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Sappanone A Ameliorates Cisplatin-induced Cytotoxicity Via Nrf2/HO-1 Signaling in HK-2 Cells
Guona Zheng1, Yang Liu1, Bingjie Li1
1Department of Pathology, Hebei General Hospital, Shijiazhuang, Hebei 050051, China.
Objective:
To explore the role of the Nrf2 signaling pathway in sappanone A (SA)-mediated protection against cisplatin (CP)-induced renal injury, we investigated the impact of Nrf2 knockdown on cell apoptosis, oxidative stress, NF-κB activation, and IL-1β release in human renal proximal tubular cell line HK-2 cells.
Materials And Methods:
siRNA-mediated knockdown of the Nrf2 gene was performed in HK-2 cells. Oxidative stress markers, including malondialdehyde (MDA) and hydroxyl radical (•OH), were quantified using colorimetric assays. Apoptotic cell death was evaluated by both TUNEL staining and flow cytometry. Western blot analysis was employed to detect protein expression levels of Nrf2, heme oxygenase-1 (HO-1), p65, and phosphorylated p65 (p-p65). The level of interleukin-1β (IL-1β) in cell culture supernatants was measured using ELISA.
Results:
Down-regulation of Nrf2 significantly suppresses SA's protective effect against CP-induced oxidative stress, as reflected by increased MDA and •OH as well as reduced the expression of HO-1 in HK-2 cells. Nrf2 depletion eliminates SA's capacity to reduce cell apoptosis. SA attenuates CP-induced phosphorylation of NF- κB p65 (p-p65), but this inhibition is reversed by Nrf2 depletion. ELISA analysis further demonstrates that Nrf2 knockdown elevates IL-1β secretion in HK-2 cells, indicating enhanced inflammatory response upon Nrf2 loss.
Discussion:
SA, an active compound from Caesalpinia sappan L. with antioxidant, anti-inflammatory, and anti-apoptotic properties, mitigates CP-induced nephrotoxicity in HK-2 cells by suppressing oxidative stress, inflammation, and apoptosis through activating the Nrf2 pathway (enhancing Nrf2 and HO-1 expression), as confirmed by experiments showing SA reduces biomarkers of these processes and that Nrf2 silencing abolishes such effects, aligning with classical Nrf2 activation patterns.
Conclusion:
Nrf2-mediated inhibition of oxidative stress, apoptosis, and inflammation contributes to the protective effect of Sappanone A against cisplatininduced cytotoxicity in HK-2 cells.
Insights
Sappanone A protects kidney cells from cisplatin damage by activating the Nrf2 pathway, reducing oxidative stress, apoptosis, and inflammation. Nrf2 gene silencing reversed these protective effects, confirming Nrf2
Area of Science:
- Cell Biology
- Molecular Biology
- Toxicology
Background:
- Cisplatin (CP) is a widely used chemotherapy agent with significant nephrotoxicity.
- The Nrf2 signaling pathway plays a crucial role in cellular defense against oxidative stress and inflammation.
- Sappanone A (SA), derived from Caesalpinia sappan L., exhibits potential therapeutic properties.
Purpose of the Study:
- To elucidate the role of the Nrf2 signaling pathway in SA-mediated protection against CP-induced renal injury.
- To investigate the impact of Nrf2 knockdown on apoptosis, oxidative stress, NF-κB activation, and IL-1β release in HK-2 cells.
Main Methods:
- siRNA-mediated Nrf2 gene knockdown in human renal proximal tubular HK-2 cells.
- Quantification of oxidative stress markers (MDA, •OH) and IL-1β levels.
- Assessment of apoptosis via TUNEL staining and flow cytometry.
- Western blot analysis for Nrf2, HO-1, p65, and p-p65 protein expression.
Main Results:
- Nrf2 knockdown significantly abrogated SA's protective effects against CP-induced oxidative stress and apoptosis.
- SA treatment reduced CP-induced p-p65 levels, an effect abolished by Nrf2 depletion.
- Nrf2 silencing increased IL-1β secretion, indicating heightened inflammation.
Conclusions:
- SA mitigates CP-induced nephrotoxicity by activating the Nrf2 pathway, thereby suppressing oxidative stress, apoptosis, and inflammation.
- The Nrf2 pathway is essential for the antioxidant, anti-inflammatory, and anti-apoptotic effects of SA in renal cells.
- Targeting the Nrf2 pathway may offer a therapeutic strategy to reduce cisplatin-induced kidney damage.
