The present study demonstrated for the first time that Falcarindiol (FAD), a natural polyacetylene compound commonly distributed in the Apiaceae species such as carrot, ameliorated kidney function in cisplatin-induced nephrotoxic mice. FAD mitigated the elevated levels of BUN and creatinine in cisplatin-treated mice, and also improved kidney injury especially tubular damage and leukocytes infiltration. Additionally, FAD exhibited anti-inflammatory and antioxidant properties through the modulation of NF-kB and Nrf2 signaling pathways, respectively.
Cisplatin is one of the most effective drugs against various solid tumors; however, it is well-known that cisplatin causes nephrotoxicity as a major side effect in dose-dependent manner. Nephrotoxicity is observed in approximately one-third of all patients that receive chemotherapeutic cisplatin. Thus, it is important to develop novel agents for alleviating this complication. Several mechanisms are attributed to this condition including DNA damage, inflammation, oxidative stress, and etc. (Oh et al., 2014, Holditch et al., 2019). We previously evaluated the efficacy of two chemical agents to reduce cisplatin-induced nephrotoxicity via the promotion of antioxidant system and the inhibition of renal inflammation (Nazari Soltan Ahmad et al., 2018, Ahmad et al., 2019). Although there are numerous studies about this issue, more researches are still needed to explore the best chemical agent with minimal toxicity.
The AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis. The active form of AMPK (phospho-AMPKαThr172) maintains normal tissues from cisplatin-mediated toxicities. AMPK controls many different cellular processes such as inflammation and oxidative stress through modulating down-stream NF-κB and Nrf2 pathways, respectively (Rashtchizadeh et al., 2019). These two main pathways play pivotal roles in the regulation of cellular inflammation and the balance between oxidant-antioxidant status. Some studies revealed that AMPK activation induces Nrf2 translocation from cytoplasm into nucleus and thereby augments its respective target genes, which are associated with antioxidant response. Meanwhile, it is indicated that activated AMPK indirectly downregulates NF-κB signaling. AMPK stimulates sirtuin1, a deacetylase enzyme that removes an acetyl group from lysine residue of NF-kB-p65 subunit, in which subsequently triggers NF-kB-p65 ubiquitination and proteasomal degradation (Mo et al., 2014, Rashtchizadeh et al., 2019).
Some investigations reported that FAD has anti-bacterial activity, and successfully inhibited the infections induced by Micrococcus luteus, Bacillus cereus and Pseudomonas aeruginosa. These findings indicate that FAD can be used as a drug candidate for treating infections (Meot-Duros et al., 2010, Zhang et al., 2020). Moreover, FAD exhibited the inhibitory effects on lipopolysaccharide (LPS)-triggered inflammatory molecules production without significant cytotoxicity in colon epithelial cells so that it reduced inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, IL-8 accompanied by an increase in the level of anti-inflammatory cytokine IL-10 (Shim et al., 2017). Similar findings were obtained in murine macrophages, and FAD compound attenuated LPS-induced pro-inflammatory cytokines including TNF-α, IL-6, IL-1β and iNOS through targeting MAPK and JAK-STAT pathways. However, FAD caused no significant alteration on LPS-stimulated activation of NF-kB and p38 pathways (Venkatesan et al., 2018). Additionally, FAD diminished NF-κβ and its respective inflammatory cytokines IL-6, TNFα, and COX-2 in colorectal neoplastic lesions obtained from rats (Kobaek-Larsen et al., 2019). In line with previous studies, we demonstrated that FAD administration declined mRNA expression of IL-1β and TNF-α together with p-NF-kB p65 protein level in cisplatin nephrotoxic mice.
Some studies indicated the ability of FAD compound to activate Nrf2/ARE signaling. In cell culture experiment, FAD provoked the activation of antioxidant enzymes, such as catalase and NAD(P)H:quinone oxidoreductase 1 (NQO-1) as a drug-metabolizing enzyme via the Nrf2/ARE pathway (Ohnuma et al., 2009). FAD can covalently alter the Cys151 residue in Keap1 protein through S-alkylation reaction, and consequently inactive it. Keap-1 traps Nrf2 protein in the cytoplasm, and also stimulates its ubiquitination and proteasomal degradation. Therefore, Keap1 inactivation by FAD stimulates Nrf2 translocation into nucleus, and binding to ARE which results in up-regulation of antioxidant enzymes (Ohnuma et al., 2010). Furthermore, it is shown that FAD treatment prevented carbon tetrachloride (CCl4)-induced hepatotoxicity through increasing the glutathione S-transferase (GST) and NQO-1 activities. FAD declined serum concentration of ALT/AST along with lipid peroxidation products in liver tissues of CCl4-treated hepatotoxic mice (Ohnuma et al., 2011). In agreement with these findings, our experiments revealed that FAD pre-treatment up-regulated NQO-1 and HO-1 mRNA expression accompanied by an increase in protein levels of nuclear Nrf2 and p-AMPK (upstream regulator of Nrf2) in kidney tissues of nephrotoxic mice. These findings suggest that FAD is a stimulator of antioxidant pathways.
Some studies demonstrated anti-cancer effects for FAD compound. FAD significantly inhibited cell growth and proliferation in different cancer types such as colorectal cancer, breast cancer, hepatocellular carcinoma (HCC) and glioblastoma cells (Jin et al., 2012, Kobaek-Larsen et al., 2017, Lu et al., 2017, Kim et al., 2018, Hong et al., 2021). FAD led to cell death through the induction of apoptosis, autophagy and endoplasmic reticulum stress. It is important to note that FAD did not interfere with cisplatin efficiency, and interestingly had synergistic effects in combination with cisplatin to suppress HCC cells (Hong et al., 2021). These results emphasize on the anti-cancer potential of FAD in addition to its anti-inflammatory and antioxidant capabilities; Nevertheless, the effects of FAD treatment on normal stem cells have not been fully evaluated yet; for example, one study exhibited deleterious effects for FAD on the neural stem cells in which impairs the balance between self-renewal and differentiation status (Kim et al., 2018).
Taken together, our results indicate that FAD ameliorated kidney injury in cisplatin nephrotoxic mice thorough down-regulation of NF-kB signaling pathway and its respective inflammatory cytokines such as TNF-α and IL-1β, as well as up-regulation of AMPK-Nrf2-ARE axis which stimulates antioxidant enzymes such as HO-1 and NQO-1. According to our results, FAD can be used clinically for the alleviation of nephrotoxicity in patients who receive cisplatin; however, further researches will be needed to clarify the effects of FAD on normal tissues homeostasis.