The Protective Effect of Marsdenia tenacissima against Cisplatin-Induced Nephrotoxicity Mediated by Inhibiting Oxidative Stress, Inflammation, and Apoptosis

Abstract

Cisplatin (Cis) is considered to be one of the most effective drugs for killing cancer cells and remains a first-line chemotherapeutic agent. However, Cis's multiple toxicities (especially nephrotoxicity) have limited its clinical use. Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), a traditional Chinese medicine (TCM) employed extensively in China, not only enhances the antitumor effect in combination with Cis, but is also used for its detoxifying effect, as it reduces the toxic side effects of chemotherapy drugs. The aim of this study was to explore the therapeutic effect of MT on Cis-induced nephrotoxicity, along with its underlying mechanisms. In this study, liquid-mass spectrometry was performed to identify the complex composition of the extracts of MT. In addition, we measured the renal function, antioxidant enzymes, and inflammatory cytokines in mice with Cis-induced nephrotoxicity and conducted renal histology evaluations to assess renal injury. The expressions of the proteins related to antioxidant, anti-inflammatory, and apoptotic markers in renal tissues was detected by Western blotting (WB). MT treatment improved the renal function, decreased the mRNA expression of the inflammatory factors, and increased the antioxidant enzyme activity in mice. A better renal histology was observed after MT treatment. Further, MT inhibited the expression of the phospho-NFκB p65 protein/NFκB p65 protein (p-p65)/p65, phospho-inhibitor of nuclear factor kappa B kinase beta subunit/inhibitor of nuclear factor kappa B kinase beta subunit (p-IKKβ/IKKβ), Bcl-2-associated X (Bax), and Cleaved Caspase 3/Caspase 3 proteins, while the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Recombinant NADH Dehydrogenase, Quinone 1 (NQO1), and B-cell lymphoma-2 (Bcl-2) was increased. The present study showed that MT ameliorated renal injury, which mainly occurs through the regulation of the Nrf2 pathway, the NF-κB pathway, and the suppression of renal tissue apoptosis. It also suggests that MT can be used as an adjuvant to mitigate the nephrotoxicity of Cis chemotherapy.

Keywords: Marsdenia tenacissima; apoptosis; cisplatin; inflammation; nephrotoxicity; oxidative stress.

Figure 1

UPLC-QTOF-based TIC curves in positive and negative ion modes: (A) negative ion mode; (B) positive ion mode. The numbers labeled in the figure represent the order and retention times of the compounds in Table 1.

Figure 2

MT improved kidney injury in mice that were exposed to Cis: (A) kidney indices for mice; (B) CRE levels in mice serum; (C) BUN levels in mice serum; (D) tubular injury score; (E) proportion of mesangial stroma (100%); (F) HE dyeing of kidney (original enlargement 200×, top row; partial enlargement image 400×, bottom row). PAS for staining of kidney slices (original enlargement 200×, top row; partial enlargement image 400×, bottom row): 1. glomerular shrinkage; 2. tubular dilation; 3. infiltration of the kidney with inflammatory cells in the interstitium; 4. detached renal tubular epithelial cells. The results are presented as the mean ± standard deviation (SD) (n = 8). * p < 0.05 vs. Cis; ** p < 0.01 vs. Cis; # p < 0.05 vs. one-way ANOVA for vehicle control; ## p < 0.01 vs. one-way ANOVA for vehicle control.

Figure 3

The influence of MT on antioxidant capacity and mRNA levels of pro-inflammatory cytokines in the renal tissue: (A) Kidney SOD; (B) GSH-Px; (C) T-AOC; (D) CAT; (E) MDA; (F) IL-1β; (G) IL-6; (H) TNF-α. Data are presented as the mean ± standard deviation (SD) (n = 8). * p < 0.05 vs. Cis; ** p < 0.05 vs. Cis; # p < 0.05 vs. vehicle control by one-way ANOVA.

Figure 4

The activation of the Nrf2-mediated antioxidant response was initiated by MT. (A) The expression of Nrf2, NQO1, and HO-1 was detected in the kidney by Western blot analysis; (B) Nrf2 protein expression; (C) HO-1 protein expression; and (D) NQO1 protein expression. Data are presented as the mean ± standard deviation (SD) (n = 3). * p < 0.05 vs. Cis; # p < 0.05 vs. vehicle control by one-way ANOVA.

Figure 5

Nrf2 protein was expressed in the nucleus and cytoplasm after MT intervention. (A) The expression of Nrf2 in the nucleus and cytoplasm of the kidney was detected by Western blot analysis; (B) nucleus Nrf2 protein expression levels; and (C) cytoplasm Nrf2 protein expression levels. Data are presented as mean ± standard deviation (SD) (n = 3). * p < 0.05 vs. Cis; # p < 0.05 vs. vehicle control by One-way ANOVA.

Figure 6

MT suppresses the NF-κB pathway in cis-induced nephrotoxic mice. (A) WB analysis of the expression of p65, p-p65; IKK β and p-IKK β in the kidney; (B) p-p65/p65 protein expression; and (C) p-IKK β/IKK β protein expression. Data are presented as the mean ± standard deviation (SD) (n = 3). * p < 0.05 vs. Cis; # p < 0.05 vs. vehicle control by one-way ANOVA.

Figure 7

MT decreased apoptosis in Cis-induced nephrotoxic mice. (A) WB analysis of the expression of Bax, Bcl-2, caspase 3, and cleaved caspase 3 in the kidney; (B) Bax protein expression; (C) Bcl-2 protein expression; and (D) cleaved caspase 3/caspase 3 protein expression. Data are presented as mean ± standard deviation (SD) (n = 3). * p < 0.05 vs. Cis; # p < 0.05 vs. vehicle control by one-way ANOVA.