Impact of Washingtonia robusta Leaves on Gamma Irradiation-Induced Hepatotoxicity in Rats and Correlation with STING Pathway and Phenolic Composition

Abstract

Exposure to ionizing radiation usually results in cellular oxidative damage and may induce liver toxicity. The efficiency of the ethanol extracts of Washingtonia filifera (EWF) and Washingtonia robusta (EWR) leaves in alleviating γ-radiation-induced oxidative hepatotoxicity was herein explored. Proximate and macronutrient composition of the leaves was determined to establish reliable quality control criteria. Colorimetric estimation of total phenolic (TPC) and flavonoid (TFC) contents revealed their occurrence in larger amounts in EWR. In vitro evaluation of the antioxidant capacity by 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays confirmed higher efficiency of EWR designating a close correlation with phenolic composition. Four phenolics, viz., naringenin, kaempferol, quercetin, and gallic acid, were isolated from EWR. In vivo assessment of the extracts' antioxidant potential was performed on γ-irradiated (7.5 Gy) female rats. EWR was found more efficient in restoring the elevated liver index, ALT, albumin, cholesterol, and reactive oxygen species (ROS) levels. Both extracts ameliorated the increase in the stimulator of interferon gene (STING) expression. Bioactivity was confirmed by immuno-histochemical examination of inflammatory and apoptotic biomarkers (TNF-α, IL-6 and caspase-3) and histopathological architecture. In addition, the interactions of the isolated compounds with STING were assessed in silico by molecular docking. Therefore, Washingtonia robusta leaves might be suggested as a valuable nutritional supplement to alleviate radiotherapy-induced hepatotoxicity.

Keywords: STING gene expression; Washingtonia filifera; Washingtonia robusta; antioxidant; hepatoprotective; phenolics; γ-irradiation.

Figure 1

Compounds isolated from the ethyl acetate extract of W. robusta leaves.

Figure 2

Effect of W. filifera and W. robusta leaves ethanolic extracts on hepatic stimulator of interferon gene (STING) relative expression in irradiated rats. Data are expressed as mean ± SD, significance was at p ≤ 0.05 by means of one-way ANOVA followed by Tukey–Kramer as a post hoc-test. ^a Significantly different as of control group. ^b Significantly different as of irradiated group.

Figure 3

Compounds’ binding mode to STING receptor and interaction maps. (a) Gallic acid; (b) Naringenin; (c) Kaempferol; (d) Quercetin.

Figure 4

Effect of W. filifera and W. robusta leaves ethanolic extracts on liver index in irradiated rats. Data are expressed as mean ± SD, significance was at p ≤ 0.05 by means of one-way ANOVA followed by Tukey–Kramer as a post hoc-test. ^a Significantly different as of control group, ^b significantly different as of irradiated group (n = 6).

Figure 5

Histopathological examination of liver in irradiated rats treated with W. filifera and W. robusta leaves ethanolic extract (H and E; ×200). Control: Liver specifying average portal tract (black arrow), average central veins (blue arrows), and average hepatocytes (yellow arrow). IR: Liver showing markedly edematous portal tract (black arrow) with markedly dilated congested portal vein (PV) and markedly dilated congested central vein (CV); another view showing markedly dilated congested CV with detached lining (black arrow) and markedly apoptotic hepatocytes (blue arrow) with moderate micro-vesicular steatosis in peri-venular area (red arrow). F100, R100, and R300: Liver showing mildly edematous portal tract (black arrow) with mildly dilated congested portal vein (PV) and average central vein (blue arrow). F300: Liver displaying mildly edematous portal tract (black arrow), moderate portal inflammatory infiltrates (blue arrow), obviously dilated congested portal vein (PV), markedly dilated congested central vein (CV), and scattered apoptotic hepatocytes in a peri-portal area (yellow arrow). FIR100: Liver showing mildly edematous portal tract (black arrow), mildly dilated congested PV, mildly dilated CV, and large areas of necrosis (blue arrow). RIR100: Liver showing mildly edematous portal tracts (black arrow) with mildly dilated congested PV and normal central vein (blue arrow). FIR300: Liver specifying markedly edematous portal tract (black arrow) with markedly dilated congested PV and average CV with detached lining (blue arrow). RIR300: Liver specifying mildly edematous portal tract (black arrow), mildly dilated congested PV and markedly dilated CV (blue arrow).

Figure 6

Immunohistochemical photographs of TNF-α expression in hepatic peri-portal (P.P.) and peri-venular (P.V.) areas in irradiated rats treated with W. filifera and W. robusta leaves ethanolic extracts (×400). Sections were taken from livers (P.P. and P.V.) of control rats showing very low expression. Sections taken from livers (P.P. and P.V.) of irradiated rats shows extensive cytoplasmic expression (brown color). Sections taken from livers (P.P. and P.V.) of irradiated rats treated with W. filifera or W. robusta showing moderate to limited expression (brown color). Data expressed as mean ± SD (n = 6), significance was at p ≤ 0.05 by means of one-way ANOVA followed by Tukey–Kramer as a post hoc-test. ^a Significantly different as of control group. ^b Significantly different as of irradiated group. ^c Significantly different as of FIR100.

Figure 7

Immunohistochemical photographs of IL-6 expression in hepatic peri-portal (P.P.) and peri-venular (P.V.) areas in irradiated rats treated with W. filifera and W. robusta leaves ethanolic extract (×400). Sections were taken from livers (P.P. and P.V.) of control rats showing rare expression. Sections taken from livers (P.P. and P.V.) of irradiated rats show extensive cytoplasmic expression (brown color). Sections taken from livers (P.P. and P.V.) of irradiated rats treated with W. filifera or W. robusta show medium to limited expression (brown color). Data conveyed as mean ± SD (n = 6), significance was at p ≤ 0.05 by means of one-way ANOVA followed by Tukey–Kramer as a post hoc-test. ^a Significantly different as of control group. ^b Significantly different as of irradiated group. ^c Significantly different as of FIR100 group. ^d Significantly different as of FIR300 group.

Figure 8

Immunohistochemical photographs of caspase-3 expression in hepatic peri-portal (P.P.) and peri-venular (P.V.) (black arrows) areas in irradiated rats treated with W. filifera and W. robusta leaves ethanolic extracts (×400). Sections taken from livers (P.P. and P.V.) of control rats showing minimal expression. Sections taken from livers (P.P. and P.V.) of irradiated rats shows extensive cytoplasmic expression (brown color). Sections taken from livers (P.P. and P.V.) of irradiated rats treated with W. filifera or W. robusta showing medium to limited expression (brown color). Data conveyed as mean ± SD (n = 6), significance was at p ≤ 0.05 by means of one-way ANOVA followed by Tukey–Kramer as a post hoc-test. ^a Significantly different as of control group. ^b Significantly different as of irradiated group.