A Phycoerythrin-SOD Fluorescent Probe Enables Detection of Oxidative Stress for Assessing Astaxanthin in NAFLD

TL;DR

A new fluorescent probe detects oxidative stress in liver disease and shows that astaxanthin can reduce liver damage.

Contribution

A novel Phycoerythrin-SOD fluorescent probe was developed for real-time SOD activity monitoring in NAFLD.

Findings

The Phycoerythrin-SOD probe effectively detected SOD activity in NAFLD models.

Astaxanthin reduced triglyceride and cholesterol levels in NAFLD-affected cells.

Astaxanthin restored 60% of SOD activity to control levels in NAFLD cells.

Abstract

Objective: To develop a superoxide dismutase (SOD) fluorescent detection probe based on Phycoerythrin (PE) from Porphyridium cruentum for real-time monitoring of SOD activity, a core biomarker of oxidative stress, in a nonalcoholic fatty liver disease (NAFLD) model, and to explore the regulatory effect of astaxanthin. Methods: Phycoerythrin and SOD were covalently coupled using the heterobifunctional cross-linker N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and the probe concentration and incubation time were optimized. A NAFLD model was established in HepG2 cells induced by free fatty acids (FFAs). The fluorescence intensity of the probe was detected by flow cytometry, and the intervention effect of astaxanthin was evaluated by measuring triglyceride (TG)/total cholesterol (TC) contents and SOD activity. Results: The optimal conditions for the Phycoerythrin-SOD probe were determined. Astaxanthin at 20 μM significantly reduced FFA-induced TG (56.8%) and TC (63.6%) contents and restored SOD activity to 60% of that in the control group. Conclusion: The Phycoerythrin-SOD probe serves as an efficient tool for dynamic monitoring of SOD activity in NAFLD. Astaxanthin alleviates liver injury by multi-target regulation of lipid metabolism and antioxidant pathways.

Figures

Number of substituents introduced by Phycoerythrin at different concentrations of SPDP. Note: Data are presented as mean ± SD (n = 3 independent derivatization reactions).

Effect of SPDP concentration on SOD antibody titer. Note: Data are presented as mean ± SD (n = 3 independent enzyme activity assays).

Shows the multi-parameter normalized comparison after treatment with 10 μM and 20 μM astaxanthin. Note: The radar chart is plotted based on the normalized mean values from three independent experiments (data from Figure 8 and Figure 9).

Full text

1. Introduction

Fluorescent probe technology, with its high sensitivity and monitoring capabilities, has become an indispensable tool in the biomedical field [1]. It is widely used in protein–protein interaction analysis [2], cellular signaling pathway tracing [3], and disease marker detection [4], among other areas. In the study of the tumor microenvironment, fluorescent probes are used to monitor real-time changes in pH and reactive oxygen species (ROS) dynamics, providing a basis for early cancer diagnosis [5,6]. In immunoassays, fluorescently labeled antibodies can precisely locate antigen expression [7], accelerating the development of precision medicine [8]. Traditional probes (e.g., FITC [9] and rhodamine derivatives [10]) have major drawbacks, including background signal interference caused by non-specific adsorption [11]; poor photostability results in photobleaching upon prolonged excitation, limiting the continuity of dynamic monitoring [12]; and some probes have cytotoxicity, affecting the reliability of experiments [13]. Therefore, the development of novel fluorescent probes has become a research hotspot in recent years. Phycoerythrin (PE), a natural fluorescent protein extracted from red algae, has become an ideal alternative to traditional probes due to its high molar extinction coefficient [14], high fluorescence quantum yield [15], and large Stokes shift, which can significantly reduce background interference. However, how to efficiently conjugate PE with functional molecules (such as enzymes, antibodies) through chemical modification while maintaining its optical properties and biological activity remains a technical challenge [16].

Non-alcoholic fatty liver disease (NAFLD), as the most common chronic liver disease globally, initiates its pathological process with excessive lipid deposition in hepatocytes [17], which subsequently triggers oxidative stress [18], mitochondrial dysfunction, and inflammatory responses, ultimately leading to liver fibrosis and even liver cancer [19]. The excessive production of reactive oxygen species (ROS) and the imbalance of the antioxidant defense system form a vicious cycle, accelerating hepatocyte apoptosis [20]. Studies have shown that SOD, as the core enzyme for scavenging superoxide anions, exhibits a significant negative correlation between its decreased activity and the level of lipid peroxidation in the livers of NAFLD patients [21,22]. Existing methods for detecting SOD activity have obvious limitations, including cumbersome operational steps, inability to monitor real-time dynamic changes within cells, and insufficient sensitivity [23,24], which cannot meet the needs of NAFLD mechanism research. Therefore, the development of a highly sensitive probe capable of real-time, in situ monitoring of SOD activity, which is a pivotal event in oxidative stress, is of great significance for elucidating the spatiotemporal dynamics of this key process in NAFLD.

In this study, a fluorescent probe combining phycoerythrin and superoxide dismutase (SOD) was prepared, combined with a fatty acid (FFA)-induced cell model of nonalcoholic fatty liver disease (NAFLD), and the regulatory effect of astaxanthin on oxidative stress response and lipid metabolism was discussed. Through the optimization of probe coupling conditions, the dynamic monitoring of SOD activity was realized. Astaxanthin can restore the activity of SOD and reduce the accumulation of lipids in cells. The preparation of fluorescent probes and the application of astaxanthin have opened up a new avenue for the precise diagnosis and treatment of NAFLD and the research and development of new drugs.

2. Materials and Methods

2.1. Experimental Materials

HepG2 human hepatoma cell line (provided by Yuchi Shanghai Cell Bank, Shanghai, China); astaxanthin (Beijing Solarbio Technology Co., Ltd., Beijing, China), DMEM high-glucose medium (Beijing Solarbio Technology Co., Ltd., Beijing, China), CCK-8 kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), PBS buffer (Beijing Solarbio Technology Co., Ltd., Beijing, China); triglyceride (TG) assay kit, total cholesterol (TCH/T-CHO) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); oleic acid (Sigma, St. Louis, MO, USA), silymarin (Sigma, St. Louis, MO, USA), palmitic acid (Sigma, St. Louis, MO, USA); SPDP (Beijing Solarbio Technology Co., Ltd., Beijing, China); phycoerythrin (PE purity > 4) was prepared in the early stages by our research group. Superoxide dismutase (SOD, Mn-SOD from human erythrocytes, Sigma-Aldrich, Catalog No. S7571, St. Louis, MO, USA) was used in this study. The enzyme exhibits an activity of ≥3000 units/mg protein and was dissolved in PBS for further coupling.

2.2. Construction and Optimization of Phycoerythrin-SOD Probes

2.2.1. Optimization of the Molar Ratio of SPDP to Phycoerythrin

High-purity Phycoerythrin was desalted, and its buffer system was exchanged using a solution containing 20 mmol/L PBS and ethylenediaminetetraacetic acid (EDTA). SPDP was dissolved in dimethyl sulfoxide (DMSO) to prepare solutions of different molar concentrations. A 0.9 mL aliquot of 0.05 mg/mL Phycoerythrin solution was selected and divided equally into nine portions of 500 µL each, placed in nine centrifuge tubes. The volume of the solution in each centrifuge tube was adjusted to 1 mL with 20 mmol/L PBS-EDTA. Ten microliters of SPDP at different molar concentrations were added to create molar ratios of SPDP to Phycoerythrin of 0 (control group), 5, 10, 20, 40, 80, 160, 320, and 640. The solutions in the centrifuge tubes were oscillated in the dark at room temperature for 2 h. Unreacted SPDP was removed by ultrafiltration centrifugation. The solution was diluted 40 times with 20 mmol/L PBS-EDTA, and the absorption spectrum and fluorescence spectrum of the Phycoerythrin solution in each tube were measured.

2.2.2. The Effect of Different Molar Ratios of SPDP on the Derivatization Efficiency or Degree of Phycoerythrin

After the protein reacts chemically with the heterobifunctional cross-linker SPDP, unreacted SPDP is removed by ultrafiltration to obtain the modified protein-PDP derivative. The formation of the derivative indicates the successful introduction of the 2-pyridylthiol group onto the protein molecule. Under specific experimental conditions, dithiothreitol (DTT) can selectively cleave the disulfide bond in the 2-pyridylthiol group, resulting in thiol modification of the protein and the release of pyridine-2-thione. Pyridine-2-thione has unique absorption characteristics at a wavelength of 343 nm, with a molar absorptivity as high as 8.08 × 10^3^. The absorbance values of the samples at a wavelength of 343 nm before and after the addition of DTT (labeled as OD343I and OD343II, respectively) are measured to quantify the molar concentration of the released pyridine-2-thione. This, in turn, assesses the degree of thiol modification of the protein.

$$[eqn]$$

When the protein concentration X is known, the number of SPDP groups substituted on the protein, S, can be calculated as S = Y/X.

The initial absorbance value of each reaction solution at a wavelength of 343 nm is measured using a spectrophotometer and recorded as OD343I. Twenty-five microliters of DTT at a concentration of 150 mmol/L is added to each reaction solution, and the solutions are allowed to react in the dark at room temperature for 30 min. The absorbance value of each reaction solution at a wavelength of 343 nm is then measured and recorded as OD343II.

2.2.3. Optimization of the Molar Ratio of SPDP to SOD

Using SOD at a concentration of 0.3 mg/mL, DMSO was used as the solvent to prepare SPDP solutions of different molar concentrations. Seven aliquots of 200 µL were taken from a 2.1 mL solution of 0.3 mg/mL SOD. SPDP solutions of different concentrations were added to the centrifuge tubes to ensure molar ratios of SPDP to SOD of 0 (control group), 25, 50, 100, 200, 400, and 800, respectively. The reactions were allowed to proceed at room temperature for 2 h. After the reactions, unbound SPDP was removed by ultrafiltration centrifugation, and the solutions in each tube were adjusted in volume and diluted for subsequent enzyme activity detection, with a dilution factor of 3000. The activity of SOD was assessed using an indirect enzyme-linked immunosorbent assay (ELISA) method. It should be noted that the above optimization is based on the assessment of the relative activity of modified SOD using the ELISA method. Since the PE-SOD complex is more relevant to subsequent cell experimental systems, and may have physical or optical interference with certain in vitro enzyme activity detection systems, this study did not directly compare the specific activity of the final PE-SOD complex. Its functional validation was mainly carried out through specific responses in subsequent cell experiments.

2.2.4. Screening of Probe Concentration and Incubation Time

The HepG2 cells were seeded in a 96-well culture plate and cultured for 24 h to allow for adherence. The medium was then replaced with serum-free medium containing different concentrations of fluorescent probes (0 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL) and incubated in the dark for 15 min, 30 min, 60 min, and 120 min. After incubation, the liquid was discarded and the cells were washed twice with PBS buffer. A medium containing 10% CCK-8 reagent was added and incubated for 2 h, followed by measurement of optical density (OD) at a wavelength of 450 nm. The average fluorescence intensity of each group of cells was detected and analyzed using flow cytometry.

2.3. Construction and Validation of NAFLD Model

2.3.1. Optimal Concentration of FFA Induces HepG2 Cells

Preparation of FFA mixture: Accurately weigh 25.6 mg of palmitic acid (PA) and 9.5 mg of oleic acid (OA), dissolve them in 1 mL of absolute ethanol, and oscillate in a 55 °C water bath until completely dissolved. Mix the FFA ethanol solution with a 10% BSA solution (without fatty acids) in a volume ratio of 1:9, and oscillate at 37 °C for 1 h to form FFA-BSA complexes. Filter the solution through a 0.22 μm membrane to remove bacteria, and store it in aliquots at −20 °C (avoid repeated freezing and thawing).

Model Induction: To investigate the optimal concentration of FFA (free fatty acid inducer), this study set up FFA solutions with different concentrations: 0 mM (control), 0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM, and 2.0 mM FFA (with a ratio of PA:OA = 2:1). Each concentration had three replicate wells, and all groups were cultured at 37 °C under 5% CO_2_ conditions for 24 h.

2.3.2. Verification of the Model

Oil Red O absorbance value: First, the cells were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Then, they were stained with 0.5% Oil Red O (prepared in isopropanol) for 30 min and washed with PBS to remove excess dye. Finally, the stained lipid droplets were dissolved in isopropanol, and the absorbance was measured at OD510 nm.

Determination of TG/TC content: Operate according to the instructions of the reagent kit and quantify using the standard curve.

2.4. Astaxanthin Intervention Experiments

Drug Treatment: Cells were incubated with astaxanthin at concentrations of 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM for 24 h. Positive control group: Cells were incubated with silymarin at concentrations of 25 μM, 50 μM, and 75 μM for 24 h. Model control group: Cells were only exposed to the culture medium. After drug treatment, the cell culture medium was removed, and the cells were gently washed twice with pre-cooled PBS solution to prevent cell detachment. One milliliter of serum-free medium containing 10 μg/mL PE-SOD fluorescent probe was added to each well, and the cells were incubated at 37 °C in the dark for 1 h. After incubation, the probe-containing medium was removed, and the cells were washed three times with PBS for 5 min each time to thoroughly remove unbound probes. The cells were then digested with 0.25% trypsin, centrifuged at 800 rpm for 5 min, and finally resuspended in PBS. Flow cytometry was used with an excitation wavelength of 488 nm and an emission wavelength of 575 nm to detect 10,000 cells per sample, and the mean fluorescence intensity (MFI) was recorded. It should be noted that in order to establish the absolute specificity of the PE-SOD probe signal for SOD enzyme activity, an ideal validation should include a competitive blocking experiment using SOD-specific inhibitors. Due to the limited research period, this crucial specificity validation experiment has not yet been completed, which is a limitation of this study and will be prioritized in future work.

2.5. Data Analysis

All data are expressed as mean ± SD. Statistical analyses were performed using SPSS 26.0. One-way ANOVA with Tukey’s post hoc test was applied for multiple comparisons, and Student’s t-test for pairwise analysis. p < 0.05 was considered significant. The ‘n’ number represents independent biological replicates unless otherwise specified as technical replicates in the figure legends.

3. Results

3.1. Construction and Optimization of Phycoerythrin-SOD Probes

3.1.1. Optimization of the Molar Ratio of SPDP to Phycoerythrin

The fluorescence properties of Phycoerythrin are highly sensitive to changes in SPDP concentration. As shown in Figure 1a, a significant change in the UV absorption peak of Phycoerythrin can be observed after different moles of SPDP solution are added to the Phycoerythrin solution. With the increase in the molar ratio of SPDP to Phycoerythrin, the original pink color of Phycoerythrin gradually weakened. At 565 nm, the absorbance of Phycoerythrin decreases gradually, and the position of the absorption peak shifts to the short band (blue-shift). There was also a slight decrease in absorbance at 490 nm, but no blue shift in the absorption peaks was observed. When the molar ratio of SPDP to Phycoerythrin reached 80, the absorbance at 565 nm wavelength decreased by about 25% compared with that of the blank control group. When the molar ratio increases to 640, the absorbance at 565 nm decreases by about 40% and the characteristic absorption peak becomes indistinct, indicating that the protein may have been significantly denatured. As shown in Figure 1b, when the molar ratio of SPDP to Phycoerythrin was less than 160, the color of the solution remained pink. When the molar ratio exceeds 160, the pink color of the solution gradually fades. When the molar ratio reaches 640, the pink color of the solution almost completely disappears, indicating that excessively high SPDP concentrations may lead to overactivation of the reaction, which can adversely affect the structure and properties of the protein. In practical applications, the use of excessively high SPDP/Phycoerythrin molar ratios should be avoided.

3.1.2. Number of Substituents Introduced by Phycoerythrin at Different Concentrations of SPDP

In Figure 2, when the molar ratio of SPDP to Phycoerythrin was 160 to 320, the number of substituents increased significantly. From the perspective of the practical application effect, the molar ratio of SPDP to Phycoerythrin should be between 80 and 200. An excessively high molar ratio of SPDP to Phycoerythrin will lead to an excessive number of substituents on Phycoerythrin, which will interfere with its biological function or affect subsequent experimental applications. The molar ratio of SPDP to Phycoerythrin is too low, and insufficient substituents cannot be introduced, which reduces the chance of binding between thiolated Phycoerythrin and derivatized antibodies, which affects the experimental effect.

Number of substituents introduced by Phycoerythrin at different concentrations of SPDP. Note: Data are presented as mean ± SD (n = 3 independent derivatization reactions).

3.1.3. Effect of Different Molar Ratios of SPDP/SOD on SOD Titer

As can be seen from Figure 3, Experiments have shown that SPDP has a significant inhibitory effect on SOD activity, with the inhibition being positively correlated with the concentration of SPDP. The optimal concentration ratio is to choose an SPDP/SOD molar ratio of 80. This finding is of great significance for understanding the role of SPDP in biological or chemical processes. In antioxidant mechanism research or related drug development, it is necessary to consider the potential impact of SPDP on SOD activity to avoid negative effects.

Effect of SPDP concentration on SOD antibody titer. Note: Data are presented as mean ± SD (n = 3 independent enzyme activity assays).

3.2. Screening of Probe Concentration and Incubation Time

When exploring the optimal concentration of the probe, an incubation time of 60 min was first selected. The results, as shown in Figure 4a, indicated that when the probe concentration ranged from 1 to 10 μg/mL, cell viability remained at 93–98% (vs. control group) with no significant toxicity (p > 0.05). When the concentration reached ≥20 μg/mL, cell viability significantly dropped below 85% (p < 0.05), suggesting that high concentrations of the probe might damage cell membranes through physical adsorption or oxidative stress. In terms of fluorescence intensity, as shown in Figure 4b, it reached 3800 MFI at 10 μg/mL and only increased to 4500 MFI at 20 μg/mL (an increase of 18% with no statistical difference). Therefore, 10 μg/mL was selected as the optimal concentration of the probe.

Next, the incubation time was investigated using a probe concentration of 10 μg/mL. As shown in Figure 4c, when the incubation time ranged from 15 to 60 min, the fluorescence intensity increased from 1200 MFI to 3800 MFI, while cell viability remained above 93%. After extending the incubation time to 120 min, the fluorescence intensity increased only slightly (to 4000 MFI), but cell viability significantly dropped to 85% (p < 0.05). The fluorescence intensity reached a plateau at 60 min, indicating that the binding of the probe to its target was nearing saturation. Moreover, prolonged incubation in the dark might lead to inhibition of cellular metabolism. Shortening the incubation time could reduce experimental errors. Therefore, 60 min was selected as the optimal incubation time.

3.3. Construction and Validation of NAFLD Model

Figure 5 illustrates the impact of free fatty acid (FFA) concentration on the viability of HepG2 cells and the absorbance of Oil Red O. The results demonstrate a dose-dependent effect of FFA on both parameters. As the FFA concentration increases from 0.25 mM to 2 mM, there is a significant and progressive decrease in HepG2 cell viability compared to the control group. Specifically, at 0.25 nM FFA, cell viability shows a slight but noticeable reduction, and this decline becomes more pronounced at higher concentrations, with statistically significant differences (* p < 0.05 vs. control; ** p < 0.01 versus control) observed at various points. Simultaneously, the absorbance of Oil Red O, which is indicative of lipid accumulation, shows a significant increase with rising FFA concentrations. This suggests that higher FFA levels lead to increased lipid deposition in HepG2 cells. The data clearly indicate that FFA exposure negatively affects cell viability while promoting lipid accumulation in a concentration-dependent manner.

Figure 6 presents the effect of FFA concentration on triglyceride (TG) and total cholesterol (TC) concentrations. The findings reveal a positive correlation between FFA concentration and both TG and TC levels. As the FFA concentration escalates, there is a marked and significant elevation in TG and TC concentrations within the cells. At each increasing FFA concentration step, the levels of TG and TC rise substantially, with statistical significance (** p < 0.01 versus control) achieved at multiple concentrations. This indicates that FFA exposure stimulates the synthesis and accumulation of both triglycerides and total cholesterol in HepG2 cells, further supporting the notion that FFA plays a crucial role in lipid metabolism dysregulation, which is a key feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). These results collectively validate the NAFLD model constructed using FFA-induced HepG2 cells, as they replicate the characteristic lipid accumulation observed in NAFLD.

3.4. Astaxanthin Intervention Experiments

3.4.1. Effect of Astaxanthin and Silymarin on the Activity of HepG2 Cells

The results, as shown in Figure 7, indicate that astaxanthin at concentrations ranging from 1 to 20 μM had no toxic effects on the growth of HepG2 cells compared to the control group, and there were no significant differences. Therefore, astaxanthin concentrations of 1–20 μM were selected for subsequent experiments. In the positive control group, silymarin had no toxic effects on HepG2 cell growth at concentrations ≤ 50 μM, and there were no significant differences. Therefore, silymarin concentrations of ≤50 μM were selected for the experiments.

3.4.2. Regulation of Astaxanthin on Oxidative Stress and Lipid Metabolism in NAFLD

After determining the optimal concentrations of the drug and probe, the activity of SOD, an oxidative stress product, was investigated in the cell model. The results, as shown in Figure 8, indicate that astaxanthin at a concentration of 10 μM reduced SOD activity to 2450 MFI (p < 0.05), and at a concentration of 20 μM, it further decreased SOD activity to 1800 MFI (p < 0.01), restoring 60% of oxidative stress damage. This is close to the effect of silymarin (50 μM) in the positive control group, which reduced SOD activity to 1550 MFI (p < 0.01), restoring 66.7% of oxidative stress damage. Although the antioxidant potential of astaxanthin is slightly weaker than that of silymarin, it still represents a substance with great application prospects.

The effect of astaxanthin intervention was evaluated by measuring the levels of TG and TC. As shown in Figure 9, astaxanthin at a concentration of 20 μM reduced TG levels from 1.87 to 0.80 mmol/mg protein, representing a decrease of 57% (p < 0.01), and decreased TC levels from 1.21 to 0.44 mmol/mg protein, representing a decrease of 63.6% (p < 0.01). In summary, astaxanthin can simultaneously reduce the levels of TG and TC and restore SOD activity, suggesting its role in regulating metabolism and oxidative stress through multiple targets.

For better visualization, the Model group is used as the baseline (100%), and other groups are shown as a percentage relative to the Model group. As can be seen in Figure 10, The radar chart of the model group shows a clear ‘expanding outward’ pattern, reflecting high levels of oxidative stress (abnormally increased SOD activity) and severe lipid accumulation (significantly increased TG and TC levels). The radar chart for the astaxanthin treatment groups (10 μM and 20 μM) shows a trend of ‘contraction’ toward the center, indicating a significant synergistic improvement in oxidative stress and lipid metabolism disorders. Among them, 20 μM astaxanthin shows the most pronounced contraction, with the smallest polygon area, suggesting that at this concentration, astaxanthin achieves the optimal overall balance in restoring SOD activity and reducing TG and TC levels. The radar chart shapes and areas of the positive control group (PC) and the 20 μM astaxanthin group are quite similar, confirming that astaxanthin achieves a comprehensive regulatory effect comparable to that of classic drugs (such as silymarin).

Shows the multi-parameter normalized comparison after treatment with 10 μM and 20 μM astaxanthin. Note: The radar chart is plotted based on the normalized mean values from three independent experiments (data from Figure 8 and Figure 9).

4. Discussion

The Phycoerythrin-SOD fluorescent probe developed in this study provides a novel method for real-time detection of SOD activity and demonstrates significant advantages in multiple aspects. Compared to traditional fluorescent probes, the natural fluorescent properties of Phycoerythrin (PE) exhibit a strong absorption peak at 565 nm [25], with a large Stokes shift that effectively avoids overlap between excitation and emission light, reducing background interference. Small molecule probes such as FITC [26] often require complex algorithms to correct signals due to spectral overlap [27]. This probe optimizes the molar ratio of SPDP to PE (80–200:1), achieving an average of 4–6 thiolated sites per PE molecule, significantly higher than the FITC-antibody coupling efficiency reported by Liang et al. [28] (average of 3.8). While an excessively high SPDP ratio (>320:1) can increase the number of substituent groups, it leads to denaturation of the PE structure, consistent with the conclusion reported by Liu et al. [29], who reported that excessive cross-linking of proteins can induce conformational changes. This indicates the need for strict control of reaction conditions during probe preparation.

In terms of SOD activity detection, the dynamic monitoring technique proposed in this study represents a significant advancement compared to traditional methods. The xanthine oxidase method relies on complex colorimetric reactions [30], whereas this probe can directly reflect changes in SOD activity through fluorescence intensity. Although ELISA-based SOD detection has strong specificity, it cannot monitor the dynamic changes in enzyme activity in live cells in real-time [31]. In contrast, this probe can complete cell incubation and signal acquisition within 60 min, providing the possibility to study the spatiotemporal regulation of oxidative stress. Compared to chemiluminescence methods, the fluorescence signal of this probe may be interfered with by intracellular autofluorescence, which can be further optimized in the future by introducing near-infrared fluorescent labels to improve the signal-to-noise ratio.

When interpreting the results of this study, it is necessary to carefully consider the biological significance of changes in superoxide dismutase (SOD) activity. In the progression of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD), the expression and activity of SOD are regulated at multiple levels and dynamically, and changes may be related to the stage of the disease, the degree of injury, and the compensatory adaptation state of the cells [32,33]. The PE-SOD probe constructed in this study has a fluorescence signal that directly reflects the functional state of SOD enzyme molecules. In the early NAFLD cell model induced by FFA used in this paper, we observed that after astaxanthin intervention, the restoration of SOD activity signals showed a trend of synergistic improvement with the alleviation of lipid deposition (TG/TC content). This strong correlation suggests that, in this specific model context, the restoration of SOD activity is a key and real-time observable biological event contributing to the beneficial effects of astaxanthin. However, it must be noted that the SOD activity reading itself is a specific indicator, rather than a comprehensive measure of the overall ‘redox balance.’ To more definitively link this signal to improvements in cellular redox homeostasis, future work will need to include parallel detection of direct oxidative damage end-products (such as malondialdehyde and protein carbonylation) for multi-parameter combined validation.

In terms of the mechanism of action of astaxanthin, the PE-SOD probe was applied to the antioxidant and lipid metabolism of NAFLD for the first time in this study. The results showed that 20 μM astaxanthin could restore the activity of SOD to 60% of the control group and reduce the TG/TC content by more than 50%, which was close to the efficacy of the positive drug silymarin [34]. This study observed that astaxanthin can synergistically improve SOD activity and lipid metabolism, suggesting its multi-target regulatory effects. Although this study primarily focused on the application of probes and phenotypic validation, without directly investigating the molecular mechanisms, we can explore potential pathways based on the existing literature. The powerful antioxidant activity of astaxanthin has been widely attributed to its ability to activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway [35]. Nrf2 is a central regulator of the cellular antioxidant response. Once activated, it translocates to the nucleus and drives the expression of a series of downstream antioxidant enzymes and phase II detoxifying enzymes [36,37]. Therefore, we speculate that the observed restoration of SOD activity and improvement in lipid profiles in this study may be partly attributed to astaxanthin’s activation of the Nrf2 pathway, thereby enhancing the cell’s endogenous antioxidant defense system.

However, there are still many limitations: The long-term stability of the probe has not been assessed, and its half-life in serum or tissue needs to be further investigated; The specific target of astaxanthin has not yet been clarified. The HepG2 cell line used in this study, although widely applied in NAFLD research, is a liver cancer cell line, which means its metabolic and stress responses may differ from those of primary hepatocytes [38,39]. For example, the intrinsic metabolic reprogramming of cancer cells may affect their response to lipid handling and antioxidant signaling pathways. The conclusions of this study are primarily applicable to this specific cell model. The existing results are only based on cell models, and the mechanism of action of astaxanthin needs to be further explored in combination with animal experiments in the future. This experiment primarily relied on the CCK-8 kit for quantitative cell viability assays and simultaneously used flow cytometry to measure mean fluorescence intensity (MFI), without systematically capturing bright-field microscopy images.

5. Conclusions

This study developed a fluorescent probe tool capable of real-time, in situ monitoring of dynamic changes in SOD activity in NAFLD cell models through multi-step systematic optimization of probe construction, toxicity, modeling, and application at the cellular level. This tool provides a new method for in-depth study of oxidative stress mechanisms in NAFLD and for drug screening at the cellular level.