Protodioscin mitigates cyclophosphamide-induced renal toxicity in HEK-293 cells by regulating endoplasmic reticulum stress, antioxidant defense, and apoptotic pathways

Figures

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  Fig 1. Cellular viability (MTT assay) of HEK-293 cells exposed to various concentrations of cyclophosphamide (CTX) for 24 hours.

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  Fig 2. Effects of Prot on the expression of apoptosis-related genes in CTX-exposed HEK-293 cells. Quantitative analysis of BAX (A), and BCL-2 (B). Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin), H2O2 (positive control for Apoptosis). GAPDH was used as a housekeeping gene. Results are presented as Mean ± SD of three independent experiments. *, **, ***, and **** denote statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

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  Fig 3. Effects of Prot on the expression of apoptosis-related proteins in HEK-293 cells exposed to CTX. (A)Western blot analysis. (B-D)Quantitative analysis of BAX, BCL-2 and Cleaved Caspase-3 proteins expression. Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin), H2O2 (positive control for Apoptosis). Protein levels were assessed by Western blot analysis. GAPDH was used as a calibrator protein. Results are presented as Mean ± SD of independent experiments. *, **, ***, and **** denote statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

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  Fig 4. Effects of Prot on apoptosis in CTX-exposed HEK-293 cells assessed by the TUNEL assay. (A) Representative fluorescence images of DAPI staining (blue), TUNEL-positive cells (green), and merged images. (B) Quantitative analysis of TUNEL fluorescence intensity. Experimental groups included: Control (untreated), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin), and H₂O₂ (positive control for apoptosis). Results are presented as mean ± SD of independent experiments. *, **, ***, and **** indicate statistical significance at P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

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  Fig 5. Effects of Prot on the expression of endoplasmic reticulum stress–related proteins in HEK-293 cells exposed to CTX. (A)Western blot analysis. (B-E) Quantitative analysis of ATF6, CHOP, PERK, and GRP78. Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin). Protein levels related to ER stress were assessed by Western blot analysis. GAPDH was used as a calibrator protein. Results are presented as Mean ± SD of independent experiments. *, **, ***, and **** denote statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

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  Fig 6. Effects of Prot on the expression of phosphorylated ER stress–related proteins in HEK-293 cells exposed to CTX. (A)Western blot analysis. (B-D) Quantitative analysis of cleaved ATF6, p-eIF2α, and p-PERK,. Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin).Protein levels related to ER stress were assessed by Western blot analysis. GAPDH was used as a calibrator protein. Results are presented as Mean ± SD of independent experiments. *, **, ***, and **** denote statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

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  Fig 7. Effects of Prot on Catalase (A), GPx (B), GSH (C), SOD (D), NO (E) and MDA (F) levels in HEK-293 cells exposed to CTX. Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin). Results are presented as Mean ± SD of three independent experiments. *, **, ***, and **** indicate statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

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  Fig 8. Effects of Prot t on Cystatin C (A) and KIM-1 (B) levels in HEK-293 cells exposed to CTX measured by ELISA. Control (untreated group), Prot (10 µM Protodioscin), CTX (54 µM Cyclophosphamide), CTX + Prot 5 (54 µM Cyclophosphamide + 5 µM Protodioscin), CTX + Prot 10 (54 µM Cyclophosphamide + 10 µM Protodioscin). Results are presented as Mean ± SD of three independent experiments. *, **, ***, and **** indicate statistical significance between groups at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

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  Fig 9. Graphical abstract illustrating the protective effects of protodioscin against cyclophosphamide-induced renal injury in HEK-293 cells through modulation of apoptosis, endoplasmic reticulum stress, and oxidative stress pathways.