Abstract
Objectives: This study aimed to investigate the protective effect of gallic acid (GA) against cyclophosphamide (CYP)-induced lung toxicity with exploring the mechanism of CYP cytotoxicity using normal human lung cells, WI-38 cell line. Methods: WI-38 cells have been categorized into three groups; first group was considered as control, untreated, cells; CYP-group, cells were treated with IC50 concentration of CYP for 24 h; GA+CYP group, cells were pretreated with a selected dose of GA for 24 h then followed by CYP for additional 24 h. After incubation times, cells were collected to evaluate cell viability by trypan blue assay, cell morphology and nuclear condensation by DAPI stain and apoptosis was assessed by AO/EB staining. Lipid peroxidation and total antioxidant capacity were determined in cell lysate biochemically. ELISA technique was applied to determine inflammatory markers (TNF-α and IL-1β), total caspase-3 and LC3-B. RAGE, NF-κB, and Beclin-1 mRNA-expressions were estimated by qRT-PCR. Results: WI-38 cells treated with CYP showed increased cell death by both necrosis and apoptosis with elevated caspase-3 level. Additionally, RAGE expression was up regulated accompanied with activated inflammatory cascade and autophagy. While, GA pretreatment suppressed oxidative stress, inflammatory and autophagy signaling induced by CYP. Conclusion: GA is considered as a promising natural therapeutic option that can halt oxidative damage and RAGE expression activation induced by CYP in lung cells thus preventing lung injury.
Highlights
Cyclophosphamide (CYP), an alkylating agent, is a widely used chemotherapeutic and immunesuppressive agent in treating different solid tumors and leukemias as well as many autoimmune disorders 1
The cytoprotective effect of gallic acid (GA) was screened by trypan blue exclusion assay, since CYP treatment reduced the percentage of viable cells to 52.1% ± 3.4 when compared with control group; pretreatment of WI-38 cells with GA, 25 μM, for 24 h before CYP significantly reduced CYP-induced cytotoxicity and cell viability was significantly enhanced to 82.6% ± 3.8 when compared with CYP group
Acridine orange/ethidium bromide (AO/EB) staining showed that CYP induced cell death by both apoptosis and necrosis when compared with control cells
Summary
Cyclophosphamide (CYP), an alkylating agent, is a widely used chemotherapeutic and immunesuppressive agent in treating different solid tumors and leukemias as well as many autoimmune disorders 1. CYP is a prodrug that is metabolized in liver to a toxic metabolite, acrolein, which is responsible for different organs toxicities via activating oxidative damage/inflammatory signaling. The receptor for advanced glycation endproducts (RAGE) activation plays an important role in inducing inflammatory signals related to CYP 5. Acts as a counter-receptor for various molecules such as damage-associated molecular patterns (DAMPs) that are released in response to injured and dying cells due to stressful conditions, playing a crucial role in modulating the lung injury response 6,7. As a result of Nuclear factor-κB (NF-κB) translocation, excessive inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) can be released in response to RAGE activation causing an extreme tissue damage 8
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