Magnesium Lithospermate B Protects Against Cisplatin-Induced Acute Kidney Injury via Alleviating Mitochondrial Dysfunction

Figures

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  Figure 1. Mlb attenuated cisplatin-induced nephrotoxicity in vivo. (A and B) Cisplatin increased the kidney index and decreased the body weight of mice, injection of Mlb reversed this change. (C and D) Kidney samples were stained with periodic acid-Schiff (PAS) and tubular injury scores were measured. Arrowheads indicate tubular cast and necrosis. Original magnification, ×200 (left) (scale bar = 200 µm) and ×400 (right) (scale bar = 100 µm). (E) Serum creatine was measured at 72 h after cisplatin injection. (F and G) Relative mRNA levels of KIM-1 and NGAL were measured. The data were presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance, n =4–6 for RT-qPCR, n = 6 for the others.

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  Figure 2. Anti-apoptosis and anti-inflammation effect of Mlb in cisplatin-treated mice. (A) images of TUNEL assay in renal sections at 72 h after cisplatin injection. Original magnification, ×400. Green staining represents TUNEL-positive cells and blue staining indicates nuclei in the tissue. (B) Statistical analysis of TUNEL-positive cells. (C and D) Protein levels of pro-caspase3, cleaved caspase3, Bax, and Bcl-2 in the kidney. (E and F) The relative mRNA expression levels of IL-6 and TNF-α. The data were shown as the mean ± SEM, *P < 0.05, **P < 0.01, ***P <0.001, n =4–6 for RT-qPCR, n = 6 for the others.

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  Figure 3. Effect of Mlb on mitochondrial dysfunction after injection of cisplatin in vivo. (A and B) Western blot for the expression of total Drp1 and mito-Drp1. (C) Representative electron micrograph images of mitochondria in the proximal tubular cells from each group (scale bar = 1μm). The data were presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n=3.

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  Figure 4. Effect of Mlb on oxidative stress indicators. (A and B) Cisplatin exposure markedly increased the MDA level and decreased the SOD level in the kidney. These effects were reversed by the Mlb pretreatment. The data were presented as the mean ± SEM. *P < 0.05, **P < 0.01, n=6.

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  Figure 5. Mlb attenuated cisplatin-induced cytotoxicity. (A–C) Cell viability was measured by the CCK-8 on different concentrations of Mlb and cisplatin. (D) Typical images of Annexin-V/PI staining in mTECs with Mlb (200 μM) under cisplatin incubation. Evaluated by flow cytometry and represented by quantitative analysis. (E and F) Representative images of Bax, Bcl-2 and Caspase3 in samples from the control, the Mlb, the Sal+Cis and the Mlb+Cis groups. The data were presented as the mean ± SEM, *P <0.05, **P <0.01, and ***P <0.001, n=3–6.

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  Figure 6. Mlb reduced cisplatin-induced drp1 expression and translocation in vitro. (A and B) Cisplatin-induced overexpression of the Drp1 protein and Mlb incubation reversed this change. (C) Co-localization of the Drp1 and Mitotracker. More Drp1 was located on fragmented mitochondria, while the Mlb reduced Drp1 migration onto mitochondria (scale bar = 100 µm). The data was presented as the mean ± SEM, *P <0.05, **P <0.01, n=3.

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  Figure 7. Representative images and quantitative analysis indicated the mitochondrial morphology and function in the mTECs. Mitochondrial morphology, mtROS and mitochondrial membrane potential were respectively measured by the Mitotracker staining (top) (scale bar = 10 µm), the MitoSOX (middle ×400) and JC-1 assay (bottom). The data was presented as the mean ± SEM, *P <0.05, **P <0.01, and ***P <0.001, n=3.