Abstract
As a pair of differential isomers, Kaji-ichigoside F1 and Rosamultin are both pentacyclic triterpenoids isolated from the subterranean root of Potentilla anserina L., a plant used in folk medicine in western China as antihypoxia and anti-inflammatory treatments. We demonstrated that Kaji-ichigoside F1 and Rosamultin effectively prevented hypoxia-induced apoptosis in vascular endothelial cells. We established a hypoxia model, using EA.hy926 cells, to further explore the mechanisms. Hypoxia promoted the phosphorylation of AKT, ERK1/2, and NF-κB. In hypoxic cells treated with Kaji-ichigoside F1, p-ERK1/2 and p-NF-κB levels were increased, while the level of p-AKT was decreased. Treatment with Rosamultin promoted phosphorylation of ERK1/2, NF-κB, and AKT in hypoxic cells. Following the addition of LY294002, the levels of p-AKT, p-ERK1/2, and p-NF-κB decreased significantly. Addition of PD98059 resulted in reduced levels of p-ERK1/2 and p-NF-κB, while p-AKT levels were increased. Pharmacodynamic analysis demonstrated that both LY294002 and PD98059 significantly inhibited the positive effects of Kaji-ichigoside F1 on cell viability during hypoxia, consistent with the results of hematoxylin-eosin (H&E) staining, DAPI staining, and flow cytometry. The antihypoxia effects of Rosamultin were remarkably inhibited by LY294002 but promoted by PD98059. In Kaji-ichigoside F1- and Rosamultin-treated cells, Bcl2 expression was significantly upregulated, while expression of Bax and cytochrome C and levels of cleaved caspase-9 and cleaved caspase-3 were reduced. Corresponding to pharmacodynamic analysis, LY294002 inhibited the regulatory effects of Kaji-ichigoside F1 and Rosamultin on the above molecules, while PD98059 inhibited the regulatory effects of Kaji-ichigoside F1 but enhanced the regulatory effects of Rosamultin. In conclusion, Kaji-ichigoside F1 protected vascular endothelial cells against hypoxia-induced apoptosis by activating the ERK1/2 signaling pathway, which positively regulated the NF-κB signaling pathway and negatively regulated the PI3K/AKT signaling pathway. Rosamultin protected vascular endothelial cells against hypoxia-induced apoptosis by activating the PI3K/AKT signaling pathway and positively regulating ERK1/2 and NF-κB signaling pathways.
Highlights
Potentilla anserina L., a medicinal herb widely distributed in western China, is commonly used as an antihypoxia and antiinflammatory treatment [1,2,3,4]
We established a model of hypoxia using EA.hy926 cells and used a phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor, LY294002, and an Extracellular regulated kinase 1/2 (ERK1/2) signaling inhibitor, PD98059, to explore (a) the correlation between the antiapoptotic effects of Kaji-ichigoside F1 and Rosamultin and the PI3K/AKT and ERK1/2 signaling pathways, (b) the interaction between PI3K/AKT and ERK1/2 signaling pathways during hypoxia, and (c) the effects of PI3K/AKT and ERK1/2 signaling on nuclear factor- (NF-)κB pathways, in order to elucidate the antiapoptotic mechanisms of action of Kaji-ichigoside F1 and Rosamultin at the cellular level
Primary antibodies against p-ERK1/2, ERK1/2, p-AKT, AKT, p-NF-κB, NF-κB, B-cell lymphoma 2 (Bcl-2), Bcl2-associated x protein (Bax), cytochrome C (Cyt C), cleaved caspase-9, and cleaved caspase-3 were bought from Cell Signaling Technology (Boston, USA)
Summary
Potentilla anserina L., a medicinal herb widely distributed in western China, is commonly used as an antihypoxia and antiinflammatory treatment [1,2,3,4]. We demonstrated that the Oxidative Medicine and Cellular Longevity n-butanol extract of Potentilla anserina L. protected primary hippocampal neurons against hypoxia-induced injury by inhibiting caspase cascade reaction [7]. Our previous studies indicated that Rosamultin activated phosphoinositide 3-kinase (PI3K)/AKT signaling pathways and had potential as a treatment for hydrogen peroxide-induced oxidative stress injury through its antioxidant and antiapoptotic effects in H9c2 cardiomyocytes [12]. Hypoxia-induced mitochondrial apoptosis is regulated by PI3K/AKT, mitogen-activated protein kinase (MAPK), nuclear factor- (NF-) κB, hypoxiainducible factor 1 (HIF-1), and other signaling pathways. PI3K/AKT and MAPK signaling pathways are activated, which can protect cells by regulating biological processes, such as apoptosis, proliferation, and differentiation [19]. Yang et al demonstrated that IGF-1 could inhibit hypoxia-induced apoptosis of retinal ganglion cells via activation of ERK1/2 and PI3K/AKT signaling pathways [36]. We established a model of hypoxia using EA.hy926 cells and used a PI3K/AKT pathway inhibitor, LY294002, and an ERK1/2 signaling inhibitor, PD98059, to explore (a) the correlation between the antiapoptotic effects of Kaji-ichigoside F1 and Rosamultin and the PI3K/AKT and ERK1/2 signaling pathways, (b) the interaction between PI3K/AKT and ERK1/2 signaling pathways during hypoxia, and (c) the effects of PI3K/AKT and ERK1/2 signaling on NF-κB pathways, in order to elucidate the antiapoptotic mechanisms of action of Kaji-ichigoside F1 and Rosamultin at the cellular level
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