Antioxidant hepatic lipid metabolism can be promoted by orally administered inorganic nanoparticles

Accumulation of inorganic nanoparticles in living organisms can cause an increase in cellular reactive oxygen species (ROS) in a dose-dependent manner. Low doses of nanoparticles have shown possibilities to induce moderate ROS increases and lead to adaptive responses of biological systems, but beneficial effects of such responses on metabolic health remain elusive. Here, we report that repeated oral administrations of various inorganic nanoparticles, including TiO2, Au, and NaYF4 nanoparticles at low doses, can promote lipid degradation and alleviate steatosis in the liver of male mice. We show that low-level uptake of nanoparticles evokes an unusual antioxidant response in hepatocytes by promoting Ces2h expression and consequently enhancing ester hydrolysis. This process can be implemented to treat specific hepatic metabolic disorders, such as fatty liver in both genetic and high-fat-diet obese mice without causing observed adverse effects. Our results demonstrate that low-dose nanoparticle administration may serve as a promising treatment for metabolic regulation.


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Forward ( White arrows indicated the nanoparticles. Scale bar, 500 nm. b, Relative concentration of nanoparticles in the blood and lymph after a single oral administration (0.72 mg/kg). * represented P < 0.05. n = 6. c, Percentage of Au nanoparticle-positive cells in the main organs of mice gavaged by Cy5.5-conjugated Au nanoparticles (0.72 mg/kg). The control group was set as mice gavaged by a same volume of the vehicle solution chosen as supernatant retrieved from Cy5.5-conjugated Au nanoparticles centrifuged twice at 600000 g for 60 minutes. n = 6. The full gating strategy was included in Fig. S6 Ces2d was not found. Region upstream of the mouse Ces2h (more similar to human CES2 than other mouse Ces2 genes) and Human CES2 (bottom). Chr represents the chromosome; TSS represents the transcriptional start site; Arrows represent binding sites of the primers used for amplification of the two AREs (s1 and s2), or nonspecific region away from the AREs. e-g, Protein expression of Nrf2 (e-f), and mRNA expression of its target Nqo1 and Ces2h (g) in liver after Nrf2 agonist treatment (Bardoxolone, 3 mg/kg). h-i, mRNA (h) and protein (i) levels of Nrf2 in wild-type mice and Nrf2 -/mice. j, Nrf2 targeted gene-Nqo1 mRNA levels in wildtype mice and Nrf2 -/mice. k, mRNA expression of Ces2h in liver after Nrf2 agonist treatment (Bardoxolone, 3 mg/kg) in wild-type mice and Nrf2 -/mice. l-m, Ces2h protein expression (l) and Ces2h mRNA expression (m) measured by RT-qPCR in Nrf2 -/mice fed with chow food and gavaged with either saline or nanoparticles (TiO2, NaYF4, and Au; 0.72 mg/kg) for 21 days. n, ChIP from liver lysates of wild-type mice using an Nrf2 antibody. Positive or negative controls were set as binding of Nrf2 to the Nqo1 ARE or to a Nqo1 ns region, respectively. Results are shown as percentage of input bound by the antibody. All data in plot represent mean ± SEM. Different letters indicate the significant difference (P < 0.05) analyzed by one-way ANOVA. n = 6. Source data are provided as a Source Data file.

Supplementary Fig. 10 | Oral nanoparticle effects on body weight, Ces2h
expression, hepatic metabolism, and survival in wild-type and Ces2h-deficient (Ces2h -/-) mice. a, Effect of nanoparticles (0.72 mg/kg/day) on the body weight (left) and liver weight (right) in the wild-type mice (n = 6). b, Functional enrichment analysis of differential expressed genes from hepatic transcriptome comparison of TiO2 group (0.72 mg/kg/day) vs control group. The differential expressed genes were defined by the |log2FC| > 1 and P < 0.05. c, Changes in mRNA expression of the carboxylesterase family (Ces1 and Ces2) in livers of wild-type mice treated with TiO2 as determined by transcriptome. d, Relative mRNA expression of Ces2h in major organs of wild-type mice determined by RT-qPCR (n = 6). e, Overview of the targeting strategy to knockout Ces2h gene. f, Relative mRNA expression of Ces2 family, canonical triglyceride lipase Atgl, and re-esterification pathways in livers of Ces2h -/mice compared with wild-type mice (n = 6). g, Ces2h protein levels in liver of Ces2h -/mice compared with wild-type mice. h, Relative mRNA expression of Ces2 family, canonical triglyceride lipase Atgl, and re-esterification pathways in intestines of Ces2h -/mice compared with wild-type mice (n = 6). i, Histological analysis of Ces2h -/mouse liver compared with wild-type mouse liver. j-k, Body and liver weight of Ces2h -/mice fed with chow food and with/without oral nanoparticle administration (0.72 mg/kg/day; n = 6). l-m, Survivorship curve (n = 12) and serum liver index (alanine transaminase, ALT; aspartate aminotransferase, AST) (n = 6) of Ces2h -/mice fed with chow food and with/without oral nanoparticle administration (0.72 mg/kg/day). n, Serum liver index (alanine transaminase, ALT; aspartate aminotransferase, AST) of wild-type mice fed with chow food and with/without oral nanoparticle administration (0.72 mg/kg/day; n = 6). Different letters indicate the significant difference (P < 0.05) analyzed by oneway ANOVA. Data in a, d, f, h, j, k, m, and n are presented as mean values ± SEM. Source data are provided as a Source Data file.  (e) were measured. f-g, Hepatic mRNA levels, measured by RT-qPCR, of fatty acid oxidation genes, and re-esterification genes influenced by nanoparticles (0.72 mg/kg/day for 21 days) in Ces2h -/mice fed with chow food. Data are presented as mean values ± SEM. n = 6. Source data are provided as a Source Data file.

Supplementary Fig. 15 | Different effects of nanoparticles on cellular ROS production, and cell survival rate between normal or Ces2h-deficient (Ces2h -/-) hepatocytes.
The normal or Ces2h -/mice were treated with either saline or nanoparticles (0.72 mg/kg/day) by gavage. The hepatocytes were identified according to the gating strategy presented in the Fig. S7. Two-sided student's t-test was applied to ROS production (a) and hepatocyte survival rate (b) with a significance define as followed: *, P < 0.05; **, P < 0.01. Different letters indicate the significant difference (P < 0.05). Data are presented as mean values ± SEM. n = 6. Source data are provided as a Source Data file.

Supplementary Fig. 16 | Chemical catalysis of esters by nanoparticles. a, Diagram
illustrating the measurement of esterase activity. b-d, Absorbance spectra of reaction supernatants sampled at selected time intervals. The reaction was catalyzed by of Au, NaYF4, TiO2 and positive control (liver lysate) at 37°C. The characteristic absorption peak was observed near 320 nm for 1-Naphthyl acetate (1-NA), near 390 nm for intermediate naphthol peak and near 450 nm for final product diazo. e, The relative conversion of 1-NA to diazo was determined for esterase activity of nanoparticles and positive control (liver lysate) at 37°C. Data in e are presented as mean values ± SEM. n = 6. 1-2-3-4-5-6-7-8: All the locations are hydrophobic pockets, which will be an important binding region for the carbon chain group of cholesterol ester. Our molecule will move around in this region, as evident in our following docking system.

Supplementary Fig. 18 | Docking analysis of molecular systems of Homo sapiens and Mus musculus carboxylesterase 2 (CES2/Ces2h)-superoxide anion (O 2 -)/hydroxyl radical (ꞏOH)-ester.
In the docking analysis, O2and ꞏOH was added at the same time, and 500 ps of non-limiting molecular dynamics analysis was performed using Amber 16. Kinetic constraint was set as followed: 1. ester, O2 -, and ꞏOH was unrestricted; 2. Docking analysis was completely flexible; 3. Proteins were kept rigid. The average structure was extracted from the trajectory of molecular dynamics and used for subsequent analysis. Preliminary optimization results of dynamics were as followed: 1. Due to the limited space of oxygen anion hole, the coexistence of O2and ꞏOH in the protein-molecule docking complex was not detected; 2. In the CES2 protein system, O2was not located at the oxyanion hole, but ꞏOH stayed at this position; 3. Similarly in the Ces2h protein system, O2was not located at oxyanion hole, but ꞏOH stayed at this position and formed a hydrogen bond interaction with the ligand.

Supplementary Fig. 19 | Molecular modeling and docking of Homo sapiens and
Mus musculus carboxylesterase 2 (CES2/Ces2h) protein with reactive oxygen species induced by nanoparticles. a, The schematic diagram of catalytic mechanism of carboxylesterases and the perturbation of nanoparticles. Supplementary Fig. 20 | Immunofluorescence staining showing catalytic ability of hepatocytes (n = 6) with normal CES2 or mutated CES2 p.G193A on ester and the perturbation of nanoparticles. a, Knockout effects of CES2 and Ces2h in LO2 and NCTC1469 cell lines, respectively. b, Ces2h p.G148A and CES2 p.G193A expression after transfecting LO2/NCTC1469 cells with the corresponding eukaryotic expression vectors (pcDNA3.1(+)-His-CES2 g.695G>C , pcDNA3.1(+)-His-Ces2h g.436G>C ) and their protein level of Ces2h/CES2 compared to their wild-type cells. c, Intracellular lipid fraction analysis of normal CES2 or mutated CES2 p.G193A hepatocyte cell lines upon TiO2, NaYF4, Au nanoparticle treatments, respectively. d, Incorporation of [1-13 C]oleate into cellular lipids. e-g, Chase experiments evaluating turnover of lipid species, including triglyceride (TG), cholesterol ester (CE), and phospholipid (PL). Statistics for the chase period were analyzed as a percentage of the pulse. The group setting was same in (c-g) as indicated in (d). Different letters indicate the significant difference (P < 0.05) analyzed by one-way ANOVA. Data in a, c-g are presented as mean values ± SEM. n = 6. Source data are provided as a Source Data file.  Fig. 27 | Safety assessment of orally administrated nanoparticles in genetically obesity (db/db) mice and high fat diet (HFD)-fed mice. a-b, Clearance of orally administrated TiO2, NaYF4, and Au nanoparticles (0.72 mg/kg/day) for each organ in db/db mice (a) and HFD-fed C57BL/6 wild type mice (b). The oral administration procedure for nanoparticle treatments was performed every two days (46 times in total) during the 3-month treatment. The clearance data was collected 21 days after administration was stopped. c, The pathological analysis of the main organs in db/db mice and HFD-fed C57BL/6 wild-type mice with 3-month orally administrated TiO2, NaYF4, and Au nanoparticles (0.72 mg/kg/day) using HE staining. Scale bar, 100 μm. d-e, Hematology analysis of db/db mice (d) and HFD-fed C57BL/6 wild-type mice with 3-month orally administrated TiO2, NaYF4, and Au nanoparticles (0.72 mg/kg/day). All data in plot represent mean ± SEM. n = 6. Source data are provided as a Source Data file.
Supplementary Fig. 28 | Clearance of TiO 2 , NaYF 4 , and Au nanoparticles after 3week low (a), middle (b), and high (c) oral administration for each organ. Day 0 represented the time when oral administration was stopped. All data in plot represent mean ± SEM. n = 6. Source data are provided as a Source Data file.
Supplementary Fig. 30 | Safety assessment of orally administrated nanoparticles in Ces2h-dificient (Ces2h -/-) mice. HE staining of main organs in Ces2h -/mice with/without oral administrated TiO2, NaYF4, and Au nanoparticles (0.72 mg/kg/day) was performed on day 21. Blue arrows indicate immune cell infiltrations; Red cells indicate aggregation of extramedullary hematopoietic cells in the spleen and tubulointerstitial congestion in the kidney; Yellow arrows indicate eosinophilic substances in the renal tubule lumen; Black arrows indicate bare lamina propria. Scale bar, 100 μm.