Z-ligustilide reduces cisplatin-induced nephrotoxicity via activation of NRF2/HO-1 signaling pathways

Purpose: To investigate the effect of Z-ligustilide (Z-lig) on cisplatin-induced nephrotoxicity and examine whether NRF2 signaling mediates the underlying mechanism of action. Methods: Human proximal tubular epithelial cells (HK-2) were pretreated with 20 or 100 μM Z-lig for 2 h, followed by 10 μM cisplatin treatment for 24 h. Cell viability was measured using (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A commercial kit was used to determine lactate dehydrogenase (LDH) release. Apoptosis was determined by flow cytometry while Western blotting was used to evaluate protein levels. Levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) were assessed by enzymelinked immunosorbent assay (ELISA). Results: Cisplatin decreased HK-2 cell viability and increased LDH release, while Z-lig increased cell viability and decreased LDH release in a dose-dependent manner (p < 0.05). Moreover, Z-lig reduced cisplatin-induced apoptosis (p < 0.01), and alleviated cellular oxidative stress caused by cisplatin (p < 0.05). Furthermore, Z-lig activated NRF2/HO-1 signaling in cells treated with cisplatin (p < 0.05). Conclusion: Z-lig reduces cisplatin-induced nephrotoxicity via activation of NRF2/HO-1 signaling. Thus, Z-lig is a potential drug for the treatment of nephrotoxicity caused by cisplatin.


INTRODUCTION
Cisplatin is a primary chemotherapy drug used to treat various cancers [1]. Nevertheless, nephrotoxicity limits the use of cisplatin [2]. Previous studies revealed that over 30% of patients present symptoms of acute renal injury following cisplatin administration [3,4]. The mechanism of cisplatin-induced nephrotoxicity is related to many factors, such as cellular oxidative stress, mitochondrial dysfunction, and apoptosis [5]. Oxidative stress is considered to be an important factor leading to cisplatin nephrotoxicity [6]. In some cell types, Nuclear factor erythroid 2-related factor 2 (NRF2) is involved in oxidative stress [7].
Z-Ligustilide (Z-lig), the main phthalide component of medicinal plants in the Apiaceae (Umbelliferae) family, exerts protective effects in diseases of the nervous system [13]. For example, Z-lig decreased cerebral infarct size and brain swelling in rats with focal ischemic injury [13].
Moreover, Wu et al found that Z-lig alleviated oxidative stress induced by ultraviolet B irradiation, reduced release of inflammatory factors via activation of NRF2/HO-1 signaling, and suppressed the Nuclear Factor Kappa B (NF-κB) pathway [14]. These results imply that Zlig also functions as an NRF2 activator. Therefore, Z-lig may alleviate cisplatin-induced nephrotoxicity via NRF2 signaling.
Hence, the goals of the present study were to determine effects of Z-lig on cisplatin-induced nephrotoxicity, and to investigate whether NRF2 signaling mediates its underlying mechanism of action.

Cell culture
Cells of the human proximal tubular epithelial cell line HK-2 were obtained from American Type Culture Collection, and maintained on Keratinocyte Serum Free Medium plus 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Grand Island, NY, USA), in a 5% CO2 atmosphere at 37°C. Cells were pretreated with 20 or 100 µM Z-lig for 2 h, followed by 10 μM cisplatin treatment for 24 h.

Assessment of lactate dehydrogenase (LDH) release
LDH release from HK-2 cells after cisplatin or Zlig treatments was determined using the CyQUANT LDH Cytotoxicity Assay (Thermo Fisher, Waltham, MA, USA), according to the protocols of the manufacturer. Absorbance at 490 nm was determined using a microplate reader.

Flow cytometry
After cisplatin or Z-lig treatments, cells were suspended, centrifuged, resuspended in 1× binding buffer, and stained with 5 μL of Annexin V-FITC and 10 μL of PI (BD, San Jose, CA, USA) for 15 min. The proportions of apoptotic cells were determined using flow cytometry (BD, San Jose, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)
After treatment of cells with cisplatin or Z-lig and harvesting, levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) were determined using ELISA assay kits (R & D Systems, Minneapolis, MN, USA), following the protocols of the manufacturer.

Statistical analysis
The SPSS statistics software (Chicago, IL, USA) was used for statistical analysis of the data. Data are presented as mean ± standard deviation (SD). The differences among multiple groups were determined by one-way ANOVA. Differences were considered statistically significant at p < 0.05.

Z-lig reduces cisplatin-induced HK-2 cell injury
To elucidate the effect of Z-lig on nephrotoxicity caused by cisplatin, we first assessed cell viability and LDH release by HK-2 cells that had been treated with Z-lig or cisplatin. The chemical structure of Z-lig is shown in Figure 1 A. Cisplatin significantly reduced cell viability (p < 0.01,

Z-lig modulates the NRF2/HO-1 signaling pathway in cisplatin-treated HK-2 cells
To explain the mechanism of modulation of cisplatin-induced cell injury by Z-lig, we determined apoptosis, cellular oxidative stress, and changes in the activation of NRF2/HO-1 signaling in cells treated with Z-lig or cisplatin.

DISCUSSION
Cisplatin is a primary chemotherapy drug for the treatment of various cancers. However, more than 30% of patients present acute renal injury symptoms following cisplatin treatment [1,3]. Therefore, nephrotoxicity induced by cisplatin limits its use in cancer chemotherapy. Hence, it is essential to search for effective drugs to prevent nephrotoxicity.
The present study investigated the protective effect of Z-ligustilide, a phthalide component of medicinal plants in the family Apiaceae (Umbelliferae), on cisplatin-induced nephrotoxicity. We found that Z-lig reversed both the loss of viability and increase in LDH leakage induced in HK-2 cells by cisplatin, consistent with previous studies [15,16]. For instance, research by Wu et al also revealed that Z-lig enhanced cell viability and reduced LDH release in neurons subjected to oxygen-glucose deprivation [15]. Therefore, Z-lig reduces cellular injury caused by cisplatin.
Because apoptosis is among the causes of cisplatin-induced neurotoxicity, we studied the effects of Z-lig on apoptosis induced in HK-2 cells by cisplatin and found that Z-lig reduced cisplatin-induced apoptosis in these cells. Antiapoptotic effects of Z-lig were also proven in neurons treated with Abeta25-35 [17]. In a study conducted by Zhang et al, Z-lig reversed the decrease in neuron viability and the increase in apoptosis caused by Abeta25-35, and prevented Abeta25-35 cytotoxicity [17]. Furthermore, a study by Bunel et al comparing protection against cisplatin toxicity by ferulic acid, Z-lig, and E-lig revealed that Z-lig reduced cisplatin-induced apoptosis [18].
Oxidative stress has been regarded as the critical factor leading to cisplatin nephrotoxicity [6]. The effect of Z-lig in modulating oxidative stress was investigated in a previous study, in which levels of primary indicators of oxidative stress were determined, including MDA, SOD, GSH, and GSH-Px [19]. Z-Lig reversed the cisplatin-induced increase in MDA formation, the reduced GSH level, and the decreased expression of SOD and GSH-Px in that study, in accordance with other previous reports [14,20].
Z-lig also functions as an antioxidant in ischemic brain tissues, suppressing MDA content and elevating SOD, GSH, and GSH-Px [20]. In addition, Wu et al concluded that Z-lig attenuates oxidative stress induced by ultraviolet B [14]. Thus, these results are consistent with reduction of cisplatin-induced oxidative stress by Z-lig.
NRF2 signaling is known to be involved in oxidative stress [21,22], and activation of this signaling pathway alleviates cisplatin-induced nephrotoxicity [7,9]. To study the underlying mechanism of Z-lig protection against cisplatin nephrotoxicity, we investigated activation of NRF2 signaling in cisplatin-treated HK-2 cells and showed that Z-lig activated the NRF2/HO-1 signaling pathway, a result consistent with previous studies [23,24].

CONCLUSION
Z-lig attenuates cisplatin-induced nephrotoxicity via activation of NRF2/HO-1 signaling. Thus, Zlig is a potential drug for the therapy of nephrotoxicity caused by cisplatin. However, further studies are required to strengthen this assertion.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

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