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Salidroside alleviates hepatic ischemia–reperfusion injury during liver transplant in rat through regulating TLR-4/NF-κB/NLRP3 inflammatory pathway


Salidroside enhances liver function and reduces inflammatory cytokines in rats after liver transplant

The rats were divided into three groups and received liver transplantation after salidroside intraperitoneally for 7 days (Fig. 1A). The chemical formula of salidroside is shown (Fig. 1B). Serum levels of AST and ALT are the main indicators in evaluating liver function. Our analysis showed that ALT and AST levels were significantly elevated after liver transplant. Pretreatment with increasing dosages of salidroside (5, 10, or 20 mg/kg) for 7 days decreased ALT and AST levels compared to the I/R group, demonstrating that salidroside treatment improves the recipients’ liver functioning (Fig. 1C). The IL-1β, IL-18, and TNF-α serum levels were markedly higher in the I/R group compared to the control group. In comparison to the I/R group, salidroside pretreatment (5, 10 or 20 mg/kg) gradually suppressed the elevated levels of the cytokines (Fig. 1D).

Figure 1
figure 1

Salidroside improves liver function and decrease inflammatory cytokines in rats after liver transplantation. (A) Schematic diagram describing the grouping of the rats. (B) Chemical structure of salidroside. (C) Serum levels of ALT and AST in different groups. (D) Serum levels of inflammatory cytokines in different groups. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Salidroside decreases histopathological changes in rats after liver transplant

To assess the function of salidroside in IRI-induced hepatocellular damage, HE staining was used examine the severity of histopathological changes in the liver from the rats in different groups. The HE staining data showed that the degree of coagulation necrosis, architectural anomalies, and hepatocyte vacuolization were milder in the I/R + Sal (5, 10 or 20 mg/kg) group. These findings showed that salidroside pretreatment significantly reduces IRI-induced hepatic pathological changes (Fig. 2A,C).

Figure 2
figure 2

Salidroside attenuates hepatic histopathological changes and hepatocyte apoptosis of the rats following liver transplantation. (A) Representative HE staining images showing hepatic histological alterations (indicated with arrowheads) in the rat liver (magnification, 200; scale bar, 200 m; n = 6). (B) Hepatic apoptosis detected by TUNEL assay (magnification, × 200; scale bar, 200 μm; n = 6). (C) Hepatic IRI grading based on suzuki’s grade. (D) TUNEL+ cells of the liver based on TUNEL assay. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Salidroside attenuates hepatocyte apoptosis in rats following liver transplant

To investigate whether salidroside prevents hepatocyte apoptosis in vivo, the levels of hepatocyte apoptosis were determined using a TUNEL assay. The data demonstrated that the sham group had few TUNEL positive cells, which substantially increased following liver transplantation, consistent with the HE staining results. In contrast, salidroside pretreatment alleviated hepatic apoptosis in a dose-dependent manner (Fig. 2B,D).

Salidroside regulates the TLR-4/NF-κB/NLRP3 inflammatory pathway in the liver of rats after liver transplantation

Liver tissues from I/R group exhibited significant increase in the expression of TLR-4, MyD88, p-IKKα, p-IKKβ, p-IKK, p-IκBα, p-P65, NLRP3, ASC, Cleaved caspase-1, IL-1β, IL-18, TNF-α and IL-6 proteins compared to those in the sham group. As expected, pretreatment with salidroside (5, 10, or 20 mg/kg) reduced activation of the TLR-4/ NF-κB /NLRP3 inflammatory pathway (Fig. 3).

Figure 3
figure 3

Salidroside inhibits TLR-4/NF-κB/NLRP3 inflammatory pathway in vivo. (A,C) Representative western blot assays showing TLR-4/NF-κB/NLRP3 inflammatory pathway associated proteins in the rat liver after liver transplantation. (B,D) Results of the Western blot experiments. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Salidroside mitigates hepatic IRI-induced inflammation and neutrophil infiltration

To establish a connection between neutrophil infiltration and liver inflammation, qRT-PCR was conducted to determine the expression levels of Pro-inflammatory cytokines and neutrophil markers, such as IL-1β, IL-18, IL-6, TNF-α, Ly6G and CD11b. Our analyses showed significantly elevated IL-1β, IL-18, IL-6 TNF-α, Ly6G and CD11b mRNA expression levels in the liver of the rats following liver transplantation, which were suppressed in the rats in the I/R + Sal (5, 10 or 20 mg/kg) group. The results of qRT-PCR indicated that salidroside pretreatment could mitigate hepatic IRI-induced inflammation and neutrophil infiltration in a dose-dependent manner (Fig. 4).

Figure 4
figure 4

Salidroside suppresses transcription of pro-inflammatory cytokinesis. (A) IL-1β mRNA expression. (B) IL-18 mRNA expression. (C) TNF-α mRNA expression. (D) IL-6 mRNA expression. (E) Ly6G mRNA expression. (F) CD11b mRNA expression. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Salidroside induced macrophage M2 polarization and alleviates the H/R-induced inflammation through the TLR-4/NF-κB/NLRP3 inflammatory pathway

Notably, our data showed that H/R exposure elevates the TLR-4, MyD88, p-IKKα, p-IKKβ, p-IKK, p-IκBα, p-P65, NLRP3, ASC, Cleaved caspase-1, IL-1β, IL-18, TNF-α and IL-6 protein levels compared to those in the control group. Treatment with salidroside (1 μM, 10 μM or 50 μM) diminished the activation of TLR-4/NF-κB/NLRP3 inflammatory pathway and decreased the secretion of the proinflammatory factors in a dose-dependent manner. Although there were significantly higher levels of IL-1β, IL-18 and TNF-α in the cell supernatant of the H/R group, treatment with salidroside (1 M, 10 M, or 50 M) decreased the levels of these cytokines in a dose-dependent manner. Western blot analysis was used to determine the expression levels of M2 polarization-related proteins (Arg-1, CD206, IL-10). As the salidroside concentration increased, the M2 polarization effect of macrophage gradually increased (Fig. 5).

Figure 5
figure 5

Salidroside inhibits inflammation in the H/R-exposed macrophages by regulating TLR-4/NF-κB/NLRP3 inflammatory pathway. (A,C) Representative Western blot data showing TLR-4/NF-κB/NLRP3 inflammatory pathway associated proteins in macrophages. (B,D) The results of western blot experiments. (E) Expression of IL-1β, IL-18 and TNF-α in macrophages was analyzed by ELISA. (F) Expression of M2 polarization markers in macrophages was analyzed by western blot. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Protective effect of salidroside is attenuated after TLR-4 activation

In comparison with the H/R + Sal (10 M) group, Western blotting analyses revealed that lipopolysaccharides (LPS) treatment significantly boosted the expression of TLR-4/NF-κB/NLRP3 inflammatory pathway related proteins (Fig. 6A–D). Similarly, compared to the H/R + Sal (10 μM) group, LPS significantly elevated the inflammatory cytokines in the cell supernatants (Fig. 6E). These data demonstrated that LPS (TLR-4 agonist) partly reversed the effects of salidroside. In addition, LPS stimulation significantly activated TLR-4/NF-κB/NLRP3 inflammatory pathway and facilitated the production of inflammatory cytokines by macrophages. Interestingly, the protective effects of salidroside in the macrophages with H/R exposure were attenuated after TLR-4 activation.

Figure 6
figure 6

LPS (TLR-4 activator) partially alleviates the anti-inflammatory ability of salidroside via regulation of the TLR-4/NF-κB/NLRP3 inflammatory pathway. (A,C) Representative Western blot data of the TLR-4/NF-κB/NLRP3 inflammatory pathway associated proteins in macrophages. (B,D) The results of western blot experiments. (E) Production of IL-1β, IL-18 and TNF-α in macrophages was assessed by ELISA. The statistical differences among groups were assessed by one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.



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