Sevoflurane attenuates hepatic ischemia reperfusion injury by the miR-122/Nrf2 pathway

Abstract

Background: Sevoflurane can protect organs from ischemia-reperfusion (IR) injury, but the mechanism is still unclear. MicroRNA-122 (miR-122) is a liver-specific microRNA (miRNA) and regulates liver function. Therefore, this study aims to elucidate the relationship between the protective effect of sevoflurane and miR-122 in liver IR injury.

Methods: Wistar rats were divided into the following groups: sham, IR, IR + sevoflurane, IR + miR-122 antagomir, and IR + miR-122 antagomir + sevoflurane. Hematoxylin and eosin (H&E) staining and Suzuki score were used to evaluate the pathological damage of the liver. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in the serum and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) in the liver homogenate supernatant were detected by using the corresponding kit. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and flow cytometry was applied to evaluate the apoptosis of liver tissues. The expression of nuclear factor E2-related factor 2 (Nrf2), miR-122, p53, and HO-1 in liver tissue was evaluated by using immunohistochemistry, qRT-PCR, and western blot as needed.

Results: Compared to the IR group, the sevoflurane post-treatment or miR-122 antagomir groups showed improved liver injury, decreased Suzuki score, inhibited the levels of AST, ALT, LDH, MDA, NO, TNF-α, IL-1β, and IL-6, increased levels of SOD, IL-10, and inhibited hepatocyte apoptosis. Regarding the molecular mechanism, sevoflurane post-treatment fostered the expression of HO-1, promoted the transport of Nrf2 from cytoplasm to the nucleus, and decreased the expression of miR-122 and p53. The combined use of miR-122 antagomir and sevoflurane enhanced the protective effect of miR-122 antagomir in liver injury in IR rats.

Conclusions: Sevoflurane protected the liver from IR damage by regulating the miR-122/Nrf2/HO-1 pathway.

Keywords: Nrf2/HO-1; Sevoflurane; liver ischemia/reperfusion; miR-122.

Figure 1

Sevoflurane ameliorated IR-induced liver damage. The rats were randomly divided into the sham group, the IR group, and the IR + sevoflurane group. Liver injury for the rats in the IR group was induced through ischemia/reperfusion, and liver injury was induced in the IR + sevoflurane group through ischemia/reperfusion and then inhalation of 2% sevoflurane. (A) H&E staining of liver tissue sections (magnification: ×200, scale bar =100 mm; ×400, scale bar =50 mm). (B) The Suzuki score of livers. (C) The levels of AST, ALT, and LDH in the serum of rats. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the sham group; ^##, P<0.01 vs. the IR group. IR, ischemia-reperfusion; H&E, hematoxylin and eosin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase.

Figure 2

Effects of sevoflurane on the antioxidant activity and inflammation induced by IR in liver tissues. (A) Levels of MDA, SOD, and NO in liver tissue. (B) The levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in the serum of rats. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the sham group; ^##, P<0.01 vs. the IR group. IR, ischemia-reperfusion; MDA, malondialdehyde; SOD, superoxide dismutase; NO, nitric oxide; TNF-α, tumor necrosis factor-α; IL, interleukin.

Figure 3

Immunohistochemistry was used to detect the expression of Nrf2 in liver tissue. (A) Immunohistochemical staining of Nrf2 (magnification: ×200, scale bar =100 mm; ×400, scale bar =50 mm). (B) Relative positive expression analysis of Nrf2. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the sham group; ^#, P<0.05 vs. the IR group. Nrf2, nuclear factor E2-related factor 2; IR, ischemia-reperfusion.

Figure 4

Sevoflurane improved IR-induced apoptosis of liver tissue. (A) Apoptosis of liver tissue in each group was detected by flow cytometry. (B) TUNEL staining was used to detect the apoptosis of liver tissue in each group (magnification, ×200). (C) Statistics of apoptosis rate were detected with flow cytometry. (D) Statistics of positive cell rate were detected with TUNEL staining. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the sham group; ^##, P<0.01 vs. the IR group. IR, ischemia-reperfusion; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Figure 5

qRT-PCR detection of the effects of sevoflurane post-treatment on miR-122 levels after liver IR injury in rats. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the sham group; ^##, P<0.01 vs. the IR group. IR, ischemia-reperfusion.

Figure 6

The effect of sevoflurane on the protein expression of p53 and Nrf2 in liver tissue induced by IR. (A) Western blot showed the expressions of p53. (B) Western blot showed the expressions of Nrf2 in the nucleus. (C) Western blot showed the expressions of Nrf2 in the cytoplasm. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲, P<0.05 vs. the sham group; ^##, P<0.01 vs. the IR group. IR, ischemia-reperfusion; Nrf2, nuclear factor E2-related factor 2.

Figure 7

MiR-122 antagomir combined with sevoflurane further reduced IR-induced liver damage. The rats were randomly divided into the IR group, the IR + miR-122 antagomir group, and the IR + miR-122 antagomir + Sevo group. Liver injury was induced in the rats in the IR group through ischemia/reperfusion. The mice in the IR + miR-122 antagomir group were pretreated with miR-122 antagomir and then liver injury was induced through ischemia/reperfusion. The mice in the IR + miR-122 antagomir + Sevo group were pretreated with miR-122 antagomir, then liver injury was induced through ischemia/reperfusion, and then the rats inhaled 2% sevoflurane. (A) H&E staining of liver tissue sections (magnification: ×200, scale bar =100 mm; ×400, scale bar =50 mm). (B) The Suzuki score of livers. (C) The levels of AST, ALT, and LDH in the serum of rats. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲, P<0.05, ^▲▲, P<0.01 vs. the IR group;^★, P<0.05, ^★★, P<0.01 vs. the IR + miR-122 antagomir group. +, this group rats were treated with indicated agent; −, this groups did not receive this indicated treatment. IR, ischemia-reperfusion; H&E, hematoxylin and eosin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase.

Figure 8

MiR-122 antagomir combined with sevoflurane further inhibited oxidative stress and inflammatory response induced by IR. (A) Levels of MDA, SOD, and NO in liver tissue. (B) The levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in the serum of rats. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲▲, P<0.01 vs. the IR group; ^★, P<0.05, ^★★, P<0.01 vs. the IR + miR-122 antagomir group. +, this group rats were treated with indicated agent; −, this groups did not receive this indicated treatment. IR, ischemia-reperfusion; MDA, malondialdehyde; SOD, superoxide dismutase; NO, nitric oxide; TNF-α, tumor necrosis factor-α; IL, interleukin.

Figure 9

MiR-122 antagomir combined with sevoflurane further promoted the expression of miR-122 and activated the Nrf2/HO-1 signaling pathway. (A) qRT-PCR showed the miR-122 levels in liver tissue. (B) Western blot showed the expressions of Nrf2 in the nucleus, and Nrf2 in the cytoplasm and HO-1. Quantified values were mean ± standard deviation of at least three independent experiments. ^▲, P<0.05, ^▲▲, P<0.01 vs. the IR group; ^★, P<0.05, ^★★, P<0.01 vs. the IR + miR-122 antagomir group. +, this group rats were treated with indicated agent; −, this groups did not receive this indicated treatment. IR, ischemia-reperfusion; Nrf2, nuclear factor E2-related factor 2.