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. 2020 Apr 12:2020:6837982.
doi: 10.1155/2020/6837982. eCollection 2020.

Kaji-Ichigoside F1 and Rosamultin Protect Vascular Endothelial Cells against Hypoxia-Induced Apoptosis via the PI3K/AKT or ERK1/2 Signaling Pathway

Affiliations

Kaji-Ichigoside F1 and Rosamultin Protect Vascular Endothelial Cells against Hypoxia-Induced Apoptosis via the PI3K/AKT or ERK1/2 Signaling Pathway

Chaofeng Shi et al. Oxid Med Cell Longev. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of Kaji-ichigoside F1 and Rosamultin. (a) Chemical structure of Kaji-ichigoside F1. Molecular formula: C36H58O10. (b) Chemical structure of Rosamultin. Molecular formula: C36H58O10.
Figure 2
Figure 2
Kaji-ichigoside F1 activated the ERK1/2 signaling pathway and inhibited the PI3K/AKT signaling pathway, and Rosamultin activated PI3K/AKT and ERK1/2 signaling pathways. (a) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the PI3K/AKT inhibitor, LY294002, on p-AKT and AKT expressions. (b) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the ERK1/2 inhibitor, PD98059, on p-ERK1/2 and ERK1/2 expressions. (c) Quantitative analyses of AKT. (d) Quantitative analyses of ERK1/2. (e) Quantitative analyses of p-AKT. (f) Quantitative analyses of p-ERK1/2. (g) Quantitative analyses of p-AKT/AKT. (h) Quantitative analyses of p-ERK1/2/ERK1/2. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 3
Figure 3
PI3K/AKT signaling positively regulated the ERK1/2 signaling pathway, while ERK1/2 signaling negatively regulated PI3K/AKT signaling. (a) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the PI3K/AKT inhibitor, LY294002, on p-ERK1/2 and ERK1/2 expressions. (b) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the ERK1/2 inhibitor, PD98059, on p-AKT and AKT expressions. (c) Quantitative analyses of ERK1/2. (d) Quantitative analyses of AKT. (e) Quantitative analyses of p-ERK1/2. (f) Quantitative analyses of p-AKT. (g) Quantitative analyses of p-ERK1/2/ERK1/2. (h) Quantitative analyses of p-AKT/AKT. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 4
Figure 4
PI3K/AKT and ERK1/2 signaling pathways positively regulated NF-κB signaling. (a) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the PI3K/AKT inhibitor, LY294002, on p-NF-κB and NF-κB. (b) Western blot showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the ERK1/2 inhibitor, PD98059, on p-NF-κB and NF-κB. (c) Quantitative analyses of NF-κB. (d) Quantitative analyses of NF-κB. (e) Quantitative analyses of p-NF-κB. (f) Quantitative analyses of p-NF-κB. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 5
Figure 5
Kaji-ichigoside F1 enhanced cell viability via activation of ERK1/2 signaling, and Rosamultin enhanced cell viability via PI3K/AKT and ERK1/2 signaling pathways during hypoxia. (a) MTT assay showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the PI3K/AKT inhibitor, LY294002, on cell viability. (b) MTT assay showing the effects of Kaji-ichigoside F1, Rosamultin, and/or the ERK1/2 inhibitor, PD98059, on cell viability. Results are displayed as mean ± SEM (n = 6). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 6
Figure 6
Kaji-ichigoside F1 reduced cell damage by activating ERK1/2 signaling, and Rosamultin reduced cell damage via PI3K/AKT and ERK1/2 signaling pathway activation during hypoxia. (a) H&E staining of EA.hy926 endothelial cells in each group. (b) DAPI staining of EA.hy926 endothelial cells in each group. Scale bar represents 50 μm.
Figure 7
Figure 7
Kaji-ichigoside F1 decreased cell apoptotic rates via ERK1/2 signaling pathway activation, and Rosamultin decreased cell apoptotic rates via PI3K/AKT and ERK1/2 signaling pathway activation during hypoxia. (a) FACS analysis of apoptosis. (b) Cell apoptotic rate by FACS. The percentage of apoptotic cells is shown as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 8
Figure 8
The results of q-PCR showed the antiapoptotic effects of Kaji-ichigoside F1 and Rosamultin, and Rosamultin inhibited hypoxia-induced apoptosis via the PI3K/AKT signaling pathway at the gene level. (a) Quantitative analyses of Cyt C mRNA expression. (b) Quantitative analyses of caspase-3 mRNA expression. (c) Quantitative analyses of Bax mRNA expression. (d) Quantitative analyses of Bcl-2 mRNA expression. (e) Quantitative analyses of caspase-9 mRNA expression. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 9
Figure 9
Kaji-ichigoside F1 and Rosamultin inhibited hypoxia-induced apoptosis by activating the ERK1/2 signaling pathway at the gene level. (a) Quantitative analyses of Cyt C mRNA expression. (b) Quantitative analyses of caspase-3 mRNA expression. (c) Quantitative analyses of Bax mRNA expression. (d) Quantitative analyses of Bcl-2 mRNA expression. (e) Quantitative analyses of caspase-9 mRNA expression. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 10
Figure 10
The results of Western blot showed the antiapoptotic effects of Kaji-ichigoside F1 and Rosamultin, and Rosamultin inhibited hypoxia-induced apoptosis via the PI3K/AKT signaling pathway at the protein level. (a) Western blot of Cyt C, cleaved caspase-3, Bax, Bcl-2, and cleaved caspase-9 in Kaji-ichigoside F1-, Rosamultin-, and/or, PI3K/AKT inhibitor LY294002-treated endothelial cells. (b) Quantitative analyses of Cyt C. (c) Quantitative analyses of cleaved caspase-3. (d) Quantitative analyses of Bax. (e) Quantitative analyses of Bcl-2. (f) Quantitative analyses of cleaved caspase-9. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 11
Figure 11
Kaji-ichigoside F1 and Rosamultin inhibited hypoxia-induced apoptosis by activating the ERK1/2 signaling pathway at the protein level. (a) Western blot of Cyt C, cleaved caspase-3, Bax, Bcl-2, and cleaved caspase-9 expression in Kaji-ichigoside F1-, Rosamultin-, and/or, ERK1/2 inhibitor PD98059-treated endothelial cells. (b) Quantitative analyses of Cyt C. (c) Quantitative analyses of cleaved caspase-3. (d) Quantitative analyses of Bax. (e) Quantitative analyses of Bcl-2. (f) Quantitative analyses of cleaved caspase-9. Results are displayed as mean ± SEM (n = 3). +P < 0.05 vs. normoxia control group, , ++P < 0.05 vs. model group, #P < 0.05 vs. Kaji-ichigoside F1 group. &P < 0.05 vs. Rosamultin group.
Figure 12
Figure 12
Schematic diagram depicting the cytoprotective effects of Kaji-ichigoside F1 and Rosamultin on hypoxia-induced apoptosis of EA.hy926 endothelial cells. Hypoxia causes mitochondrial apoptosis through activation of caspase-3. Rosamultin inhibits apoptosis induced by hypoxia by activating PI3K/AKT and ERK1/2 signaling pathways in EA.hy926 endothelial cells. Kaji-ichigoside F1 inhibits apoptosis via ERK1/2 signaling pathway activation. The PI3K/AKT signaling pathway positively regulates ERK1/2 and NF-κB signaling pathways, while ERK1/2 signaling positively regulates NF-κB signaling and negatively regulates the PI3K/AKT signaling pathway.

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References

    1. Guo T., Wei J. Q., Ma J. P. Antitussive and expectorant activities of Potentilla anserina. Pharmaceutical Biology. 2016;54(5):807–811. doi: 10.3109/13880209.2015.1080734. - DOI - PubMed
    1. Zhao B., Zhang J., Yao J., Song S., Yin Z., Gao Q. Selenylation modification can enhance antioxidant activity of Potentilla anserina L. polysaccharide. International Journal of Biological Macromolecules. 2013;58:320–328. doi: 10.1016/j.ijbiomac.2013.04.059. - DOI - PubMed
    1. Wei W., Li G. C., Gong H. Y., Li Y., Li J. Y., Li L. Z. The anti-hypoxia effects of potentilla anserine L. polysaccharide. Wu Jing Yi Xue Yuan Xue Bao. 2010;19(5):345–347.
    1. Paduch R., Wiater A., Locatelli M., Pleszczyńska M., Tomczyk M. Aqueous extracts of selected Potentilla species modulate biological activity of human normal colon cells. Current Drug Targets. 2015;16(13):1495–1502. doi: 10.2174/1389450116666141205160444. - DOI - PubMed
    1. Li J. Y., Li Y., Gong H. Y., Zhao X. B., Li L. Z. Protective effects of n-butanol extract of potentilla anserina on acute myocardial ischemic injury in mice. Zhong Xi Yi Jie He Xue Bao. 2009;7(1):48–52. doi: 10.3736/jcim20090107. - DOI - PubMed

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