Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide

Abstract

Silibinin, the bioactive compound of milk thistle (), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor N-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

The milk thistle (Silybum marianum) compound Silibinin stimulates leukopoiesis from mouse embryonic stem cells

“…Moreover, VEGFR2 as well as VE‐cadherin expression stimulated upon treatment with 10‐μM Silibinin was blunted by L‐NAME treatment. Taken together, these data corroborate our previous results on a stimulation of vasculogenesis by Silibinin (Ali et al, ) and link leukopoiesis to vasculogenic processes in differentiating ES cells.…”

“…This assumption would implicate that the anti‐inflammatory action of Silibinin is not just due to the radical scavenging chemical structure of this flavonolignan but to the initiation of receptor‐regulated anti‐inflammatory signaling cascades, which indirectly results in antioxidative action. Our previous observations that Silibinin activates NO generation by eNOS stimulation, which was inhibited upon interference with STAT3 and PI3K/AKT (Ali et al, ) and the data of the present study that Silibinin raised NO in CD68 + cells point in this direction. To underscore the notion that Silibinin is stimulating the differentiation of cells expressing leukocyte markers through STAT3 and PI3K/AKT signaling pathways, we applied pharmacological inhibitors.…”

“…We recently demonstrated that Silibinin raised intracellular NO by stimulation of eNOS in differentiating ES cells (Ali et al, ). To specify this observation, we determined NO generation in CD68 + as well as CD68 − cells differentiated in 14‐day‐old embryoid bodies using the NO‐sensitive fluorescence indicator DAF‐FM.…”

“…However, the pharmacological action of Silibinin may well extend beyond its antioxidative properties, which per se should not offer a unique feature of this compound because antioxidative properties have been described for many polyphenolic plant ingredients (Adegbola, Aderibigbe, Hammed, & Omotayo, ; Umeno, Horie, Murotomi, Nakajima, & Yoshida, ). In this respect, we have recently demonstrated that stimulation of differentiating ES cells with Silibinin results within few minutes in activation of specific signaling pathways, that is, the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway as well as the phosphatidylinositide 3‐kinase/AKT (PI3K/AKT) pathway and downstream in the activation of eNOS (Ali, Sharifpanah, Wartenberg, & Sauer, ). Stem cells are the key elements in regenerative processes, and stem cell activation and differentiation may contribute to the organ protective effects described for Silibinin in numerous studies (Milic, Milosevic, Suvajdzic, Zarkov, & Abenavoli, ).…”

“…Silibinin‐C‐2′,3‐dihydrogen succinate is an intravenously injectable solution of silybin A and silybin B, which is generally named Silibinin and sold under the trade name Legalon SIL® (Polyak et al, ; Figure S1). We have recently demonstrated that Silibinin stimulated vasculogenesis of ES cells (Ali et al, ). Vasculogenesis and hematopoiesis are closely linked events, and vascular and hematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs (Reischauer et al, ).…”