Benzyl salicylate from the stems and stem barks of Cornus walteri as a nephroprotective agent against cisplatin-induced apoptotic cell death in LLC-PK1 cells

Abstract

Nephrotoxicity currently plagues the therapeutic use of cisplatin. Its major side effects are predominantly associated with renal tubular cell apoptosis. In this study, we examined the potential nephroprotective constituents from C. walteri that can exert effects against cisplatin-induced oxidative stress and apoptosis in renal tubular epithelial (LLC-PK1) cells. Phytochemical investigation of the MeOH extract of the stems and stem barks of C. walteri led to the isolation of the potential nephroprotective constituents (1-15). Among the isolates, treatment with benzyl salicylate (BS) (15) (50 and 100 μM) significantly attenuated LLC-PK1 cell apoptosis and potently blocked cisplatin-induced nuclei condensation, intracellular ROS generation, and apoptosis, as indicated by Hoechst 33342 and H₂DCFDA staining. Furthermore, the expression of apoptosis-related proteins such as MAPK, Bax/Bcl-2 protein ratio, and cleaved caspase-9 and -3, were significantly decreased by BS. These findings provide that benzyl salicylate can be a nephroprotective agent against cisplatin-induced oxidative stress and apoptosis.

Fig. 1. Chemical structures of compounds 1–14 (A) and benzyl salicylate (BS) (15) (B). Protective effects of 6β-hydroxysitostenone (HS) (8) and BS (15), and quercetin (positive control, (E)) on the viability of cisplatin-damaged LLC-PK1 cells for 24 h, evaluated via the MTT assay (C and D). *p < 0.05 vs. the cisplatin-treated cells.

Fig. 2. Effects of benzyl salicylate (BS) on cisplatin-induced morphological changes and nuclear condensation in LLC-PK1 cells. (A) Representative phase contrast microscopy images of cells. (B) Representative fluorescence microscopy images of cells stained with Hoechst 33342. Scale bar, 50 μm.

Fig. 3. Effect of BS on cisplatin-induced oxidative stress in LLC-PK1 cells. (A) Accumulation of ROS visualized with H₂DCFDA. Scale bar, 50 μm. (B) Bar graph showing the fold increase in the intracellular ROS accumulation. *p < 0.05 vs. the cisplatin-treated cells.

Fig. 4. Effect of BS on cisplatin-induced apoptosis in LLC-PK1 cells. (A) Visualization of apoptotic (green circles), live, and necrotic cells (red circles) cells (40× magnification). (B) Bar graph showing percentage of percentage of apoptotic, live, and necrotic cells. *p < 0.05 vs. cisplatin-treated cells.

Fig. 5. Effects of BS on the levels of p-JNK, p-p38, and p-ERK in LLC-PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) p-JNK, p-p38, and p-ERK detected using western blot analyses. (B) Quantitative analyses of relative protein levels. *p < 0.05 vs. the cisplatin-treated cells.

Fig. 6. Effects of BS on the levels of Bcl-2, Bax, cleaved caspase-3, -9, and -8 in LLC PK1 cells with cisplatin-induced damage. Immunoreactive bands of (A) Bcl-2, Bax, and cleaved caspase-8, -9, and -3 detected using western blot analyses. (B) Quantitative analyses of relative protein levels. *p < 0.05 vs. the cisplatin-treated cells.