SS-31 Protects Liver from Ischemia-Reperfusion Injury via Modulating Macrophage Polarization

Abstract

Ischemia-reperfusion injury (IRI) is a common complication in liver surgeries. It is a focus to discover effective treatments to reduce ischemia-reperfusion injury. Previous studies show that oxidative stress and inflammation response contribute to the liver damage during IRI. SS-31 is an innovated mitochondrial-targeted antioxidant peptide shown to scavenge reactive oxygen species and decrease oxidative stress, but the protective effects of SS-31 against hepatic IRI are not well understood. The aim of our study is to investigate whether SS-31 could protect the liver from damages induced by IRI and understand the protective mechanism. The results showed that SS-31 treatment can significantly attenuate liver injury during IRI, proved by HE staining, serum ALT/AST, and TUNEL staining which can assess the degree of liver damage. Meanwhile, we find that oxidative stress and inflammation were significantly suppressed after SS-31 administration. Furthermore, the mechanism revealed that SS-31 can directly decrease ROS production and regulate STAT1/STAT3 signaling in macrophages, thus inhibiting macrophage M1 polarization. The proinflammation cytokines are then significantly reduced, which suppress inflammation response in the liver. Taken together, our study discovered that SS-31 can regulate macrophage polarization through ROS scavenging and STAT1/STAT3 signaling to ameliorate liver injury; the protective effects against hepatic IRI suggest that SS-31 may be an appropriate treatment for liver IRI in the clinic.

Figure 1

SS-31 treatment ameliorated liver ischemia-reperfusion injury. (a) Representative H&E staining of the liver section in different groups after IR (n = 4‐5 per group), scale bars, 200 μm. (b, c) Serum ALT and AST of vehicle- and SS-31-treated mice were measured after IR procedure (n = 4‐5 per group). (d) Quantitative assessment of levels of liver injuries by using Suzuki's score. (e, f) Representative TUNEL staining of liver sections to assess the rate of apoptosis after IR with the administration of SS-31 (n = 4‐5 per group), scale bars, 100 μm. Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 2

SS-31 treatment decreased oxidative stress in the liver after IRI. (a, b) Representative images of DHE staining in liver sections undergoing IRI treated with or without SS-31 (n = 4‐5 per group). (c) Representative 8-OHDG immunohistochemistry of liver sections with or without IRI (n = 4‐5 per group). (d) The mRNA levels of genes related to liver oxidative stress were measured. (e) The activity of MnSOD was detected by using the Superoxide Dismutase (SOD) Activity Assay Kit in the liver to assess liver oxidative stress (n = 4‐5 per group). (f) Immunoblot analysis of HO-1 and SOD2 expression in the liver (n = 4 per group). Scale bars, 100 μm. Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 3

The production of inflammation-related cytokines is inhibited by SS-31 administration. (a–d) The levels of TNFα, IL6, IL1β, and IL10 in the serum of mice undergoing liver IRI were detected by the ELISA kit (n = 4 per group). (e–h) qPCR analysis of mRNAs encoding inflammation-related cytokines, including TNFα, IL6, IL1β, and IL10 in livers after IRI treated with or without SS-31 (n = 4 per group). Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 4

SS-31 inhibited the M1 polarization of KCs in the liver. (a) Representative immunofluorescence of iNOS (red) and F4/80 (green) in liver sections after IRI treated with or without SS-31 (n = 4‐5 per group). (b) Levels of mRNAs encoding TNFα, iNOS, IL1β, and CCL2 (M1 markers) in KCs (n = 4 per group). (c) Levels of mRNAs encoding PMP22, KLF4, IrF4, and Mgl1 (M2 markers) in KCs (n = 4 per group). Scale bars, 20 μm. Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 5

SS-31 downregulated LPS-induced macrophage polarization in vitro. (a) Representative images of the morphology of Raw264.7 cells stimulated with LPS and SS-31. Scale bars, 100 μm. (b, c) Immunoblot analysis of inflammation-related proteins TNFα, IL1β, and iNOS in Raw264.7 cells treated with LPS and SS-31. (d–f) Levels of mRNAs encoding IL1β, IL6, and CCL2 (M1 markers) in Raw264.7 cells treated with LPS and SS-31. (g) Representative images of immunofluorescence of iNOS (green) and F4/80 (red) in Raw264.7 cells. Scale bars, 100 μm. Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 6

SS-31 preserved mitochondrial function and decreased the level of mtROS. (a) The changes of ROS levels of Raw264.7 cells were assessed by DCFH-DA staining, scale bars, 200 μm. (b, f) Immunoblot analysis of Nrf2, HO-1, and SOD2 in Raw264.7 cells to evaluate the oxidative stress-related signaling changes. (c, d) Flow cytometry analysis of mtROS levels in Raw264.7 cells treating with LPS and SS-31 by staining with MitoSox. (e) The ATP levels were detected by the ATP Assay Kit to evaluate the mitochondrial function. (f) The staining of JC-1 in Raw264.7 cells was conducted to show the changes of mitochondrial membrane potential, scale bars, 20 μm. Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 7

SS-31 regulated STAT1 and STAT3 to govern macrophage polarization. (a, b) Immunoblot analysis of STAT1 and p-STAT1 (Tyr701) in Raw264.7 treated with LPS with or without SS-31. (c, d) Immunoblot analysis of STAT3 and p-STAT3 (Tyr705) in Raw264.7 treated with LPS with or without SS-31. (e, f) Immunoblot analysis of STAT1 and p-STAT1 (Tyr701) in the liver after IRI with SS-31 pretreatment (n = 4 per group). (g, h) Immunoblot analysis of STAT3 and p-STAT3 (Tyr705) in the liver after IRI with SS-31 pretreatment (n = 4 per group). Data are mean ± SEM, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 by unpaired Student's t-test.

Figure 8

The diagram of the mechanism that SS-31 protects the liver against IRI. As a mitochondrial-targeted peptide, SS-31 preserves the function of mitochondrial and decreases the production of mtROS. With the reduction of mtROS, the phosphorylation process of STAT1 and STAT3 is inhibited, which in turn affects macrophage polarization to proinflammatory phenotype. Macrophages play a vital role in the inflammatory response caused by ischemia reperfusion, and inhibiting M1 polarization will reduce the release of inflammatory factors such as TNFα and IL1β. In general, SS-31 protects the liver from ischemia-reperfusion injury by regulating oxidative stress and inflammatory damage.