Protective effects of hydrogen enriched saline on liver ischemia reperfusion injury by reducing oxidative stress and HMGB1 release

Yantao Liu, Liqun Yang, Kunming Tao, Marcela P Vizcaychipi, Dafydd M Lloyd, Xuejun Sun, Michael G Irwin, Daqing Ma, Weifeng Yu, Yantao Liu, Liqun Yang, Kunming Tao, Marcela P Vizcaychipi, Dafydd M Lloyd, Xuejun Sun, Michael G Irwin, Daqing Ma, Weifeng Yu

Abstract

Background: The nuclear protein high-mobility group box 1 (HMGB1) is a key trigger for the inflammatory reaction during liver ischemia reperfusion injury (IRI). Hydrogen treatment was recently associated with down-regulation of the expression of HMGB1 and pro-inflammatory cytokines during sepsis and myocardial IRI, but it is not known whether hydrogen has an effect on HMGB1 in liver IRI.

Methods: A rat model of 60 minutes 70% partial liver ischemia reperfusion injury was used. Hydrogen enriched saline (2.5, 5 or 10 ml/kg) was injected intraperitoneally 10 minutes before hepatic reperfusion. Liver injury was assessed by serum alanine aminotransferase (ALT) enzyme levels and histological changes. We also measured malondialdehyde (MDA), hydroxynonenal (HNE) and 8-hydroxy-guanosine (8-OH-G) levels as markers of the peroxidation injury induced by reactive oxygen species (ROS). In addition, pro-inflammatory cytokines including TNF-α and IL-6, and high mobility group box B1 protein (HMGB1) were measured as markers of post ischemia-reperfusion inflammation.

Results: Hydrogen enriched saline treatment significantly attenuated the severity of liver injury induced by ischemia-reperfusion. The treatment group showed reduced serum ALT activity and markers of lipid peroxidation and post ischemia reperfusion histological changes were reduced. Hydrogen enriched saline treatment inhibited HMGB1 expression and release, reflecting a reduced local and systemic inflammatory response to hepatic ischemia reperfusion.

Conclusion: These results suggest that, in our model, hydrogen enriched saline treatment is protective against liver ischemia-reperfusion injury. This effect may be mediated by both the anti-oxidative and anti-inflammatory effects of the solution.

Figures

Figure 1
Figure 1
Schematic illustration of the experimental protocol. Sham: laparotomy and dissection of the portal vein but not clamping; I/R group: Ischemia was induced in the median and left lateral hepatic lobes for 1 hr, followed by 2 hour period of reperfusion; Normal saline + I/R group: Saline 10ml/kg injected intraperitoneally 10 minutes before hepatic reperfusion, followed by 2, 6, 12, 24 hour period of reperfusion; Hydrogen-enriched saline + I/R group: 1% hydrogen enriched saline (2.5, 5 or 10ml/kg) injected intraperitoneally 10 minutes before hepatic reperfusion, followed by 2, 6, 12, 24 hour period of reperfusion.
Figure 2
Figure 2
Hydrogen-enriched saline treatment protects liver against I/R injury. Male Sprague Dawley rats were subject to partial warm liver ischemia/reperfusion injury with intraperitoneal injection of either normal saline or hydrogen enriched saline at dose of 2.5, 5 or 10 ml/kg 10 minutes before reperfusion. Liver damage was assessed 2 hours after reperfusion with paraffin sections stained with H&E (original magnification × 200) and serum ALT level measurement. An example of microphotograph from (A) a sham-operated animal, (B) an animal undergoing 60 minutes of ischemia followed by 2 hours of reperfusion without any treatment, (C) with normal saline (10 ml/kg), or (D) hydrogen-enriched saline (10 ml/kg). (E) The dose-response (2.5-10 ml/kg) of hydrogen-enriched saline treatment and 10 ml/kg saline treatment on serum ALT release. Sham-operated animals underwent laparotomy only. Mean ± SEM (n = 8), *p < 0.05. (F) The time-course of liver damage assessed with ALT measurement at 2, 6, 12 and 24 hours after reperfusion with normal saline or hydrogen enriched saline treatment (10 ml/kg).
Figure 3
Figure 3
Hydrogen-enrich saline inhibited hepatic peroxidation induced by ischemia/reperfusion. Male Sprague Dawley rats were subjected a partial warm liver ischemia/reperfusion injury with intraperitoneal injection of either normal saline or hydrogen enriched saline at dose of 10 ml/kg 10 minutes before reperfusion. Liver damage was assessed 2 hours after reperfusion using immunohistochemistry staining of paraffin sections for HNE or 8-OH-G (original magnification × 400), and hepatic tissue MDA measurement. An example of microphotograph stained for HNE (brown precipitation at cellular membrane) and nucleus (blue) from (A) a sham-control, (B) 2 after reperfusion with normal saline, or (C) hydrogen enriched saline treatment. An example of microphotograph stained for 8-OH-G (brown precipitation at nucleus) and nucleus (blue) from (D) a sham-control, (E) 2 hours after reperfusion with normal saline, or (F) hydrogen enriched saline treatment. (G). Percentage of peroxidative cells was significantly lower in 10 ml/kg hydrogen enriched saline treatment in comparsion of 10 ml/kg saline treatment. (*P < 0.05) (H) MDA contents obtained from liver sections subjected to reperfusion for 2, 6, 12, and 24 hours after 60 minutes ischemia with normal saline or hydrogen enriched saline treatment. Sham-operated animals underwent laparotomy only. Mean ± SEM (n = 8), *p < 0.05.
Figure 4
Figure 4
Hydrogen-enriched saline prevents liver ischemia/reperfusion-induced HMGB1 over-expression and release. Male Sprague Dawley rats were subject to partial warm liver ischemia/reperfusion injury with either intraperitoneal injection of normal saline or hydrogen enriched saline at a dose of 10 ml/kg, 10 minutes before reperfusion. HMGB1 over expression in the liver were assessed by immunostaining and western blot. Serum levels were assayed by western blot. Examples of microphotographs stained for HMGB1 (seen as a brown precipitation both within the cytoplasm and nucleus, original magnification × 400): (A) sham-control, (B) 2 hours after reperfusion with normal saline, or (C) 2 after reperfusion with hydrogen enriched saline. (D) Western blot analysis for HMGB1 was performed on both liver sections and (E) serum samples collected from rats 2, 6, 12 and 24 hours after reperfusion. Sham-operated animals underwent laparotomy only. Mean ± SEM (n = 6); *p < 0.05.
Figure 5
Figure 5
Hydrogen-enriched saline attenuates TNFα and IL-6 mRNA expression in the liver and reduces serum TNFα and IL-6 levels in serum following liver ischemia reperfusion injury. Male Sprague Dawley rats were subject to partial warm liver ischemia/reperfusion injury with intraperitoneal injection of either normal saline or hydrogen enriched saline at dose of 10 ml/kg 10 minutes before reperfusion. (A) TNF-α and (B) IL-6 mRNA expression in the liver were measured by quantitative RT-PCR analysis at 2, 6, 12 and 24 hours after reperfusion and compared to the baseline levels prior to IRI. (C) Serum TNF-α and (D) IL-6 proteins levels were assessed by enzyme-linked immunosorbent assay. Mean ± SEM (n = 6 -8); *p < 0.05.

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