Swainsonine induces autophagy via PI3K/AKT/mTOR signaling pathway to injure the renal tubular epithelial cells
Introduction
Locoweed (it's name originated from Spanish “loco” meaning crazy) commonly refers to any plant, usually plants from the genera Astragalus and Oxytropis, which produces Swainsonine (SW), a phytotoxin which induces toxic responses in grazing livestock [1]. Currently, locoweed has been found throughout the world and has become the major toxic plant affecting the livestock production in pasturelands [2]. Consumption of locoweeds by the livestock may cause toxic responses and one salient toxic response at cellular levels is characterized by extensive cellular vacuolar degeneration of multiple tissues/organs including the epithelium of renal tubules [3], and in some cases may cause death [[4], [5], [6]]. The estimated economical loss was 20–100 million dollars which severely hampered the development of the grassland [7,8].
Swainsonine (Fig. 1), an indolizidine alkaloid, is the principal toxic component of locoweeds that is produced by fungi living within the plants [8,9]. SW which resembles mannose in chemical structure and has high affinity to the α-mannosidase and is a competitive inhibitor for the lysosomal α-mannosidase and Golgi α-mannosidase II [10,11]. The Golgiα-mannosidase II is an enzyme for N-glycosylation of proteins, that plays an important role in the formation of functional proteins. Inhibition of the α-mannosidase directly affects the synthesis of proteins leading to the dysfunction of the multiple cellular activities including physiological activity controlled by adhesion and receptors [12,13]. Inhibition of lysosomal α-mannosidase results in disruption of the metabolism of mannose leading to the accumulation of the oligosaccharides. Thus, SW has been shown to disrupt the glycoprotein formation and accumulation of oligosaccharides [[14], [15], [16]]. However, the cellular signaling processes involved in the SW-induced toxicity needs investigation.
Previous research indicated SW induces apoptosis in neuron cells [17,18], trophoblast cells [19], and the corpus luteum of goats [20]. However, xenobiotic compounds can cause both apoptosis and autophagy [21,22] and the two processes are interconnected [23,24]. For example, the apoptotic protein Bax interferes with the association between Bcl-2 and Beclin1 and induces autophagy [25], the apoptosis specific protein (ASP) gene and autophagy gene 5 (ATG5) are highly homologous, thus ATG5 plays a role in apoptotic pathway and also plays an important role in autophagy [[26], [27], [28]].
Autophagy is a process in which cells transport the damaged, denatured and aged macromolecules/organelles into lysosomes for degradation [[29], [30], [31]]. Interference with the processes of autophagy leads to pathological vacuolar degeneration. Zhang et al. [32] showed that the fluoride-induced vacuolar degeneration in testicular tissue is due to the blockage of the degradation in autophagosomes. In the Chloroquine Myopathy mouse model, there is evidence for the accumulation of the autophagosome around the vacuolar pathological lesion [33]. Additionally, the genetic disease Danon is characterized by vacuolar lesion in the muscle, and there is an extensive accumulation of the autophagosome in the muscle tissues [34,35]. The increase in the autophagosomes is the results from either increases in the formation of the autophagosome or inhibition of the degradation of the autophagy processes [36]. The pathological accumulation of the autophagosome is correlated with dysfunctions of the lysosome in the above examples.
Collectively, increasing evidence strongly suggests that autophagy may play a role in vacuolar degeneration observed in the SW-induced toxicity. Based on this, we used the mouse TCMK-1 cell line model to investigate the effects of SW on autophagy-related proteins and to delineate the mechanism of SW-induced toxic responses in the TCMK-1 cells to investigate SW-induced nephrotoxicity and manage the intoxication cured by locoweed consumption.
Section snippets
Reagents and antibodies
SW was isolated from Oxytropis kansuensis Bunge (a locoweed widely distributed in western China) and identified by interpretation of spectral data (MS, 1H NMR, 13C NMR, 2D NMR) as described previously [37]. Its purity was 98.17%. Chloroquine (CQ) and Monodansylcadaverine (MDC) were obtained from Sigma-Aldrich, USA. Beclin-1 (#3495, Rabbit, anti-mouse), SQSTM1 (p62) (#5114, Rabbit, anti-mouse), PI3K(#4249, Rabbit, anti-mouse), Akt (#4691, Rabbit, anti-mouse), mTOR (#2983, Rabbit, anti-mouse),
SW inhibits proliferation in TCMK-1 cells
SW has been shown to induce cytotoxicity in many different cell types and tissues/organs. We first determine the SW-induced cytotoxicity using a cell proliferation assay (CCK-8 kit). Briefly, cells were seeded in 96 well culture plate at 103 cell/well and proliferation assays were performed every 24 h and cell proliferation characteristics was determined without SW (Fig. 2A). The cytotoxicity due to SW was determined by measuring the dose-dependent inhibition of the cell proliferation (Fig. 2
Discussion
Swainsonine is the principal toxic component of locoweeds and it causes injury of multiple tissues/organs of grazing livestock. The extensive vacuolar degeneration is major pathological manifestation [3,38]. Because of the high resemblance between the SW and mannose in chemical structure, similar pathological manifestation between the SW poisoning and genetic lysosomal storage diseases, the SW-induced toxicity is attributed to the inhibition of the mannosidase activity leading to the
Author contributions
Conceived and designed the experiments: Hao Lu, Shuai Wang, Jinglong Wang, Qingyun Guo, Baoyu Zhao. Performed the experiments: Shuai Wang, Lin Yang, Rong Guo, Enxia Huang. Analyzed the data: Hanqi Yang, Yajing Zhang, Lu Sun, Runjie Song. Wrote the manuscript: Shuai Wang, Jingshu Chen, Yanan Tian. All authors read and approved the final manuscript.
Conflicts of interest
The authors declare that they have no competing interests.
Competing interests
The authors declare no competing financial interests.
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation of China (No. 31201958) and the Key project of Natural Science Foundation of Shaanxi province (No. 2017JZ004) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201203062) and Tibet Academy of Agricultural and Animal Husbandry Sciences.
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These authors contributed equally to this work.