Salidroside ameliorates acute liver transplantation rejection in rats by inhibiting neutrophil extracellular trap formation

Abstract

Acute rejection is an important factor affecting the survival of recipients after liver transplantation. Salidroside has various properties, including anti-inflammatory, antioxidant, and hepatoprotective properties. This study aims to investigate whether salidroside can prevent acute rejection after liver transplantation and to examine the underlying mechanisms involved. An in vivo acute rejection model is established in rats that are pretreated with tacrolimus (1 mg/kg/d) or salidroside (10 or 20 mg/kg/d) for seven days after liver transplantation. In addition, an in vitro experiment is performed using neutrophils incubated with salidroside (1, 10, 50 or 100 μM). Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, immunosorbent assays, immunofluorescence analysis, Evans blue staining, and western blot analysis are performed to examine the impact of salidroside on NET formation and acute rejection in vitro and in vivo. We find that Salidroside treatment reduces pathological liver damage, serum aminotransferase level, and serum levels of IL-1β, IL-6, and TNF-α in vivo. The expressions of proteins associated with the HMGB1/TLR-4/MAPK signaling pathway (HMGB1, TLR-4, p-ERK1/2, p-JNK, p-P38, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, IL-1β, TNF-α, and IL-6) are also decreased after salidroside treatment. In vitro experiments show that the release of HMGB1/TLR-4/MAPK signaling pathway-associated proteins from neutrophils treated with lipopolysaccharide is decreased by salidroside. Moreover, salidroside inhibits NETosis and protects against acute rejection by regulating the HMGB1/TLR-4/MAPK signaling pathway. Furthermore, salidroside combined with tacrolimus has a better effect than either of the other treatments alone. In summary, salidroside can prevent acute liver rejection after liver transplantation by reducing neutrophil extracellular trap development through the HMGB1/TLR-4/MAPK signaling pathway.

Keywords: HMGB1; acute rejection; liver transplantation; neutrophil extracellular traps; salidroside.

Figure 1

The experimental design (A) Flow chart of the experimental design of the animal experiment. (B) Schematic diagram showing the grouping of the rats. The rats were divided into 6 groups. The sham group received an abdominal incision, exposing the hepatic portal vein (n=6); the AR group received no treatment after liver transplantation; the AR+TAC group received intraperitoneal administration of TAC (1 mg/kg/d) for 7 days after liver transplantation; the AR+Sal group received intraperitoneal administration of salidroside (10 or 20 mg/kg/d) for 7 days after liver transplantation; and the AR+TAC+Sal group received intraperitoneal administration of TAC (1 mg/kg/d) and salidroside (20 mg/kg/d) for 7 days after liver transplantation.

Figure 2

Salidroside inhibits NET formation and alleviates acute rejection after liver transplantation (A) Chemical structure of salidroside. (B) Serum levels of ALT and AST in the different groups. (C) Serum levels of IL-1β, IL-6, and TNF-α in the different groups. (D) Representative images of the hepatic histopathological changes in the livers of rats observed after HE staining (magnification, ×200; scale bar: 200 μm; n=6). (E) RAI scores of different groups according to the Banff scheme. (F) NET production in the serum of rats in different groups. (G) Increased levels of circulating H3Cit in the serum of rats in different groups. The significant differences among groups were compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Salidroside inhibits hepatocyte apoptosis in rats after liver transplantation (A) Hepatic apoptosis detected by TUNEL in different groups (magnification, ×200; n=6). (B) TUNEL+ cells of the liver based on the TUNEL assay. (C) Representative western blots showing the levels of apoptosis-associated proteins in the different groups. (D) The quantitative results of the western blots. Statistical differences among groups were determined using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. ns, not significant.

Figure 4

Salidroside suppresses liver inflammation via the HMGB1 signaling pathway after liver transplantation (A) Representative western blots showing HMGB1/TLR-4/MAPK signaling pathway-related proteins in the different groups. (B) Quantitative analysis of the western blots. The significant differences among groups were compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

Figure 5

Salidroside improves LPS-induced NET formation (A) The expression levels of HMGB1/TLR-4/MAPK signaling pathway-associated proteins were detected by western blot analysis. (B) Quantitative analysis of the western blots. (C,D) The separated neutrophils were pretreated with or without Sal (1 μM, 10 μM, or 50 μM) for 60 min and then activated with 25 μg/μL LPS for 1 h. Neutrophils in the control group received no treatment. The structure of the NETs (red arrowheads) was observed using laser scanning confocal microscopy (magnification, ×200; scale bar: 200 μm), and quantitative NET formation by neutrophils was analyzed. (E) Neutrophils were incubated with different concentrations of lipopolysaccharide (0.1–25 μL/μg) for 24 h. Cell viability was determined using MTT. Statistical differences among groups were compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. ns, not significant.

Figure 6

Salidroside suppresses LPS-induced NET formation by inhibiting the activation of the HMGB1 signaling pathway (A) Immunofluorescence analysis of neutrophil extracellular trap formation induced by Sal and HMGB1. (B) Representative western blots showing proteins associated with the HMGB1/TLR-4/MAPK signaling pathway. (C) Quantitative analysis of the western blots. Differences among groups were compared using one-way ANOVA. **P<0.01, ***P<0.001.

Figure 7

Synergistic effects of salidroside and tacrolimus on acute rejection after liver transplantation (A, B) Pathological liver alterations after salidroside and tacrolimus administration. (C) Immunofluorescence analysis of neutrophil extracellular trap formation after salidroside and tacrolimus treatment. (D,E) Liver vascular leakage analysis. (F) Serum levels of TNF-α, IL-6, and IL-1β in the different groups. (G) Serum levels of ALT on days 1, 3, 5, 7, and 14 after liver transplantation. (H) Serum levels of AST on days 1, 3, 5, 7, and 14 after liver transplantation. (I) Survival analysis of rats in different groups. Differences among groups were compared using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.