Silicon dioxide nanoparticles inhibit the effects of cold exposure on metabolism and inflammatory responses in brown adipocytes

2.1 Sample preparation

SiO₂ NPs (shape, crystal structure; size, 20.2 ± 6.4 nm; composition, SiO₂ > 99.0%), purchased from Shanghai Run He Nano Co. Ltd. (Shanghai, China), were prepared in 2 mg/mL phosphate buffered saline (PBS), followed by ultrasonication at < 50°C for 8 h. Using transmission electron microscopy (JEM-100CX II, JEOL Co., Tokyo, Japan), the shape and size of the SiO₂ NPs were measured (Fig. 1). After sonication, aliquots of the stock suspensions were immediately serially diluted in cell culture medium to obtain the desired test concentrations. The zeta (ζ) potential of SiO₂ NPs (-11.76 ± 0.58 mV) was analyzed by dynamic light scattering (Malvern Zeta sizer Nano ZS, Worcestershire, UK).

Fig. 1

Image of SiO₂ NPs by transmission electron microscopy

2.2 Cell isolation and culture

Brown adipocytes were isolated from the BAT near scapular area of 1-2-day-old Sprague-Dawley (SD) newborn rats. The parental SD female and male rats were housed in stress free, clean and animal friendly conditions in a standard environment (relative humidity 60% ± 10%, room temperature 21 ± 2°C) with a 12 h dark/ light cycle and 10-15 air changes per hour. Food and water were freely accessible. All newborn rats lived with their parental female rats until they were used for experiment. The tissue was cut into 1 mm × 1 mm pieces and digested using 2 mg/mL collagenase and 20 g/L bovine serum albumin (BSA) in Dulbecco’s modified eagle medium (DMEM) in a shaking water bath at 37°C for 1 h. After digestion, the digested tissue was filtered through a 100-μm nylon screen, and cells were centrifuged at 300 × g for 5 min. The pellet consisting of precursor cells was washed once in isolation buffer and centrifuged again. Cells were resuspended in 2 mL of culture medium (DMEM containing 25 mmol/L glucose, 20 mmol/L HEPES, 20% FBS, 100 units/mL penicillin/streptomycin), seeded on 35 mm plates, and grown in a humidified atmosphere of 5% CO₂ and 95% air with the medium changed every day. Preadipocytes were grown to confluence for differentiation in culture medium supplemented with 20 nmol/L insulin and 1 nmol/L T3 (differentiation medium). Confluent cells were incubated in differentiation medium further supplemented with 0.125 mmol/L indomethacin, 0.5 μmol/L dexamethasone, and 0.5 mmol/L isobutylmethylxanthine (induction medium) for 24 h. Subsequently, the cells were maintained in differentiation medium for 4-5 days until a fully differentiated phenotype with massive accumulation of multilocular fat droplets had emerged.All experiments involving the care and use of animals were performed in accordance with the guidelines and regulations concerning the ethics of science research in the Institute of Environmental and Operational Medicine and approved by the Ethics Review Board of Institute of Environmental and Operational Medicine (AMMS-04-2020-017). The animals, which were provided by the Laboratory Animal Center of Institute of Environmental and Operational Medicine, were treated humanely and with regard for alleviation of suffering.

2.3 Oil red O staining

After washed twice with PBS, the culture dishes were fixed with 10% buffered formalin at room temperature for at least 1 h. Cells were then stained at room temperature for 2 h with the filtered oil red O solution (0.5 g of oil red O in 100 mL of isopropyl alcohol), washed twice with water, and visualized.

2.4 Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR)

To quantify the relative mRNA levels of the genes of interest in brown adipocyte, qRT-PCR was performed using primers from SYBR GREEN Gene Expression Assays and the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression levels were established using the comparative threshold cycle (CT) method outlined in the manufacturer’s instructions. All expression levels were normalized to the expression level of housekeeping gene (β-actin), and reported as a relative fold change over the control. Relative mRNA expression was determined by the ΔΔ-Ct method using the expression level of the gene encoding the TATA-binding protein levels as the endogenous control. The brown adipocytes for qRT-PCR were cultured at 37°C and at 31°C with the concentrations of SiO₂ NPs 0 and 100 μg/mL, respectively^[31]. The brown adipocytes were divided into four groups, such as the control group(37°C, SiO₂ NPs 0 μg/mL), the SiO₂ NPs group(37°C, SiO₂ NPs 100 μg/ mL), the cold exposure group(31°C, SiO₂ NPs 0 μg/mL) and the combined group(31°C, SiO₂ NPs 100 μg/mL). The primer pairs were as follows:

• UCP1, 5’-TGTGCAATGACCATGTACACCAA-3’ (forward) and 5’-GCACACAAACATGATGACGTTCC-3’ (reverse);

• PGC-1α, 5’-CACCGTAAATCTGCGGGATG-3’ (forward) and 5’-TATCCATTCTCAAGAGCAGCGAAAG-3’ (reverse);

• Dio2, 5’-CTGTGGTTGGATGTAGTCACACGA-3’ (forward) and 5’-CTTTGCACCAGGACCCAAATG-3’ (reverse);

• PRDM16, 5’-CAAAGGGAAGCCAGCAGAG-3’ (forward) and 5’-GAGGCGGGAAGAAGGAATG-3’ (reverse);

• TNF-α, 5’-TTCCAATGGGCTTTCGGAAC-3’ (forward) and 5’-AGACATCTTCAGCAGCCTTGTGAG-3’ (reverse);

• IL-1β, 5’-CCCTGAACTCAACTGTGAAATAGCA-3’ (forward) and 5’-CCCAAGTCAAGGGCTTGGAA-3’ (reverse);

• IL-6, 5’-ATTGTATGAACAGCGATGATGCAC-3’ (forward) and 5’-CCAGGTAGAAACGGAACTCCAGA-3’ (reverse);

• ADPN, 5’-CAAGGCCGTTCTCTTCACCT-3’ (forward) and 5’-CCCCATACACTTGGAGCCAG-3’ (reverse);

• Leptin, 5’-TGACAAACAGGTTACAGGACCAGA-3’ (forward) and 5’-ACGAAACCCGATCCAGTTCA-3’ (reverse);

• β-actin, 5’-CCTAAGGCCAACCGTGAAAA-3’ (forward) and 5’-CAGAGGCATACAGGGACAACAC-3’ (reverse).

2.5 Statistical analyses

All quantitative variables are expressed as the mean ± standard error of mean (SEM). Statistical analyses were conducted using the SPSS software (IBM SPSS Statistics version 21.0, IBM Co., New York, USA). Analysis of variance (ANOVA) was used to establish the statistical significance of differences between the experimental groups and control. One-way ANOVA with Bonferroni’s correction was used for multiple comparisons. UNIANOVA was used to analyze the main effect and the interaction between SiO₂ NPs and cold exposure. P values < 0.05 were considered significant and < 0.01 highly significant.

3.1 SiO₂ NPs antagonize the effect of cold exposure on the metabolism genes and plasticity genes in brown adipocytes

The brown adipocytes, stained with oil red O (Fig. 2), verified that the isolation, differentiation, and culture from BAT were successful. With SiO₂ NPs treatment or cold exposure, the expression of adipogenic genes significantly changed in brown adipocytes (Fig. 3). In Fig. 3A, the expression of PRDM16, a marker gene for brown adipocytes, was downregulated significantly (P < 0.05) in the SiO₂ NPs group, but upregulated significantly (P < 0.01) in the cold exposure group, compared with the control group. As expected based on the opposing effects between SiO₂ NPs and cold exposure, co-treatment with two rendered a loss of the effects seen with SiO₂ NPs or cold exposure alone. Similar pattern of changes of the mRNA levels of Dio2, PGC-1α and UCP1 genes was consistently observed. Specifically, Dio2, a gene critical for thermogenesis in response to dropping temperature, sympathetic stimulation, or overfeeding in BAT, was downregulated (P < 0.05) by SiO₂ NPs alone but upregulated (P < 0.05) by cold exposure, whereas SiO₂ NPs/cold exposure combination produced relatively moderate but statistically significant (P < 0.05) effect with a smaller magnitude of downregulation compared to SiO₂ NPs alone and less upregulation compared to cold exposure alone. As illustrated in Fig. 3C and 3D, the expression of PGC-1α and UCP1 was significantly downregulated by SiO₂ NPs alone (UCP1: P < 0.01; PGC-1α: P < 0.05) but increased (P < 0.05) by cold exposure alone, and the effects of SiO₂ NPs or cold exposure alone were neutralized by their combination (UCP1: P < 0.01, PGC-1α: P < 0.05). The factorial analysis showed that there were antagonistic effects between SiO₂ NPs and cold exposure on the plasticity genes and metabolism genes in brown adipocytes, where the main effect of SiO₂ NPs or cold exposure on the plasticity genes and metabolism genes were significant (P < 0.05).

Fig. 2

The oil red O staining of brown adipocytes

The brown adipocytes were isolated from the brown adipose tissue, induced in differentiation medium, and stained with oil red O.

Fig. 3

Adipogenic gene expression determined by qRT-PCR in brown adipocytes

(A) PRDM16. (B) Dio2. (C) PGC-1a. (D) UCP1. Values represent mean ± SEM, and significant differences compared with controls are indicated by ^*P < 0.05, ^**P < 0.01.

3.2 SiO₂ NPs antagonize the effect of cold exposure on the inflammatory response genes in brown adipocytes

After SiO₂ NPs or cold exposure, the expression of proinflammatory cytokine genes changed significantly in brown adipocytes (Fig. 4). The expression of IL-1β, IL-6 and TNF-α was remarkably (P < 0.05) upregulated by either SiO₂ NPs alone or cold exposure alone; yet, co-treatment with SiO₂ NPs and cold exposure markedly mitigated the upregulation of these proinflammatory cytokine genes. The factorial analysis indicated that the main effects of SiO₂ NPs and/ or cold exposure on the inflammatory responses genes were significant (P < 0.05), Moreover, SiO₂ NPs and cold exposure have mutually antagonistic effects on the expression of proinflammatory genes in brown adipocytes.

Fig. 4

Expression of inflammatory cytokines determined by qRT-PCR in brown adipocytes

(A) IL-1β. (B) IL-6. (C) TNF-α. Values represent mean ± SEM, and significant differences compared with controls are indicated by ^*P < 0.05, ^**P < 0.01.

3.3 Antagonistic effects between SiO₂ NPs and cold exposure on the expression of secretion genes in brown adipocytes

Fig. 5A demonstrated that the expression of ADPN gene, which correlates with adipocyte differentiation and modulates a number of metabolic processes, was decreased significantly (P < 0.01) by SiO₂ NPs alone, but increased significantly (P < 0.01) by cold exposure, compared with non-treated control. Similarly, leptin, which plays a pivotal role in inflammatory responses and in the regulation of energy expenditure and appetite, was considerably decreased (P < 0.01) by SiO₂ NPs alone but increased (P < 0.01) by cold exposure (Fig. 5B). As anticipated, SiO₂ NPs/cold exposure combination compromised the effects produced by SiO₂ NPs alone or cold exposure alone. There were antagonistic effect between SiO₂ NPs and cold exposure on leptin and ADPN.

Fig. 5

Effect of cold exposure or/and SiO₂ NPs on the expression of secretion-related genes in brown adipocytes

(A) ADPN. (B) Leptin. Values represent mean ± SEM, and significant differences compared with controls are indicated by ^**P < 0.01.