Nonlethal dose of silver nanoparticles attenuates TNF-α-induced hepatic epithelial cell death through HSP70 overexpression

Abstract

Silver nanoparticles (Ag-nps) have been widely used in various biomedical products. Compared with its hazardous effects extensively being studied, rare attention has been paid to the potential protective effect of Ag-nps to human health. The present study was designed to evaluate the protective effects of Ag-nps and heat shock treatment on tumor necrosis factor-α (TNF-α)-induced cell damage in Clone 9 cells. Clone 9 cells were pretreated with nonlethal concentration of Ag-nps (1 μg/ml) or heat shock, and then cell damages were induced by TNF-α (1 ng/ml). Protective effects of Ag-nps administration or heat shock treatment were determined by examining the TNF-α-induced changes in cell viabilities. The results showed that the intensity of cytotoxicity produced by TNF-α was alleviated upon treatment with nonlethal concentration of Ag-nps (1 μg/ml). Similar protective effects were also found upon heat shock treatment. These data demonstrate that Ag-nps and heat shock treatment were equally capable of inducing heat shock protein 70 (HSP70) protein expression in Clone 9 cells. The results suggest that clinically Ag-nps administration is a viable strategy to induce endogenous HSP70 expression instead of applying heat shock. In conclusion, our study for the first time provides evidence that Ag-nps may act as a viable alternative for HSP70 induction clinically.

silver nanoparticles (Ag-nps), widely used in medicine, industry, material science, and household products, have recently gained considerable attention due to their strong antimicrobial activities (17). Several studies have demonstrated the benefits of Ag-nps, including Ag-nps that deposited on the surface of bacteria caused destruction and lysis of cell walls, prevention of microbial DNA replication, and impediment of bacterial function via binding of silver particles to the thiol group of bacterial proteins (7, 17). Another advantage is that the Ag-nps have potent anti-inflammatory effects that are capable of accelerating wound healing through reduction of inflammatory mediators, i.e., interleukin-1 (IL-1), IL-6, IL-10, and interferon-γ (IFN-γ) (23, 27).

Despite their widespread application, mechanisms of protective action have not been fully elucidated. Although nanoparticle exposure occurs through various routes, i.e., inhalation, ingestion, injection, and direct dermal contact (3), circulating Ag-nps are deposited in certain major organ, including liver (20, 28). Therefore, the Clone 9 immortal rat hepatic epithelial cell line was chosen as a target cell line in this study.

Tumor necrosis factor-α (TNF-α) is one of the most important proinflammatory cytokines that is induced in response to the early phase of injury. There are several reports regarding the TNF-α-induced apoptotic pathways via receptor-free complex II, reactive oxygen species (ROS) production, mitochondrial dysfunction, and caspase activation (13, 21). Thus TNF-α is commonly used as an experimental model to evaluate the cytotoxicity.

Cells have many different ways of combating off foreign invaders, such as the induction of antioxidant machinery, wherein the highly conserved heat shock proteins (HSP) are a specific group of proteins that play an important role in protecting cells at the molecular and cellular levels against different kinds of stress-induced damage (26). The stress-inducible form of the 70-kDa HSP (HSP70) has been shown to be one of the important species of these conserved proteins (25, 26) and always serves as an indicator of stress induction. Moreover, it has been reported that HSP70 can protect against TNF-α-induced lethal inflammatory shock and hepatocyte apoptosis (25, 30). Accordingly, the primary aim of this study was to test the hypothesis that Ag-nps treatment can protect TNF-α-induced cell death, and the protective mechanism is related to the overexpression of endogenous HSP70.

MATERIALS AND METHODS

Cell culture and nanoparticle treatment.

Rat hepatic epithelial cell line, Clone 9 cell, was purchased from Food Industry Research and Development Institute, Taiwan. Clone 9 cells were maintained in growth medium [Ham's F-12K medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Hyclone, Logan, CT) and 1% penicillin streptomycin (Gibco, Paisley, UK)] at 37°C in 5% CO₂ humidified atmosphere.

Ag-nps were purchased from Sigma-Aldrich (St. Louis, MO). The physical characteristics of the nanoparticles according to the manufacturer's data are as follows: size (≤100 nm), purity (99.5% trace metals basis), surface area (5.0 m²/g), density 10.49 g/cm³ (liter), with polyvinylpyrrolidone as dispersant. Nanoparticles (10 mM) were homogeneously dispersed in deionized water and diluted to the required concentrations using the cell growth medium. At ∼80% of confluence, various concentrations of Ag-nps were added, and cells were subjected to viability determination and Western blot analysis. After the treatment, the various effect end points were evaluated in control and Ag-nps-treated cells.

Transmission electron microscopy studies.

Nanoparticles were suspended in deionized water, subsequently deposited on carbon film-coated Cu grids, and then allowed to dry in air. The morphology of nanoparticles was examined under transmission electron microscopy (JEM2000 EXII; Jeol, Tokyo, Japan), and then sizes were analyzed by ImageJ 1.42q software. All images were taken at an accelerating voltage of 100 kV at 200,000 times magnification.

Cell viability assay.

Cell viability of the Ag-nps was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction] assay. The assay depends on the reduction of MTT to form a purple color formazan dye by mitochondrial succinate dehydrogenase, a mitochondrial enzyme present in the living cells. Briefly, cells were seeded at a density of 1 × 10⁴/ml in 96-well plates and then incubated with different concentrations of Ag-nps (0, 0.5, 1, 1.5, 2, 2.5, 5, 10, and 20 μg/ml) for 24 h; the medium was removed and replaced with 100 μl fresh medium containing MTT solution in an amount equal to 10% of original culture volume. The mixture was incubated for 1 h at 37°C until the purple colored formazan product developed. The precipitated formazan was dissolved by addition of 100 μl of DMSO, and the absorbency was measured at 595 nm by using an ELISA Reader (BioTek Instrument Winooski, VT). The relative cell viability (%) related to control well was calculated using the following equation: (mean optical density of treated cells/mean optical of control cells) ×100. A similar procedure was used for cytotoxicity determination involving TNF-α treatment (0.5, 1, 2, 4, 8, 16, and 32 ng/ml), except that 1) cells were pretreated with actinomycin D (400 ng/ml) for 30 min before the addition of TNF-α; and 2) TNF-α treatment time was 10 h. For some experiments, the cytoprotective effect analysis of Ag-nps and heat shock TNF-α dosing time is 2 h before the time of Ag-nps- and heat-induced HSP70 at maximum level.

Western blot analysis.

For analysis of HSP70 protein expression, samples of cell homogenate containing an equal amount of protein extract were denatured and separated to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes by electroblotting for 1 h (100 V). The membranes were blocked with the Tris-buffer saline (TBS) containing 5% nonfat dry milk for 30 min, and then washed with t-TBS (TBS containing 0.5% Tween 20). The blots were incubated overnight at 4°C with primary antibody against HSP70 (1:2,000) (Enzo Life Science). After that, membranes were incubated for 1 h with the secondary antibody (1:5,000) at room temperature and then washed with the t-TBS. The membrane was also probed with β-actin as an internal housekeeping control. Protein bands were visualized by enhanced chemiluminescence (Amersham), and the relative densities were quantified. All values were normalized to β-actin expression.

Statistical analysis.

All values were expressed as means ± SD of at least three independent experiments. Statistical analysis was performed using Student's t-test. P values of <0.05 were considered as significant difference.

RESULTS

Transmission electron microscopic observation of Ag-nps characterization.

Figure 1 shows the size and morphology of Ag-nps following dispersion in water. The majority of the dispersed particles appears to be in spherical shape (Fig. 1, top), with a size distribution ranging from 13.1 to 79.3 nm in diameter and a mean diameter of 35.3 ± 13.85 nm (Fig. 1, bottom). These data indicate that the Ag-nps used in this study was confined to nano-sized form based on conventional consensus of ≤100 nm (8).

Fig. 1.

Size distribution and morphology of silver nanoparticles (Ag-nps) following dispersion in water. Top: transmission electron microscopy (TEM) image of the Ag-nps. Particles were processed for TEM evaluation, as described in materials and methods. Scale bar is 100 nm. Image was taken at ×200,000 magnification. Bottom: size distribution of Ag-nps.

Determination of nonlethal dose of Ag-nps.

Figure 2 depicts cell viabilities of Clone 9 hepatic epithelial cells following Ag-nps exposure at various concentrations. Ag-nps concentrations <1.5 μg/ml did not affect cell viabilities, whereas the concentrations ranging from 2 to 20 μg/ml decreased the viabilities in a dose-dependent manner ranging from 12 to 58% (Fig. 2). Based on these data, a nonlethal concentration of 1 μg/ml was chosen for the entire experiments in this study.

Fig. 2.

Cell viability of Clone 9 cells after exposure to Ag-nps. Cells were exposed to various concentrations of Ag-nps (0.5, 1, 1.5, 2, 2.5, 5, 10, and 20 μg/ml) for 24 h, and cell viabilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction (MTT) assay, as described in materials and methods. Values are means ± SD; n = 4 experiments for each time points. *P < 0.05 and **P < 0.01 vs. control.

Upregulation of HSP70 expression by nonlethal dose of Ag-nps and heat shock treatment.

Figure 3 shows levels of the HSP70 protein in Clone 9 cells following exposure to nonlethal dose of Ag-nps (1 μg/ml) were determined at various time points (4, 8, 16, and 24 h). HSP70 proteins were overexpressed at 8 h and, thereafter, up to 24 h (by 19.7 times at 8 h, P < 0.01; by 29.3 times at 16 h, P < 0.01; by 19.6 times at 24 h, P < 0.01) (Fig. 3A). A time point of exposure of 16 h was selected for the entire study. These data indicate that a nonlethal dose of Ag-nps was capable of inducing HSP70 expression. A similar trend was also found in heat shock treatment, indicating that HSP70 was overexpressed at 8 h (Fig. 3B).

Fig. 3.

Effects of Ag-nps and heat shock treatment on the induction of heat shock protein 70 (HSP70) level in Clone 9 cells. A: changes in HSP70 protein expression at various time points following exposure to nonlethal concentration of Ag-nps (1 μg/ml). B: changes in HSP70 protein expression after Ag-nps and heat shock treatment. Experiments were carried out as described in materials and methods. A and B, top: representative immunoblots of HSP70. A and B, bottom: densitometric analysis of immunoblots. β-Actin was used as a loading control. Values are means ± SD; n = 6 experiments for each column. *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 vs. Ag-nps group.

Effect of different concentrations of TNF-α on the viabilities of Clone 9 hepatic epithelial cells.

Figure 4 depicts the effects of different concentrations of TNF-α on the viabilities of Clone 9 cells. After 10 h of exposure to TNF-α, the viabilities of Clone 9 cells were decreased in a dose-dependent manner; the decrease was 40% at 0.5 ng/ml (P < 0.01), and the extents of decreases were expanded from 40 to 83% when the concentrations of Ag-nps were increased from 0.5 to 32 ng/ml (P < 0.01). Based on these data, a fix concentration of 1 ng/ml was selected for the entire experiment in this study.

Fig. 4.

Effect of different concentrations of tumor necrosis factor (TNF)-α on the viabilities of Clone 9 cells. Cells were exposed to various concentrations of TNF-α (0.5, 1, 2, 4, 8, 16, and 32 ng/ml) for 10 h, and cell viabilities were measured by MTT assay, as described in materials and methods. Values are means ± SD; n = 4 independent experiments. **P < 0.01 vs. control.

Cytoprotective effect of Ag-nps in the TNF-α-induced cytotoxicity of Clone 9 cells.

Ag-nps concentration at 1 μg/ml did not affect cell viability (Fig. 5A, column 1 vs. column 3), whereas TNF-α alone (1 ng/ml) decreased viability by 58% (P < 0.01) (column 1 vs. column 2). The TNF-α-induced reduction in cell viability was ameliorated from 58 to 37% (comparison between differences in columns 1 and 2 vs. differences in columns 3 and 4). These data indicate that Ag-nps was capable of protecting TNF-α-induced cytotoxicity on Clone 9 cells (Fig. 5A).

Fig. 5.

Protective effects of Ag-nps and heat shock treatments on the TNF-α-induced cytotoxicity of Clone 9 cells. A: changes of cell viabilities in Clone 9 cells with (+) or without (−) Ag-nps (1 μg/ml) and TNF-α (1 ng/ml) treatment. B: changes of cell viabilities in Clone 9 cells with (+) or without (−) heat shock and TNF-α (1 ng/ml) treatment. MTT assay for measurement of cell viability was carried out as described in materials and methods. NC, no change. Values are means ± SD; n = 7 experiments. **P < 0.01 vs. control.

As shown in Fig. 5B, heat pretreatment alone did not affect cell viability (column 1 vs. column 3). TNF-α decreased cell viability by 58% (column 1 vs. column 2) in nonheated cells. The TNF-α-induced reduction in cell viability (nonheated) was ameliorated from 58 to 41% (heated) (comparison between differences in columns 1 and 2 vs. differences in columns 3 and 4). These data indicate that heat pretreatment was capable of protecting TNF-α-induced cytotoxicity on Clone 9 cells (Fig. 5B). By pulling results together from Ag-nps (Fig. 5A) and heat pretreatment (Fig. 5B), it is clear that the protective effects of Ag-nps against TNF-α-induced cytotoxicity are comparable with the potential from heat shock treatment.

DISCUSSION

Ag-nps have been widely reported to be hazardous to human health, but relatively few studies have been undertaken to determine the protective effects of Ag-nps in addition to its antimicrobial function. In this study, we revealed that the potential protective effects of Ag-nps against TNF-α-induced cytotoxicity are comparable with that of heat shock treatment.

There is strong evidence that extensive uptake of Ag-nps into cells induces proinflammatory and toxic activities, which are initiated by the production of ROS (5, 10). The ROS-induced oxidative stress is believed to be a trigger for DNA damage, cell cycle arrest, mitochondrial dysfunction, as well as cellular apoptosis (1, 4, 9). Being a marker of oxidative stress, HSP70 was suggested to be involved in toxicity mechanism of Ag-nps (12). Recently, Lim et al. (15) reported a low level (2.5 μg/ml) of exposure at the early stage to Ag-nps-induced HSP70, heme oxygenase-1, and IL-8, indicating that nonlethal concentrations of Ag-nps can induce HSP. In the present study, we found that HSP70 was induced by Ag-nps at concentrations as low as 1 μg/ml, and the effect was comparable to heat shock treatment.

The role of HSP protein family has been widely described in many articles. Among several members of the HSP family, stress-inducible HSP70 is most intensively studied for its function as a molecular chaperone and antiapoptotic activity in preventing cellular damage under a variety of physiological and pathological conditions (6, 16). Many studies have shown that HSP play a critical role in modulating apoptotic cascades. Van Molle et al. (25) reported that heat shock treatment of mice leads to a strong induction of HSP70, prevention of IL-6 and nitric oxide-induced severe damage, and reduction of apoptosis in enterocytes. Another report by Arya et al. (2) demonstrated that HSP70 interacts with both the intrinsic and extrinsic apoptosis pathways at several junctions and inhibits cell death. The apoptotic signaling cascade triggered by nitric oxide and heat stress stimulates translocation of Bax from cytoplasm to mitochondria and release of cytochrome c from mitochondria. Further downstream in the intrinsic pathway, HSP70 also inhibits formation of a functional apoptosome through direct interaction with apoptotic protease activating factor-1 (Apaf-1). HSP70 inhibits late caspase-dependent events, such as activation of cytosolic phospholipase A2 and changes in nuclear morphology, and protect cells from forced expression of caspase-3 (2). In this study, we showed that Ag-nps exerted a significant antiapoptotic effect in Clone 9 cells associated with the overexpression of HSP70; furthermore, its potency was as obvious as that of heat shock.

Ag-nps have been reported to be able to translocate in the blood circulation and distribute to various tissues, including liver, lungs, spleen, brain, heart, kidneys, and testes, in which the liver is shown to have the highest accumulation (20, 28), indicating a high affinity of Ag-nps to hepatocytes. Therefore, a hepatic cell line, Clone 9 cells, was chosen as the target cells in this study. Similar studies have been conducted in other cell types, including skin epithelial A431, lung epithelial A549, murine macrophages RAW264.7 (12), and human macrophage cell line U937 (15). However, our results demonstrated the lowest dose of Ag-np was capable of inducing the expression of HSP70 in cultured cells. Whether or not other cell types could be triggered by such a low dose of Ag-np needs further investigation.

With the more convincing data of HSP-related protective effects published, finding a viable clinical strategy to induce endogenous HSP70 instead of applying heat shock has been well searched. Geranylgeranylacetone has been reported to be an inducer of HSP70 that prevents acoustic injury in guinea pig (18). Compound of paeoniflorin was shown to induce HSP70 synthesis by affecting various genes (29). A small molecule, TRC051384, is shown to be beneficial in ischemic stroke by inducing HSP70 (19). Oversynthesis of HSP70 by arimoclomol has been revealed to be effective for various neurodegenerative diseases (11, 24). Our data provide evidence that Ag-nps has the potential to be developed as a pharmacological agent.

One of the most critical issues in clinical application concerns the absorption, the accumulation, the metabolism, and the elimination of Ag-nps. Most of the administered nanoparticles have been reported to be excreted from kidney or hepatobiliary pathways within 15 days (14, 22). In case of Ag-nps, AshaRani et al. (3) reported that a long incubation period of 48 h was taken to expel 66% of the particles endocytosed in 2 h, implying a slow rate of exocytosis than that of endocytosis. Nevertheless, that Ag-nps can be excreted is an indisputable fact: it is speculated that even with excessive accumulation, cell damage will not occur at low doses following Ag-nps treatment.

The limitation of the study is that other HSP members besides HSP70 were not all evaluated. HSP90 is one of the most abundant and highly conserved HSP. It is induced in response to different apoptotic stimuli, such as heat stress, UV, doxorubicin, and sodium arsenite. These observations support the role of HSP90 in preserving cell viability. HSP90 has been reported to suppress TNF-α-induced apoptosis by preventing the cleavage of Bid (31). In addition, HSP90 forms a cytosolic complex with Apaf-1, inhibits cytochrome c-mediated oligomerization of Apaf-1, and inactivates procaspase-9. These events lead to eventual inhibition of apoptosis. The mechanisms by which HSP90 prevent apoptosis from occurring are reasonable, compared with those for HSP70, as described in the preceding paragraph, imply that the underlying mechanism of protection for HSP70 would be applicable to HSP90.

Conclusions.

In conclusion, our results show that administration of nonlethal dose of Ag-nps leads to induction of HSP70, which in turn protected cell apoptosis against TNF-α. TNF-α is one of the key regulators during inflammation. Agents such as Ag-nps with the ability to block TNF-α action may be developed to become a therapeutic agent for treating inflammatory diseases.

GRANTS

This work was supported by a grant from National Science Council (NSC 102-2320-B-037-023).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: T.-N.T., J.-J.H., L.-J.H., C.-J.L., T.-J.C., and R.-C.Y. conception and design of research; T.-N.T. and J.-J.H. performed experiments; T.-N.T., J.-J.H., and R.-C.Y. analyzed data; T.-N.T., J.-J.H., L.-J.H., C.-J.L., T.-J.C., and R.-C.Y. interpreted results of experiments; T.-N.T., T.-Y.L., and J.-J.H. prepared figures; T.-Y.L. and M.-S.L. drafted manuscript; T.-Y.L., M.-S.L., and R.-C.Y. edited and revised manuscript; T.-Y.L. and R.-C.Y. approved final version of manuscript.