A Hydrodistillate of Gynostemma pentaphyllum and Damulin B Prevent Cisplatin-Induced Nephrotoxicity In Vitro and In Vivo via Regulation of AMPKα1 Transcription

Figures

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  Figure 1. Preventive effects of GPHD on cisplatin-induced cytotoxicity in HEK293 cells. HEK293 cells were treated with the indicated concentration of cisplatin and/or GPHD for 24 h, then cell viability assays (A,B), FACS analysis of 7-AAD and annexin V double-positive stained cells (C), and Western blotting (D) were performed. The results shown are representative of the three independent experiments. p-value < 0.05 was considered statistically significant; individual p-values (* p < 0.05; ** p < 0.01) are indicated in figures.

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  Figure 2. GPHD effectively prevents cisplatin-induced inhibition of AMPKα1 transcription. HEK293 cells were treated with cisplatin and/or GPHD for 24 h, and then Western blot analysis or RT-PCR was performed (A,C). HEK293 cells were transfected with a pGL3 luciferase reporter vector containing a human AMPKα1 promoter (~1.7 kb). After the indicated treatment for 24 h, luciferase activity was measured (B,D). WB, Western blotting; RT-PCR, reverse transcriptase-polymerase chain reaction. The results shown are representative of the three independent experiments. p-value < 0.05 was considered statistically significant; individual p-values (** p < 0.01) are indicated in figures.

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  Figure 3. AMPKα1 plays a critical role in cell survival in the context of cisplatin and GPHD treatment. HEK293 cells were transfected with an expression vector for HA-tagged AMPKα1 (A,B) or siRNA for AMPKα1 (C,D), and then were treated with cisplatin and/or GPHD for 24 h. Apoptosis was analyzed by Western blotting (A,C) and FACS analysis (B,D). The results shown are representative of the three independent experiments. p-value < 0.05 was considered statistically significant; individual p-values (* p < 0.05; ** p < 0.01) are indicated in figures.

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  Figure 4. GPHD suppresses cisplatin-generated ROS and ROS-induced apoptosis via AMPKα1, a cellular anti-oxidant factor. HEK293 cells were treated with cisplatin (20 μM) and/or NAC (10 μM) for 24 h. Thereafter, intracellular ROS levels were measured (A), and FACS analysis of 7-AAD and annexin V double-positive cells (B), Western blot analysis (C), and luciferase reporter assays for AMPKα1 promoter activity (D), were performed. HEK293 cells were transfected with an expression vector for HA-tagged AMPKα1 (E) or siRNA for AMPKα1 (F), then treated with cisplatin and/or GPHD for 24 h, after which cellular ROS levels were measured. The results shown are representative of the three independent experiments. p-value < 0.05 was considered statistically significant; individual p-values (** p < 0.01) are indicated in figures.

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  Figure 5. Effects of GPHD on cisplatin-induced AKI in mice. Four-week-old mice were fed a normal diet and GPDH or distilled water (~0.9 mL/d) for 28 days prior to intraperitoneal injection of cisplatin (20 mg/kg). Four days after cisplatin injection, mice were sacrificed (A). Mice were weighed (B), blood urea nitrogen (BUN), creatinine, neutrophil gelatinase-associated lipocalin (NGAL) in serum were measured (C), tissues were histologically examined by H&E, PAS, and TUNEL staining (D), and kidney tissue extracts were analyzed by Western blotting (E). p-value < 0.05 was considered statistically significant; individual p-values (* p < 0.05; ** p < 0.01) are indicated in figures.

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  Figure 6. LC-MS spectrum of GPHD. GPHD was analyzed by UHPLC-Q/TOF MS in negative ion mode. Several types of gypenosides, hydroxylated dammarane-type glycosides, and damulins were detected as major components and tentatively identified based on their LC elution order and exact mass measurements.

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  Figure 7. Damulin B protects HEK293 cells from cisplatin-induced cytotoxicity. HEK293 cells were treated with the indicated concentration of damulin B or gypenoside XLVI or XVII in the presence or absence of cisplatin for 24 h, and then analyzed. (A,B) Cell viability assay; (C) apoptosis assay; (D) Western blotting and RT-PCR analysis; (E), cellular ROS level; (F) luciferase assay. The results shown are representative of the three independent experiments. p-value < 0.05 was considered statistically significant; individual p-values (* p < 0.05; ** p < 0.01) are indicated in figures.

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  Figure 8. Damulin B prevents cisplatin-induced AKI in mice. Mice were intraperitoneally injected with damulin B (25 or 50 mg/kg) or vehicle every day prior to intraperitoneal injection of cisplatin (20 mg/kg). Four days after cisplatin injection, animals were sacrificed. (A) Serum BUN, creatinine, and NGAL levels; (B) histological examination of kidney tissue via H&E, PAS, and TUNEL staining; (C) Western blot analysis of kidney extracts. p-value < 0.05 was considered statistically significant; individual p-values (* p < 0.05; ** p < 0.01) are indicated in figures.