Exogenous melatonin alleviates hemorrhagic shock‑induced hepatic ischemic injury in rats by inhibiting the NF‑κB/IκBα signaling pathway

Abstract

Melatonin (MT) is an indoleamine hormone that can counteract ischemia‑induced organ injury through its antioxidant effects. The aim of the present study was to investigate the protective effects of exogenous MT against hemorrhagic shock (HS)‑induced hepatic ischemic injury in rats, and the role of the nuclear factor (NF)‑κB signaling pathway in this process. A rat model of HS‑induced hepatic ischemic injury was established. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), tumor necrosis factor (TNF)‑α, interferon (IFN)‑γ, interleukin (IL)‑6 and IL‑1β were measured every 6 h, and the 24‑h survival rate of the rats was analyzed. All surviving rats were sacrificed after 24 h. Pathological changes in the liver and the hepatocyte apoptosis rate were observed by hematoxylin and eosin staining and TUNEL assay, respectively, and the expression levels of NF‑κB p65 and NF‑κB inhibitor α (IκBα) were analyzed by reverse transcription‑quantitative PCR analysis and western blotting. The results demonstrated that the serum levels of ALT, AST, LDH, GDH, TNF‑α, IFN‑γ, IL‑6 and IL‑1β gradually increased after HS compared with those in rats subjected to a sham procedure, but this increase was attenuated by MT. Furthermore, the survival rate of the MT group was significantly higher compared with that of the HS group. The degree of pathological hepatic injury, the hepatocyte apoptosis rate, and the hepatic levels of TNF‑α, IFN‑γ, IL‑6 and IL‑1β were significantly decreased in the MT group compared with the HS group. In addition, the mRNA expression of NF‑κB p65 was significantly decreased and the mRNA expression of IκBα was significantly increased in the MT group compared with the sham group. Furthermore, the NF‑κB p65 protein levels in the MT group were significantly increased in the cytosol but decreased in the nucleus, and the IκBα protein levels were increased while those of phosphorylated IκBα were decreased compared with those in the HS group. Therefore, it may be inferred that exogenous MT alleviates HS‑induced hepatic ischemic injury in rats via the inhibition of NF‑κB activation and IκBα phosphorylation.

Keywords: hemorrhagic shock; hepatic ischemia injury; melatonin; NF‑κB/IκBα.

Figure 1.

Serum levels of ALT, AST, LDH and GDH in each group at 6-h intervals as measured using an automatic biochemical analyzer. Serum (A) ALT, (B) AST, (C) GDH and (D) LDH levels. **P<0.01 as indicated. ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; GDH, glutamate dehydrogenase; HS, hemorrhagic shock; MT, melatonin.

Figure 2.

Serum levels of TNF-α, IFN-γ, IL-6 and IL-1β in each group at 6-h intervals as measured by ELISA assay. Serum (A) TNF-α, (B) IFN-γ, (C) IL-6 and (D) IL-1β levels. **P<0.01 as indicated. TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; HS, hemorrhagic shock; MT, melatonin.

Figure 3.

Survival rate of the rats in each group. HS, hemorrhagic shock; MT, melatonin.

Figure 4.

Morphological changes of hepatic tissue were detected by H&E staining. Representative images of H&E staining in the (A) sham, (B) HS and (C) MT groups. (D) Suzuki score in each group. Magnification, ×200. **P<0.01 as indicated. Black arrows indicate inflammatory infiltration and green arrows indicate edema. H&E, hematoxylin and eosin; HS, hemorrhagic shock; MT, melatonin.

Figure 5.

Hepatocyte apoptosis rate as detected by TUNEL assay. Representative images of TUNEL staining in the (A) sham, (B) HS and (C) MT groups. Hepatocyte apoptosis rate. Arrows indicate apoptotic cells. Magnification, ×400. *P<0.05, **P<0.01 as indicated. HS, hemorrhagic shock; MT, melatonin.

Figure 6.

Hepatic tissue levels of TNF-α, IFN-γ, IL-6 and IL-1β in each group after sacrifice were measured by ELISA. Hepatic tissue levels of (A) TNF-α, (B) IFN-γ, (C) IL-6 and (D) IL-1β. *P<0.05, **P<0.01 as indicated. TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; HS, hemorrhagic shock; MT, melatonin.

Figure 7.

Transcriptional activity of the NF-κB/IκBα pathway. Relative expression of (A) NF-κB p56 and (B) IκBα mRNA in hepatic tissues. **P<0.01 as indicated. HS, hemorrhagic shock; MT, melatonin; NF, nuclear factor; IκBα, NF-κB inhibitor α.

Figure 8.

Hepatic levels of NF-κB p65, IκBα and p-IκBα proteins. NF-κB p56 protein in the (A) cytosol and (B) nucleus. (C) Relative ratio of p-IκBα/IκBα proteins. *P<0.05, **P<0.01 as indicated. HS, hemorrhagic shock; MT, melatonin; p, phosphorylated; NF, nuclear factor; IκBα, NF-κB inhibitor.