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.

1. Introduction

Liver transplantation is the mainstay treatment for end-stage liver disease and acute liver failure [1]. Acute rejection (AR), a common cause of poor prognosis after liver transplantation, occurs in approximately 20%‒30% of patients within the first 12 months [2]. Although immunosuppressive protocols have improved the prognosis of patients after liver transplantation, some studies have shown that immunosuppressive drugs have some adverse effects, including metabolic disorders, severe infection, and tumor recurrence, which limits their application in liver transplantation [3,4]. Therefore, more studies are required to elucidate AR pathogenesis to establish novel therapeutic targets for AR.

The nuclear protein high-mobility group box-1 (HMGB1) is highly conserved and is an immunomodulatory factor involved in hepatic ischemia-reperfusion injury (IRI) and acute rejection after liver transplantation [5,6]. Recent studies have revealed that the HMGB1-TLR-4 signaling pathway promotes the pathophysiology of liver IRI by activating the inflammatory response. Moreover, HMGB1 can induce acute rejection after liver transplantation by activating dendritic cells [7,8]. Many studies have shown that HMGB1 participates in the pathological processes of many inflammation-related diseases by regulating neutrophil activation and NET formation [9,10].

Neutrophil extracellular traps are extracellular DNA fibers decorated with histones and granular proteins [11]. NETs can neutralize and kill bacteria. However, NET dysregulation may also induce immune-related adverse events, such as hepatic IRI and acute liver rejection after liver transplantation [12,13]. Numerous investigations have demonstrated that NETosis exacerbates inflammation and contributes to brain ischemia through HMGB1 signaling. Necrotic hepatocytes induce NET formation and exacerbate hepatic IRI by releasing HMGB1 [14,15]. An earlier study demonstrated that excessive neutrophil accumulation, neutrophil hyper-reactivity, and the uncontrolled formation of neutrophil extracellular traps (NETs) after liver transplantation promote the creation of a local liver inflammatory microenvironment and acute rejection after liver transplantation [16,17]. These studies indicate that HMGB1-induced NET formation promotes the incidence and development of acute rejection after liver transplantation.

Salidroside, which is extracted from various Rhodiola plants, can treat ischemic stroke, Alzheimer’s disease, and cardiovascular diseases [18,19]. Salidroside has broad pharmacological effects, including inhibiting hypoxia, inflammation, and oxidation [20]. Moreover, salidroside can trea