Cytoprotective effect and antioxidant activities of... : Journal of Advanced Pharmaceutical Technology & Research (2025)

INTRODUCTION

Cytoprotection involves shielding cells from harmful agents to maintain their function and survival, crucial in biomedical research, especially amid treatments causing cellular damage (Marcantonini et al., 2022).^[1] Cisplatin (Cisp), a common chemotherapy drug, often triggers severe side effects by harming fibroblast cells. One approach to alleviate these effects explores natural compounds like hesperidin (HSD) from citrus fruits. HSD, a flavanone glycoside, has garnered attention for its potential protective effects against oxidative stress and cellular damage (Ahmadi and Shadboorestan, 2016).^[2] This study examines how HSD shields fibroblast cells against Cisp-induced damage to uncover its therapeutic potential in reducing chemotherapy’s adverse effects.

MATERIALS AND METHODS

DPPH scavenging assay

DPPH scavenging activity was assessed as described by Siregar et al., 2019. Samples (50 µL) at 0.5–20 µM concentration in 1% dimethyl sulfoxide were added to 96-well plates, combined with 200 µL DPPH solution, and set for 30 min in darkness at room temperature. The absorption value was detected using a microplate reader at 517 nm. The activity of scavenging was reported as a percentage.

Cell culture

Professor Masashi Kawaichi of Japan’s Nara Institute of Science and Technology provided us with NIH-3T3 (passage number 3) (ATCC^® CRL-1658) fibroblast cells. These cells were cultivated under standard conditions, specifically in Dulbeccos Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (Sigma) and 1% penicillin–streptomycin (Sigma), and incubated at 37°C with 5% CO₂.

MTT cytotoxicity assay

The MTT assay was used to determine cytotoxicity. NIH-3T3 cells (1 × 10⁴) were implanted in a 96-well plate. Cells were treated with HSD and Cisp at various concentrations for 24 h. After treatment, the well was added with 100 µL of 0.5 mg/mL MTT reagent. It was incubated in 2–4 h. After incubation, a stopping solution containing 0.01N hydrochloric acid (HCl) and sodium dodecyl sulfate (SDS) was added. Cell viability was calculated using an ELISA reader by measuring the absorbance at 595 nm. IC₅₀ values were derived from linear regression analysis of sample concentration versus cell viability (Blumenthal, 2005).^[3] For IC₅₀ calculations, a nonlinear curve-fitting model, such as a sigmoid dose–response model, is recommended (Lyles et al., 2008).^[4]

Senescence-associated β-galactosidase senescence assay

The experiment included incubating cells (2 × 10⁴ per well in a 24-well plate) for 24 h. After that, it was rinsed two times using 1X phosphate-buffered saline (PBS). Subsequently, a fixative solution was inserted, and the cells were allowed to settle for a while. The cells were then rinsed again with 1X PBS. A further X-Gal solution was inserted 1–2 mL and incubated at 37°C. During 72 h, the cells were examined with a microscope (Olympus CKX41) at ×200 magnification. Green cells indicated the existence of β-galactosidase-positive cells, indicative of senescent cells (Artanti et al., 2023).^[5]

Gelatin zymography

Gelatin zymography was used to analyze matrix metalloproteinase (MMP)-9 and MMP-2 secretion. Cells (5 × 10⁵) were plated in a 12-well plate and then incubated in 24 h. They were then treated with ½ IC₅₀ of the samples, alone or combined in a serum-free medium for 24 h. The medium was collected. The samples were subjected to 10% Sodium Deodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) with 0.1% gelatin. After running in SDS buffer, they were washed in 2.5% Triton X-100 and incubated in buffer (150 mM NaCl, 50 mM Tris-HCl, and 10 mM CaCl₂) for 20 h at 37°C. Gels were stained using 0.5% Coomassie brilliant blue, destained, and scanned (Cahyono et al., 2023).^[6]

Statistical analysis

Data were analyzed for significant differences between sample concentrations using one-way ANOVA and Tukey’s test in SPSS 20.0 (IBM corporation, USA). IC₅₀ values were determined from standard curves, with P values (*P < 0.05; **P < 0.01) [Figures 1-5].

RESULTS

Antioxidant activity of hesperidin was evaluated using the DPPH assay

The antioxidant activities of HSD and ascorbic acid were found to be concentration dependent, meaning their activity increased with higher concentrations. The IC₅₀ values, as determined by the DPPH scavenging assays, were 20.96 µM for HSD and 1.46 µM for ascorbic acid [Figure 1].

Cytotoxic effect of hesperidin and its combination with cisplatin on NIH-3T3 cells

This study examines the effects of HSD on fibroblast selectivity in vitro. Cisp, a chemotherapeutic drug, can be toxic to both cancer and fibroblast cells. We assessed HSD and Cisp cytotoxicity on NIH-3T3 cells. HSD reduced cell viability in a dose-dependent manner, with an IC₅₀ of 621 µM, indicating low cytotoxicity as IC₅₀ values exceeded 500 µM. In contrast, Cisp was more cytotoxic to NIH-3T3 cells, highlighting HSD’s greater selectivity (selectivity index >2).

Detection of cellular senescence: Substantiation in NIH-3T3 cells after hesperidin treatment

To evaluate HSD’s senescence profile, we tested its effects on NIH-3T3 fibroblasts. HSD at 50 and 100 µM did not increase β-galactosidase-positive cells, indicating that it is safe for these cells. In contrast, Cisp significantly increased β-galactosidase-positive cells, reflecting its ability to induce senescence through oxidative stress (P < 0.01). When HSD was applied to Cisp-treated cells, β-galactosidase-positive cells significantly decreased (P < 0.01), suggesting that HSD has antisenescence properties and is nontoxic to fibroblasts.

Inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression following hesperidin treatment on NIH-3T3 cells

The exact mechanism by which HSD inhibits cellular senescence beyond its antioxidant effects is unclear. We investigated HSD’s impact on expression of MMP-9 in NIH-3T3 cells, focusing on MMP-2’s role in extracellular matrix (ECM) degradation, which is linked to tissue damage and aging (Cheng et al., 2017).^[7] Increased expression of MMP-2 is associated with heightened cellular senescence activity (Hassona et al., 2014).^[8] In our study, we used Cisp at 3 µM as an inducer of cellular senescence, which led to elevated MMP-2 expression, serving as a model for chemotherapy-induced senescence. The white band on the gel indicated that MMP-2, with a molecular weight of 72 kDa, effectively degraded gelatin in the gel, simulating ECM breakdown [Figure 2]. HSD at concentrations of 50 µM and 100 µM significantly reduced MMP-2 activity in NIH-3T3 cells treated with Cisp [Figure 2a and c]. Similar inhibitory effects were observed with HSD at these concentrations in NIH-3T3 cells [Figure 5b and d]. Therefore, the inhibition of MMP-2 activity may explain the cytoprotective mechanism of HSD, as it helps prevent cellular senescence.

DISCUSSION

HSD has been extensively explored for its various pharmacological benefits, and understanding the critical threshold at which HSD exerts its effects in preserving healthy cells is vital. Concentrating on this aspect, we assessed the evidence-based antisenescence properties of the physiological changes induced by HSD in normal fibroblast cells, as demonstrated by NIH-3T3 cells. Cellular senescence, frequently caused by oxidative stress, can be reduced by antioxidants, which neutralize damaging reactive oxygen species and help prevent cells from becoming senescent (Liu et al., 2022).^[9] These studies have investigated HSD as an antioxidant. The antioxidant capacity of HSD was determined to have an IC₅₀ value of 20.96 µM [Figure 1]. This level of antioxidant activity is comparable to that of ascorbic acid, which was used as a reference and has a range of 0–1100 µM. These results indicate that HSD antioxidant effects are primarily due to its ability to scavenge free radicals. This research aims to evaluate their effectiveness not only as antioxidants but also as cytoprotective agents in vitro. In addition, these compounds were assessed for their ability to neutralize free radicals and prevent cellular senescence.

Cellular senescence refers to the irreversible cell cycle arrest that contributes to aging. Senescent cells stop dividing but are alive, active in metabolism, and eventually apoptotic (Marin et al., 2023).^[10] As a result, the development of antisenescence agents with cytoprotective effects could offer a promising strategy for mitigating the aging process.

In this study, we initially conducted a cytotoxicity test on NIH-3T3 cells to represent skin tissue (Nugraheni and Ahlina, 2021).^[11] The results revealed that HSD exhibited no cytotoxic effects in either cell type, even at a concentration of 500 µM [Figure 2]. Additional observations on the cotreatment of HSD with Cisp in fibroblast cells revealed that this combination produced combination index (CI) values ranging from 58.58 to 104.53 in NIH-3T3 cells (CI >1.1) [Figure 3a and b]. This suggests that the combined treatment was more effective as a cytoprotective agent while also minimizing side effects. These findings suggest the safety of HSD, especially for fibroblast cells and skin tissue. In normal cells, an antagonistic effect is expected when combined with chemotherapy, as it may help reduce toxicity and minimize damage to healthy tissues.

Moreover, the senescence-associated β-galactosidase assay was used to evaluate the antisenescence effect in vitro. Surprisingly, a single HSD treatment did not affect senescence markers in NIH-3T3 cells. However, when combined with Cisp, HSD significantly reduced the incidence of senescence in fibroblast cell types (P < 0.05) [Figure 4]. Cisp, known to expand senescence in fibroblast cells and human cardiac (Pan et al., 2019),^[12] was used to accentuate the antisenescence potential of HSD. These results provide a more nuanced understanding of HSD’s ability to act as a senescence inhibitor, particularly in fibroblastic cells and skin tissue. In this regard, the potential of HSD as a senescence inhibitor may be related to its ability to decrease MMP expression.

MMP-2 plays a vital role in breaking down the ECM, leading to damage in various body tissues, including the skin and kidneys, which can result in premature aging and loss of function (Cheng et al., 2020).^[13] It is well documented that MMP-2 expression increases with heightened cellular senescence (Lee et al., 2018).^[14] In our study, we used 3 µM of Cisp as an inducer of cellular senescence, increasing MMP-2 and MMP-9 expression, serving as a model for chemotherapy. The appearance of white bands on the gel indicated that MMP-2 and MMP-9 were capable of degrading gelatin, representing the ECM model [Figure 3]. HSD at concentrations of 50 µM and 100 µM was effective in reducing MMP-9 activity in NIH-3T3 cells induced by Cisp [Figure 5a and c]. Similarly, HSD at the same concentrations also reduced MMP-2 activity in these cells [Figure 5b and d]. This cotreatment of HSD and Cisp also did not alter the expression levels of MMP-9 and MMP-2, indicating that this cotreatment effectively maintains the health of fibroblast cells. Antisenescence strategies often focus on inhibiting MMP activity to prevent excessive ECM degradation. By reducing MMP activity, the structural integrity of tissues is preserved, which helps prevent the onset or progression of senescence-related tissue damage. In cancer cells, HSD has been shown to inhibit nuclear factor-κB (NF-κB) activation, which can downregulate MMP expression, particularly MMP-2 and MMP-9 where MMP inhibition reduces tumor invasion, metastasis, and inflammatory cytokine production and thereby exerts anti-inflammatory effects (Aggarwal et al., 2020).^[15] HSD can modulate the MAPK pathway, affecting cell survival, proliferation, and inflammation. The PI3K (phosphoinositide 3-kinase)/Akt pathway is involved in cell survival and growth. HSD has been reported to activate this pathway, leading to enhanced cell survival and protection against oxidative stress-induced damage (Rahmani et al., 2023).^[16] These pathways collectively contribute to HSD’s antioxidant properties by regulating oxidative stress, inflammation, and cell survival mechanisms. Furthermore, the inhibition of MMP-9 and MMP-2 activities may contribute to the mechanism by which HSD exerts its cytoprotective effects by preventing cellular senescence in normal cells. This selective action makes HSD an interesting candidate for potential therapeutic applications against cancer while sparing normal cells, which is a crucial aspect in cancer treatment to minimize side effects.

CONCLUSION

In summary, HSD is safe and inhibits senescence in fibroblast cells. However, the mechanisms, especially the link between antioxidants and senescence, need further investigation. HSD’s ability to inhibit senescence suggests it could counteract aging and cytoprotective processes. HSD has demonstrated antioxidant, anti-inflammatory, and cytoprotective properties through pathways such as NF-κB, MAPK, and PI3K/Akt; direct evidence of its ability to counteract aging at a systemic level is still lacking. Many studies suggest that HSD can mitigate oxidative stress and inflammation as key contributors to aging, but this does not necessarily mean it can extend lifespan or directly influence aging pathways.

Financial support and sponsorship

The study was funded by “the Indonesian Research Collaboration” 2024 grant with contract no. 1907/UN1/DITLIT/PT, March 01, 2024, from the Ministry of Research and Technology, Republic of Indonesia.

Conflicts of interest

There are no conflicts of interest.

Acknowledgment

Acknowledgments for “the Indonesian Research Collaboration” 2024 grant with contract no. 1907/UN1/DITLIT/PT. March 01, 2024, from the Ministry of Research and Technology, Republic of Indonesia, for providing financial support for the experiment.

REFERENCES