Total saponins of panaxnotoginseng promotes lymphangiogenesis by activation VEGF-C expression of lymphatic endothelial cells

Abstract

Ethnopharmacological relevance: Lymphatic system plays an important role in maintaining the fluid homeostasis and normal immune responses, anatomic or functional obstruction of which leads to lymphedema, and treatments for therapeutic lymphangiogenesis are efficiency for secondary lymphedema. Total saponins of panaxnotoginseng (PNS) are a mixture isolated from Panaxnotoginseng (Burkill) F.H.Chen, which has been used as traditional Chinese medicine in China for treatment of cardio- and cerebro-vascular diseases. The aim of this study was to determine the effect and mechanism of PNS on lymphangiogenesis.

Methods: The Tg (fli1: egfp; gata1: dsred) transgenic zebrafish embryos were treated with different concentrations of PNS (10, 50, 100μM) for 48h with or without the 6h pretreatment of the 30μM Vascular endothelial growth factors receptor (VEGFR)-3 kinase inhibitor, followed with morphological observation and lympangiogenesis of thoracic duct assessment. The effect of PNS on cell viability, migration, tube formation and Vascular endothelial growth factors (VEGF)-C mRNA and protein expression of lymphatic endothelial cells (LECs) were determined. The role of phosphatidylinositol-3 (PI-3)-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2 pathways, c-Jun N-terminal kinase (JNK) and P38 mitogen activated protein kinases (MAPK) signaling in PNS-induced VEGF-C expression of LECs by using pharmacological agents to block each signal.

Results: PNS promotes lymphangiogenesis of thoracic duct in zebrafish with or without VEGFR3 Kinase inhibitor pre-impairment. PNS promotes proliferation, migration and tube formation of LECs. The tube formation induced by PNS could be blocked by VEGFR3 Kinase inhibitor. PNS induce VEGF-C expression of LEC, which could be blocked by ERK1/2, PI3K and P38MAPK signaling inhibitors.

Conclusion: PNS activates lymphangiogenesis both in vivo and in vitro by up-regulating VEGF-C expression and activation of ERK1/2, PI3K and P38MAPK signaling. These findings provide a novel insight into the role of PNS in lymphangiogenesis and suggest that it might be an attractive and suitable therapeutic agent for treating secondary lymphedema or other lymphatic system impairment related disease.

Keywords: Acrylamide (PubChemCID: 6579); Ammonium Persulfate (PubChemCID:62648); Chloroform (PubChemCID:6212); Dimethyl sulfoxide (DMSO) (PubChem CID: 679); Ethanol (PubChem CID: 702); Glycine (PubChemCID:750); Lymphangiogenesis; Lymphedema; MAZ51, VEGF Receptor 3 Kinase inhibitor (PubChemCID:9839842); Methanol (PubChemCID:887); PD98059 (PubChemCID: 4713); SB203580 (PubChemCID: 176155); SP600125 (PubChemCID: 8515); Saponins of panaxnotoginseng; Sodium chloride (PubChemCID:5234); Vascular endothelial growth factors C; Wortmannin (PubChemCID: 312145).

Fig. 1

The lymphatic thoracic duct formation of zebrafish was increased by PNS in concentration dependent manner. The 48 hpf zeberafish (fli1:egfp; gata1:dsred) was treated with different concentrations of PNS (10, 50, 100μM) for 48 hours. Embryos treated with 0.2% DMSO served as a vehicle control. (A) Confocal image of the 96 hpf. zebrafish (fli1:egfp) vascular system. White boxed region indicates ten segment of thoracic duct length for quantitation in C; Red boxed region shows approximate location of regions imaged in B. (B) Representative confocal images show that PNS increased lymphatic thoracic duct formation of zeberafish, white arrow indicates lymphatic thoracic duct, and white star indicates lack of lymphatic vessel. Scale bars, 100 μm. (C) Quantitation of the length of lymphatic thoracic duct. Values are mean ± SD of 9–11 zeberafishes. *P<0.05 vs. vehicle control group.

Fig. 2

Impaired lymphatic thoracic duct formation induced by VEGFR-3 kinase inhibitor (MAZ51) was rescued by PNS in concentration dependent manner. The 48 hpf zeberafish (fli1:egfp; gata1:dsred) was treated with 30μM MAZ51 for 6 hours and then changed to be treated with different concentrations of PNS (10,50, 100 μM) for 48 hours. Embryos treated with 0.2% DMSO served as a vehicle control. (A) Representative confocal images show that PNS increased lymphatic thoracic duct formation of zeberafish, white arrow indicates lymphatic thoracic duct, and white star indicates lack of lymphatic vessel. Scale bars, 100 μm. (B) Quantitation of the length of lymphatic thoracic duct. Values are mean ± SD of 9–11 zeberafishes. *P<0.05 vs. control group; # P<0.05 versus MAZ51 treated group.

Fig. 3

PNS increased proliferation and cell migration of lymphatic endothelial cells (LECs). VEGF-C was considered as positive control. The group treated with PBS was considered as negative control. (A) Cell migration was assessed by wound healing assay. (B) Quantitation of migration length. Values are mean ± SD of 3 wells/treatment. *P<0.05 vs. control group. (C) LECs was treated with different concentrations of PNS (10, 50, 100μM) and VEGF-C (0.34nM) for 72 hours. Cell growth was determined by MTT assay. The values are the mean + SD of 4 wells. *P<0.05 vs. control group.

Fig. 4

PNS induced tube formation of LECs, which was blocked by VEGFR3 Kinase inhibitor. (A) LECs cultured on 3-dimensional Matrigel in treatment of PNS (10, 50, and 100 μM) or VEGF-C (0.34nM). Cells receiving 0.1% DMSO served as vehicle control, and receiving VEGF-C served as positive control. (B) Number of branching points/well in different concentrations of PNS-treated LECs was calculated. Results are expressed as mean±SD (n=3 independent experiments), *P<0.05 versus control. (C) LECs on Matrigel in treatment of PNS (100 μM) or VEGF-C (0.34nM), with or without VEGFR3 Kinase inhibitor (250nM). (D) Number of branching points/well was expressed as mean±SD (n=3 independent experiments), *P<0.05 versus control group; # P<0.05 versus PNS group; ‡ P<0.05 versus VEGF-C group.

Fig. 5

PNS stimulated VEGF-C expression of LECs, which was blocked by PI3K, ERK, and P38MAPK signaling inhibitor. (A) LECs were treated with different concentrations of PNS (10, 50, and 100 μM) for 24 hours, the mRNA expression of VEGF-C, VEGFR-3, FOXC2, NRP2 and Prox1 was analyzed by qPCR, Cells receiving 0.1% DMSO served as vehicle control. Results are represented as mean±SD (n = 3 independent experiments), * P<0.05 versus control. (B) The protein level of VEGF-C was assessed by westernblot. (C) LECs were incubated with or without PNS (100 μM) ± 25μM ERK inhibitor (PD98059), 50nM PI3K inhibitor (Wortmannin), 10μM P38MAPK inhibitor (SB203580), or 20μM JNK inhibitor (SP600125) for 24 hours. LECs treated with 0.1% DMSO served as a vehicle control. The mRNA expression of VEGF-C was examined by qPCR and the results are present as mean±SD (n = 3 independent experiments), * P<0.05 versus control group, and # P<0.05 versus PNS (100 μM) treated group.