The Protective Effect of Anethole against Renal Ischemia/Reperfusion: The Role of the TLR2,4/MYD88/NFκB Pathway

Abstract

Background: Anethole is the principle essential oil component of anise and fennel. Renal ischemia/reperfusion (RIR) is one of the utmost imperative reasons for acute kidney injury and often associated with high mortality rate. The aim of this study is to investigate the protective effect of anethole on RIR status, exploring the involved mechanisms.

Methods: RIR was accomplished by bilateral renal pedicle clamping for 45 min, after which the clamps were removed to achieve the reperfusion phase. Rats were randomized into five groups; Sham, Sham + anethole, RIR, and finally RIR + anethole (125 mg/kg or 250 mg/kg) groups. Animals were given anethole (in specified groups in doses) for 14 days before RIR.

Results: RIR-experienced animals developed renal injury evidenced by diminished renal function and histopathological alteration. RIR induced severe oxidative, inflammatory, and apoptotic status within renal tissue. Pre-RIR management with anethole enhanced renal morphology and improved renal function. Anethole amplified GSH content and SOD, CAT, and GPx activities and lowered MDA. Anethole reduced gene and protein expression levels of HMGB1, TLR2, TLR4, MYD88, and NFκB. Anethole distinctly dropped TNF-α, IFN-γ, and MCP-1 levels, increased IL-10, and diminished caspase 3 and 9, reflecting its anti-inflammatory and anti-apoptotic actions.

Conclusion: Anethole displayed anti-inflammatory, anti-oxidant, and anti-apoptotic actions against RIR-induced injury. Anethole exhibited renal protective actions, which could be through inhibiting the HMGB1/TLR2, 4/MYD88/NFκB pathway. These results could suggest anethole as a protective agent against RIR.

Keywords: anethole; anti-apoptotic; anti-inflammatory; anti-oxidant; in silico; renal ischemia/reperfusion.

Figure 1

(a) Anethole chemical structure, (b) quantitative assessment of renal damage score, (c,d) histopathological analysis displaying the protective action of anethole (125 and 250 mg/kg) on the renal tissues challenged through RIR stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) stain, respectively. The black arrows show glomeruli collapsed with focal necrosis. The yellow arrow shows hyaline accumulation. The green arrow illustrates the necrotic tubule.

Figure 2

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia/perfusion (RIR) on the renal function assessment, including (a) creatinine, (b) BUN, (c) uric acid, (d) Kim-1, and (e) LDH in RIR-induced injury. All values are stated as mean ± SD. # designates statistically significant compared to sham group, * designates statistically significant compared to RIR group, and @ designates statistically significant compared to RIR + anethole 125 mg/kg group (p < 0.05) using one-way ANOVA followed by Tukey’s post hoc test.

Figure 3

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia/perfusion (RIR) on the renal oxidative stress (a) SOD, (b) CAT, (c) GSH, (d) GPx, and (e) MDA in RIR-induced injury. All values are stated as mean ± SD. # designates statistically significant compared to sham group, * designates statistically significant compared to RIR group, and @ designates statistically significant compared to RIR + anethole 125 mg/kg group (p < 0.05) using one-way ANOVA followed by Tukey’s post hoc test.

Figure 4

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia perfusion (RIR) on the renal gene (mRNA) expression of (a) HMGB1, (b) TLR2, (c) TLR4, (d) MYD88, and (e) NFκB in RIR-induced injury. All values are stated as mean ± SD. # designates statistically significant compared to sham group, * designates statistically significant compared to RIR group, and @ designates statistically significant compared to RIR + anethole 125 mg/kg group (p < 0.05) using one-way ANOVA followed by Tukey’s post hoc test.

Figure 5

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia/perfusion (RIR) on the renal protein expression of (a) HMGB1, (b) TLR2, (c) TLR4, (d) MYD88, (e) NFκB and (f) protein expression of HMGB1,TLR2,4, MYD88 and NFκB in RIR-induced injury. All values are stated as mean ± SD. # designates statistically significant compared to sham group, * designates statistically significant compared to RIR group, and @ designates statistically significant compared to RIR + anethole 125 mg/kg group (p < 0.05) using one-way ANOVA followed by Tukey’s post hoc test.

Figure 6

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia/perfusion (RIR) on the renal immunohistochemical assay of (a) NFκB and (b) HMGB1 in RIR-induced injury. All values are stated as mean ± SD.

Figure 7

TLR4 complex with MD-2 exhibiting the docking site anethole. (A) The docking site of anethole in MD-2. The second MD-2 molecule was left bound with LPS for demonstration. (B) Surface depiction of MD-2 showing the docked anethole in the deep hydrophobic cavity of MD-2. (C) The docking site of anethole revealing its hydrophobic site comprising PHE119, PHE121, and PHE151. Stacking interaction is reported with PHE119.

Figure 8

Effects of anethole (125 and 250 mg/kg) administration for 14 days prior to renal ischemia perfusion (RIR) on the renal inflammatory mediators (a) TNF-α, (b) IFN-γ, (c) MCP-1, and (d) IL-10, and on apoptosis, including (e) caspase 3 and (f) caspase 9, in RIR-induced injury. All values are stated as mean ± SD. # designates statistically significant compared to sham group, * designates statistically significant compared to RIR group, and @ designates statistically significant compared to RIR + anethole 125 mg/kg group (p < 0.05) using one-way ANOVA followed by Tukey’s post hoc test.