Antidiabetic nephropathy effects of synthesized gold nanoparticles through mitigation of oxidative stress

2.1 Materials

HAuCl₄, dodecyl trimethylammonium bromide (DTAB), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased form Sigma Co. (USA). All other materials were of analytical grade.

2.2 Synthesis of AuNPs

AuNPs were fabricated through a hydrothermal method. Briefly, 50 mL aqueous solutions of a fixed concentrations of DTAB (2.5 mM) were mixed with 1 mL of HAuCl₄ 1with a concentration 10 mM and 200 µL of aqueous solution of sodium citrate with a concentration of 200 mM. Afterwards, the solution was mixed and added into autoclaves and the samples were heated up to 150 °C for 10 h at 110 °C followed by centrifugation at 10,000 rpm for 40 min. The samples were finally washed with deionized water for three times.

2.3 Characterization of AuNPs

The diameter and morphology of as-synthesized AuNPs were characterized by TEM (EM10C, 100 KV, Zeiss, Germany) study. The samples were dissolved in ethanol and sonicated for 15 min. Afterwards, and a drop of the sample was deposited on a carbon-coated copper grid and allowed to be dried in air. The crystalline pattern of as-fabricated AuNPs (0.1 g) was explored by X-ray defecation (XRD) (Philips PW 1730, Amsterdam, Netherlands). The hydrodynamic radius and zeta potential values of AuNPs were also assessed by employing dynamic light scattering (DLS) [Brookhaven instruments 90Plus particle size/zeta analyzer (Holtsville, NY, USA)]. All experiments for DLS analysis were done in distilled water and the pH of the medium was adjusted using 0.1 M NaOH and 0.1 M HCl.

2.4 Cell culture

The human renal tubular epithelial cell line (HK-2) was cultured in DMEM, supplemented with D-glucose (5.5 mM), FBS (10%), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Sigma, USA) in a 5% CO₂ incubator at 37 °C. After pretreatment of cells with AuNPs (5 µg/ml) for 24 h, 50 mM glucose (HG) was added to the samples and incubation was continued for another 24 h. Vitamin C (Vit C) with a concentration of 5 µg/ml was used as the positive control. Also, for MTT assay, the number of cells was 1 × 10⁴ and the for the rest of experiments the number of cells was 1 × 10⁶.

2.5 MTT assay

Cell viability was done by the MTT assay through reading the absorbance of the samples at 570 nm using a microplate reader (BIO-RAD microplate reader-550). Briefly, the treated samples were mixed by MTT stock solution (5 mg mL⁻¹) for 4 hr. After removing the supernatants, 150 μL DMSO solution was added for 2 min and the absorbance of the samples was then read. The data were expressed in comparison with negative control cells.

2.6 Reactive oxygen species (ROS) assay

The generation of intracellular ROS was investigated through the dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Briefly, the treated cells were washed and incubated with a DCFH-DA probe (20 μM) for 30 min. Then, the fluorescence intensity of the samples was measured at λ_ex/λ_em of 485 nm/530 nm using a spectrofluorometer (Gemini EM). The fluorescence images were taken employing a Zeiss fluorescence microscope (Zeiss, Germany).

2.7 Quantitative PCR (qPCR) analysis

The expression level of Bax, Bcl-2, and Caspase-3 mRNA was evaluated using qPCR technique. The total RNAs of cells were extracted using the RNA Isolation Kit (Roche Life Science) followed by quantification assays using a UV-3600 spectrophotometer (Shimadzu, Japan). Afterward, cDNA was done with 1 µg RNA and a first strand cDNA synthesis kit (Fermentas, Thermo scientific, USA) according to manufacturer’s protocols. The qPCR assay was carried out using SYBR Green bonding with an qPCR system (San Diego, CA 92122, USA). Quantitative gene expression was assessed by comparative CT (ΔΔCT) method (Schmittgen and Livak, 2008), using GAPDH as a housekeeping control gene.

2.8 Protein extraction

Protein was extracted from cells using a mild lysis buffer, including 50 mM Tris-HCl pH 8, 100 mM NaCl, 1.2 mM DTT, 4 mM MgCl₂, 0.5 mM EDTA, 7% glycerol, and 0.1% triton). The protein concentration was then calculated using the Bradford assay (Gotham et al., 1988).

2.9 ELISA assay

The level of AGE, and expression of Bax, Bcl-2 and Caspase-3 proteins were determined using ELISA assay using AGE Assay Kit (ab238539), human Bax ELISA Kit (ab199080), human Bcl-2 ELISA Kit (ab119506), and human Caspase-3 ELISA Kit (ab181418), respectively. After addition of the primary and secondary antibody, the final step was done by adding 100 µL stop solution and the absorbance was read at 450 nm using a microplate reader (BIO-RAD microplate reader-550).

2.10 SOD2 activity

Quantification of SOD2 activity in the cell was done using SOD Activity Kit StressXpress® (SKT-214) based on the manufacturer’s instructions. Briefly, after treatment of the cells and sample preparation, the substrate was added followed by xanthine oxidase reagent and incubated at room temperature for 20 min. The colored product was read at 450 nm using a microplate reader (BIO-RAD microplate reader-550).

2.11 SIRT3 activity

SIRT3 activity was determined using the SIRT3 Activity Assay Kit (Fluorometric, ab156067). according to the manufacturer’s instructions. HK-2 cells were treated with or without 30 μM SIRT3 inhibitor (3-TYP) for 12 h followed by incubation with AuNPs for 24 h, and finally treated with 50 mM HG for another 24 h.

Briefly, after treatment of the cells and homogenization, the fluoro-substrate peptide as a substrate was mixed with lysed samples and the fluorescence intensity at 2 min interval was measured at λ_ex/λ_em of 340–360 nm/440–460 nm using a spectrofluorometer (Gemini EM).

2.12 Statistical analysis

Statistical analysis was done using Student’s t-test and one-way analyses of variance (ANOVA) and post-hoc procedures based on Newman-Keuls tests. Results were expressed as means ± standard errors (SEs) of three (n = 3) independent experiments. P < 0.05 was considered significant.

3.1 Synthesis and characterization of AuNPs

The AuNPs were synthesized by hydrothermal method and characterized by different approaches such as TEM, DLS, and XRD investigations. As shown in Fig. 1a, the as-synthesized AuNPs provide hexagonal shapes with a diameter of <30 nm. To discuss the colloidal stability of AuNPs, a DLS study was run to determine the hydrodynamic radius and zeta potential values. As depicted in Fig. 1b, the hydrodynamic radius of AuNPs was observed to be around 157.33 nm with a PDI of 0.294, revealing the good colloidal stability of AuNPs. Zeta potential value was determined to be around −35.66 mV, indicating the presence of a potential repulsive electrostatic force between the NPs to limit the tendency of NPs to agglomeration. XRD study was also carried out to analyze the crystalline phase of fabricated NPs. ion was used to confirm the crystalline nature of the particles. Fig. 1c displayed a representative XRD pattern of the AuNPs fabricated by the hydrothermal method after the successful reduction of Au³⁺ to Au⁰. Several Bragg reflections were observed at 2ϴ = 37.99° (1 1 1), 44.02° (2 0 0), 63.11° (2 2 0) and 77.85° (3 1 1) which are in accordance with those reported for the standard AuNPs (Au⁰) (Joint Committee on Powder Diffraction Standards-JCPDS, USA). Thus, the XRD study indicated that the AuNPs were intrinsically crystalline.

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Fig. 1

TEM image (a), DLS histogram (b), and XRD pattern of as-synthesized AuNPs by hydrothermal method.

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3.2 Colloidal stability of AuNPs

To measure the colloidal stability of AuNPs in the medium with different pH values and glucose concentrations, DLS study was run. It was shown that the AuNPs had the smallest hydrodynamic radius in the medium with a pH value of 7.5 (Fig. 2a) and glucose concentration of 1 mM (Fig. 2b). However, it was seen that the hydrodynamic radius of AuNPs was almost constant in the medium with glucose concentration in the range of 1–50 mM (Fig. 2b). Moreover, zeta potential study (Fig. 2c, d) showed the same trend with hydrodynamic radius analysis, indicating that different pH values and glucose concentrations changes the charge distribution on the surface of AuNPs and the charge distribution in a medium with pH value of 7.5 (Fig. 2c) and glucose concentrations with a range of 1 to 50 mM (Fig. 2d) was in the most optimum state.

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Fig. 2

DLS study of AuNPs in a medium with different pH values (a) and different glucose concentrations (b), zeta potential study of AuNPs in a medium with different pH values (c) and different glucose concentrations (d).

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3.3 MTT assay

MTT assay was done to measure the vitality of HK-2 cells in the presence of varying concentrations of AuNPs ranging from 1 to 30 µg/mL for 48 h. As shown in Fig. 3a, the viability of cells remarkably decreased after addition of 10, 20, and 30 µg/mL of AuNPs for 48 h, however, AuNPs with concentrations of 1 and 5 µg/mL did not induce a remarkable change on the viability of HK-2 cells. Therefore, AuNPs with a concentration of 5 µg/mL was used to study the protective effect of AuNPs against HG-induced oxidative stress in HK-2 cells (Fig. 3 b). It was displayed that HG induced a remarkable decrease (***P < 0.001) in cell viability compared to the control group (Fig. 3b). However, pretreatment of the cells with AuNPs resulted in the significant recovery (^##P < 0.01) of cell viability in comparison with HG-induced cell mortality. Also, it was shown that the protective effect of AuNPs was more pronounced (^&P < 0.05) than Vit C (Fig. 3b). This data indicated that AuNPs can serve as potential agents in the protection of the cells against HG-stimulated stress.

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Fig. 3

Protective effect of AuNPs on HG-stimulated cytotoxicity in HK-2 cells. The cells were incubated with different concentrations (1–30 μg/ml) of AuNPs for 48 h (a). HK-2 cells were pretreated with a fixed concentration of AuNPs (5 μg/ml) for 24 h, and subsequently, the pretreated cells then were added by 50 mM glucose (HG) for an additional 24 h, and viability of cells was examined by MTT assay. Vit C (5 μg/ml) was used as the positive control. Data represent mean ± SEM. of three independent runs. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control; ^#P < 0.05, ^## P < 0.01 relative to HG- treated cells; ^&P < 0.05 relative to Vit C- treated cells.

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3.4 ROS assay

HG-stimulated intracellular ROS production was assessed in HK-2 cells using by a DCFH-DA assay. As exhibited in Fig. 4a, incubation with HG for 24 h caused an increase in DCF fluorescence intensity, which is related to the content of ROS produced. Indeed, the treatment of cells with HG significantly enhanced (***P < 0.001) intracellular ROS level in HK-2 cells (Fig. 4a) compared with the negative control cells. However, pretreatment with AuNPs potentially decreased (^###P < 0.001) HG-induced ROS production, as indicated by the lower DCF fluorescence intensity in AuNPs pretreated cells (Fig. 4a). The data from fluorescence microscope also indicated that a significant increase in the ROS level was detected in HK-2 cells treated with HG relative to the control cells, whereas pretreatment with AuNPs determined a remarkable decrease in the number of green fluorescence cells as an indicator of ROS production (Fig. 4b). Therefore, these results indicated that protective effect of AuNPs against HG-triggered ROS production was more pronounced than Vit C.

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Fig. 4

AuNPs mitigate HG-induced ROS production in HK-2 cells. The cells were pretreated with AuNPs (5 μg/ml) for 24 h, and then were incubated with 50 mM HG for another 24 h. Intracellular ROS levels (a) and the fluorescence image of cells (b) were then determined by DCF fluorescence intensity. Vit C (5 μg/ml) was used as the positive control. Data represent means ± SEM, n = 3. ***P < 0.001 relative to control; ^##P < 0.01, ^##P < 0.01 relative to HG- treated cells; ^&P < 0.05 relative to Vit C- treated cells.

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3.5 qPCR assay

To explore the protective mechanism of AuNPs on HG-induced apoptosis, the expression of Bax, Bcl-2, and Caspase-3 mRNAs were assessed by qPCR assay. Fig. 5 demonstrates the mRNA expression of Bax (Fig. 5a), Bcl-2 (Fig. 5b), and Caspase-3 (Fig. 5c) with or without AuNPs pretreatment in HK-2 cells exposed to 50 mM HG. It was observed that the pro-apoptotic Bax mRNA was remarkably increased (**P < 0.01) (Fig. 5a), while the anti-apoptotic Bcl-2 mRNA was decreased (^**P < 0.01) by HG treatment (Fig. 5b), However, pretreatment with AuNPs significantly decreased Bax mRNA (^##P < 0.01) and increased Bcl-2 mRNA (^#P < 0.05) expressions. These data indicated that AuNPs inhibited HG-stimulated apoptosis by decreasing the ratio of Bax/Bcl-2. As the alterations of Bax/Bcl-2 levels result in the initiation of Caspase-dependent signaling pathways, we examined the Caspase-3 in this study. As depicted in Fig. 5c, compared to the control group, HG triggered a remarkable increase in the expression of Caspase-3 mRNA (^**P < 0.01). However, AuNPs revealed a reduced level in the expression of Caspase-3 compared with the HG-treated group (^#P < 0.05) (Fig. 5c). Also, it was shown the protective effects of AuNPs against HG-induced apoptosis was more pronounced than Vit C. These data determined that AuNPs reduced Caspase-3 expression to inhibit HG-induced apoptosis.

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Fig. 5

AuNPs reduce HG-induced apoptosis in HK-2 cells as determined by qPCR assay. The cells were pretreated with AuNPs (5 μg/ml) for 24 h and then were incubated with 50 mM HG for another 24 h. The expression level of Bax mRNA (a), Bcl-2 mRNA (b), and Caspase-3 mRNA were then explored by qPCR assay (a). Vit C (5 μg/ml) was used as the positive control. Data represent means ± SEM, n = 3. **P < 0.01 relative to control; ^#P < 0.05, ^##P < 0.01 relative to HG- treated cells; ^&P < 0.05 relative to Vit C- treated cells.

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3.6 ELISA assay

ELISA assay was done to assess the level of AGEs and Bax, Bcl-2 and Caspase-3 proteins. Fig. 6 shows that the level of AGEs (Fig. 6a), and expression of Bax (Fig. 6b), Bcl-2 (Fig. 6c) and Caspase-3 (Fig. 6d) proteins in the absence and presence of AuNPs pretreatment in HK-2 cells exposed to 50 mM HG. It was deduced that the level of AGEs (Fig. 6a) and the pro-apoptotic Bax protein (Fig. 6b) was significantly enhanced (***P < 0.001), however the level of anti-apoptotic Bcl-2 protein was reduced (*^**P < 0.001) after treatment of the HK-2 cells with HG. However, pretreatment with AuNPs remarkably reduced the levels of AGEs (Fig. 6a) and Bax protein and increased the level of Bcl-2 protein. These results also showed that AuNPs mitigated HG-stimulated glycation of proteins and apoptosis. Furthermore, as shown in Fig. 6d, compared to the control group, HG stimulated a significant enhancement in the expression of Caspase-3 protein (^***P < 0.01). While, AuNPs induced a reduction in the level of Caspase-3 protein compared with the HG-treated sample (^##P < 0.01) (Fig. 6d). Moreover, it was revealed the protective effects of AuNPs against HG-triggered production of AGEs and apoptosis was more significant than Vit C.

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Fig. 6

AuNPs mitigate HG-induced AGEs production and apoptosis in HK-2 cells as determined by ELISA assay. The cells were pretreated with AuNPs (5 μg/ml) for 24 h and then were incubated with 50 mM HG for another 24 h. The level of AGEs production (a), expression level of Bax protein (b), Bcl-2 protein (c), and Caspase-3 protein (d) were then explored by ELISA assay. Vit C (5 μg/ml) was used as the positive control. Data represent means ± SEM, n = 3. **P < 0.01 relative to control; ^#P < 0.05, ^##P < 0.01 relative to HG- treated cells; ^&P < 0.05 relative to Vit C- treated cells.

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3.7 Investigating the mechanism of protective effects of AuNPs

To examine the molecular mechanism involved in the protective effect of AuNPs against oxidative stress and mitochondria-mediated apoptosis, activity assay relevant to the SIRT3-SOD2 was analysed (Bagul et al., 2018; Zhou et al., 2019; Tang et al., 2020). Indeed, to explore the involvement of SIRT3 in the mitigation of HG-induced HK-2 cell death and induction of apoptosis by AuNPs, the SIRT3 inhibitor 3-TYP was utilized, and enzyme activity, cell viability, ROS production, and Caspae-3 assays were measured. The outcomes indicated that the addition of 3-TYP together with HG aggravated the enzyme inactivation (Fig. 7a, b), cell mortality (Fig. 7c), ROS production (Fig. 7d), and Caspae-3 activation (Fig. 7e) and almost inhibited the protective impacts of AuNPs on HG-induced cytotoxicity in HK-2 cells. Therefore, these data determined that 3-TYP addition dramatically inhibited the AuNPs-triggered increase of SIRT3 activity and the activity of the downstream protein SOD2, indicating that AuNPs alleviated HG-stimulated HK-2 cell mitochondria-dependent ROS production and apoptosis through SIRT3-dependent pathway.

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Fig. 7

AuNPs inhibit oxidative damage and mitochondria-mediated cell damage though the SIRT3-SOD2 signaling pathway. The effect of AuNPs on the SIRT3 activity (a) and SOD2 activity (b). The influence of AuNPs and cotreatment with 3-TYP on cell viability (c), ROS production (d), and Caspase-3 activity (e). Data are presented as the means ± SD (n = 3) and the data shown with the same letters are not significantly different (P < 0.05).

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