d_P_ < 0.01 vs the I/R procedure plus N.S. at 6 h time point.

Figure 4

Figure 4

Hepatic I/R-induced depletion of reduced form of glutathione and enhanced lipid peroxidation, and effects of apocynin and allopurinol. A: Effect of apocynin or allopurinol on liver GSH levels with the hepatic I/R procedure. Liver GSH content was determined spectrophotometrically and expressed as nanomoles per milligram of protein of the tissue; B: Effects of apocynin or allopurinol on liver MDA levels with I/R procedure. Liver MDA content was determined spectrophotometrically and expressed as nanomoles per milligram of tissue protein (mean ± SEM). b_P_ < 0.01 vs Sham controls; c_P_ < 0.05, d_P_ < 0.01 vs the hepatic I/R procedure plus N.S. at 6 h time point (n = 6 in each group).

Figure 5

Figure 5

Effects of apocynin or allopurinol on serum TNF-α levels. Serum TNF-α in mice with hepatic I/R-induced liver injury was measured by an enzyme-linked immunosorbent assay kit. b_P_ < 0.01 vs Sham controls (mean ± SEM); c_P_ < 0.05; d_P_ < 0.01 vs I/R procedure plus N.S. at 6 or 24 h time point (n = 6 in each group).

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