Skimmianine attenuates liver ischemia/reperfusion injury by regulating PI3K-AKT signaling pathway-mediated inflammation, apoptosis and oxidative stress

Abstract

Liver ischemia/reperfusion (I/R) injury is a common injury after liver transplantation and hepatectomy. Skimmianine (Ski) has antibacterial, antiviral pharmacological effects. However, it is not clear whether Ski has a protective effect against liver I/R injury. In the present study, we established a mouse liver I/R model and an AML12 cell hypoxia-reoxygenation (H/R) model, both pretreated with different concentrations of Ski. Serum transaminase levels, necrotic liver area, cell viability, inflammatory factors, oxidative stress and apoptosis-related levels were measured to assess the protective effect of Ski against liver I/R injury. Western blotting was used to detect apoptosis-related proteins and PI3K-AKT pathway-related proteins. Mice and cells were also treated with PI3K inhibitor LY294002 to assess changes in indicators of liver injury. The results showed that Ski significantly reduced transaminase levels, liver necrosis area, oxidative stress, and apoptosis levels in mice with I/R. Ski also inhibited cell injury and apoptosis after H/R. Moreover, Ski activated phosphorylation of PI3K-AKT pathway-related proteins after liver I/R and cell H/R. Importantly, the PI3K inhibitor LY294002 effectively reversed the alleviation of I/R injury caused by Ski. These results confirm that Ski exerts a protective effect against liver I/R injury through activation of the PI3K-AKT pathway.

Figure 1

Skimmianine attenuates liver I/R injury in mice. (A and B) Serum ALS and AST contents of mice in different treatment groups. (C) Representative H&E staining and (D) necrotic area statistical analysis of liver tissue. ^**P < 0.01 versus sham group; ^##P < 0.01 versus I/R group.

Figure 2

Skimmianine alleviates inflammation in mouse liver I/R injury. (A–D) The mRNA expression of inflammatory cytokines IL-1β, IL-6, TNF-α, and CXCL10 of mice in different treatment groups. (E) Representative MPO immunohistochemical staining of liver tissues and (F) statistical analysis from different groups. ^**P < 0.01 versus sham group; ^##P < 0.01 versus I/R group.

Figure 3

Skimmianine alleviates oxidative stress after liver I/R in mice. (A) MDA content, (B) SOD activity and (C) GSH-Px activity in liver tissues of mice in different treatment groups (D) Representative DHE staining and (E) fluorescence intensity statistical analysis of liver tissues in different treatment mice. ^**P < 0.01 versus sham group; ^#P < 0.05 and ^##P < 0.01 versus I/R group.

Figure 4

Skimmianine alleviates apoptosis in mouse liver after I/R. (A, B) TUNEL staining and statistical analysis of liver tissue in different treatment mice. (C) The protein expression of Bax, Bcl-2 and Cleaved-caspase 3 and (D) protein quantitative analysis of liver tissue in different treatment mice. ^**P < 0.01 versus sham group; ^##P < 0.01 versus I/R group.

Figure 5

Skimmianine alleviates H/R-induced cell injury. (A) Effect of different concentrations of Ski on the activity of AML12 cells under normoxia. (B) Effects of different concentrations of Ski on the activity of AML12 cells under H/R. (C and D) Apoptosis rate detection and statistical analysis by flow cytometry. (E) The protein expression of Bax, Bcl-2 and Cleaved-caspase 3 and (F) protein quantitative analysis in different treatment cells. ^$$P < 0.01 versus 0 μM group; ^**P < 0.01 versus control group; ^##P < 0.01 versus H/R group.

Figure 6

Skimmianine activates PI3K–AKT pathway after liver I/R. (A) The docking results of Ski and PI3k molecules. (B) The protein expression of p-PI3K, PI3K, p-AKT, AKT and (C) protein quantitative analysis in different treatment mice. (D) The protein expression of p-PI3K, PI3K, p-AKT, AKT and (E) protein quantitative analysis in different treatment cells. ^**P < 0.01 versus sham group; ^##P < 0.01 versus I/R group; ^$$P < 0.01 versus control group; ^&&P < 0.01 versus H/R group.

Figure 7

Skimmianine inhibits liver I/R injury via PI3k–AKT pathway. (A and B) Serum ALS and AST contents of mice in different treatment groups, (C) representative H&E staining and (D) necrotic area statistical analysis of liver tissue, (E–H) The mRNA expression of inflammatory cytokines IL-1β, IL-6, TNF-α, and MCP-1 of mice in different treatment groups; (I–K) MDA content, SOD activity and GSH activity in liver tissues of mice in different treatment groups. (L, M) TUNEL staining and statistical analysis of liver tissue in different treatment mice. ^**P < 0.01 versus sham group; ^##P < 0.01 versus I/R group; ^&&P < 0.01 versus I/R + Ski group.

Figure 8

Schematic diagram of the role of Skimmianine in regulating the PI3k–AKT pathway against I/R-induced liver injury.