The Potential Effects of Quercetin-Loaded Nanoliposomes on Amoxicillin/Clavulanate-Induced Hepatic Damage: Targeting the SIRT1/Nrf2/NF-κB Signaling Pathway and Microbiota Modulation

Abstract

Amoxicillin/clavulanate (Co-Amox), a commonly used antibiotic for the treatment of bacterial infections, has been associated with drug-induced liver damage. Quercetin (QR), a naturally occurring flavonoid with pleiotropic biological activities, has poor water solubility and low bioavailability. The objective of this work was to produce a more bioavailable formulation of QR (liposomes) and to determine the effect of its intraperitoneal pretreatment on the amelioration of Co-Amox-induced liver damage in male rats. Four groups of rats were defined: control, QR liposomes (QR-lipo), Co-Amox, and Co-Amox and QR-lipo. Liver injury severity in rats was evaluated for all groups through measurement of serum liver enzymes, liver antioxidant status, proinflammatory mediators, and microbiota modulation. The results revealed that QR-lipo reduced the severity of Co-Amox-induced hepatic damage in rats, as indicated by a reduction in serum liver enzymes and total liver antioxidant capacity. In addition, QR-lipo upregulated antioxidant transcription factors SIRT1 and Nrf2 and downregulated liver proinflammatory signatures, including IL-6, IL-1β, TNF-α, NF-κB, and iNOS, with upregulation in the anti-inflammatory one, IL10. QR-lipo also prevented Co-Amox-induced gut dysbiosis by favoring the colonization of Lactobacillus, Bifidobacterium, and Bacteroides over Clostridium and Enterobacteriaceae. These results suggested that QR-lipo ameliorates Co-Amox-induced liver damage by targeting SIRT1/Nrf2/NF-κB and modulating the microbiota.

Keywords: Co-Amox-induced hepatotoxicity; SIRT1, Nrf2 and NF-κB targeting; gut dysbiosis; quercetin antioxidant activity; quercetin liposomes.

Figure 1

Schematic diagram summarizing the experimental design.

Figure 2

Characterization of QR-Lip for (a) particle size, (b) zeta potential, (c) transmission electron microscopy, and (d) in vitro QR release from QR-lipo. The ethanol injection technique was successful in the preparation of QR-lipo. The characterization of QR-lipo confirmed that it had a spherical nature, adequate zeta potential, and nano-size dimensions. In vitro release studies showed enhanced drug release upon formulation into nanoliposomes.

Figure 3

Effect of QR-lipo pretreatment on liver function tests in Co-Amox treated rats. The serum levels of (a) ALT, (b) AST, and (c) albumin were quantified for the different experimental groups. Data are expressed as the mean ± SD (n = 7). ns p > 0.05 and *** p < 0.001.

Figure 4

Effect of QR-lipo pretreatment on liver antioxidant status in Co-Amox-treated rats. The liver antioxidant status was determined via the quantification of (a) MDA, (b) GSH, (c) CAT, and (d) TAC. Data are expressed as the mean ± SD (n = 7). ** p < 0.01, and *** p < 0.001.

Figure 5

Effect of QR-lipo pretreatment on the mRNA expression level of ROS regulation genes within hepatocytes in Co-Amox-treated mice. The level of expressed mRNA for (a) keap1 and (b) GPx genes were detected using qRT-PCR. Data are expressed as the mean ± SD (n = 7). ns p > 0.05, *** p < 0.001.

Figure 6

Effect of QR-lipo pretreatment on mRNA level of inflammatory-related genes in liver tissues of Co-Amox-treated rats. The relative mRNA expression level of proinflammatory mediators, including (a) IL-6, (b) IL-1β, (c) TNF-α, (d) iNOS, and (e) Nf-κB, and anti-inflammatory mediator (f) IL-10 was quantified using RT-PCR for the different experimental groups. Data are expressed as the mean ± SD (n = 7). ns p > 0.05, * p < 0.05, and *** p < 0.001.

Figure 7

Effect of QR-lipo pretreatment on microbial populations in cecal contents of Co-Amox-treated rats. The Graph represented the counts of bacterial species expressed as log10 CFU/g of the sample; (a) Enterobacteriaceae, (b) Bacteroides, (c) Bifidobacterium, (d) Lactobacillus, and (e) Clostridium. Data are obtained from rat experiments and expressed as the mean ± SD (n = 7). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 8

Effect of QR-lipo pretreatment on histological changes in liver tissue in Co-Amox-treated rats. (a) Photomicrographs of the liver tissue stained with hematoxylin-eosin. (i,ii) Control group: showing normal hepatic architecture. A small central vein in the center of the hepatic lobule (CV). Plates of hepatocytes (Hc) are arranged radially around the small central vein. Each hepatocyte has an acidophilic cytoplasm and central vesicular nucleus (arrow). Hepatic sinusoids (s) run between the cords of hepatocytes. The portal tract (PT) at the periphery of each lobule is composed of an area of connective tissue with a small branch of the hepatic artery (a), portal vein (v), and lymphatic vessel (L). (iii,iv) QR-lipo group: nearly the same histological structure as the control group. (v,vi) Co-Amox group: shows lost normal hepatic architecture. Widespread fatty degeneration appeared as excessively small intracytoplasmic droplets with nuclear pyknosis (arrowheads). Some droplets coalesce against each other, forming cellular ballooning (circle). Chronic cholangitis in the portal tract (star) with dilated portal vein and bile stasis (red arrow) is also found. Perivascular oedema within the portal area (curved arrow) and increased thickness of the hepatic artery (black thick arrow) can be seen. (vii,viii) Co-Amox and QR-lipo group: nearly normal anastomosing plates of hepatocytes (Hc), central vein (CV), and blood sinusoids (s). Most hepatocytes appear normal with a central vesicular nucleus and eosinophilic cytoplasm (arrow), except for a few focal areas of vacuolated hepatocytes with small lipid droplets can be seen (double arrows). Improved portal area with mild inflammatory reaction (star) can be seen. (H and E; Scale bar; 20 μm). The magnification power is set at 400×. (b) Liver injury score. ns p > 0.05 and *** p < 0.001.

Figure 9

Photomicrographs of sections in the liver of rats stained with (a–d) anti-Nrf2 antibody and (f–i) anti-SIRT1 antibody. (a–d) Expression of Nrf2 mainly in the hepatocyte cytoplasm of (a) control group: moderate expression and brown stained cytoplasm indicating positive reaction (arrows). (b) QR-Lipo group: similar to the control group. (c) Co-Amox group: downregulated Nrf2 expression in hepatocytes cytoplasm and most cells are showing weak reactions. (d) Co-Amox and QR-lipo: high increase in Nrf2 expression in the cytoplasm of the hepatocytes (scale bar; 50 μm and magnification power; 400×). (f–i) Expression of SIRT1 in the hepatocyte nuclei of (f) control group: moderate expression and brown-stained nuclei indicate positive reaction (arrows). (g) QR-lipo group: moderate expression similar to the control group. (h) Co-Amox group: weak expression of SIRT1 in hepatocyte nuclei. (i) Co-Amox and QR-Lipo group: strong increase in SIRT1 expression (Scale bar; 50 μm and magnification power; 400×). (e,j) Mean area percentages of Nrf2 and SIRT1 immune expression, respectively, for the different experimental groups. *** p < 0.001.

Figure 10

Three-dimensional orientation and surface mapping against SIRT-1 target site of (a) Resveratrol (reference) and (b) QR.

Figure 11

Principal component analysis of all variables in the study. Principal component analysis for all variables was conducted to summarize the data. (a) Contribution of principal components to the total variance. (b) PC scores plot showing the different groups’ dimensions based on the PC1 scale. (c) PC loading plot showing the different variables’ dimensions based on the PC1 scale. (d) PC biplot combining the PC scores and PC loading plots.