Berberine protects endothelial progenitor cell from damage of TNF-α via the PI3K/AKT/eNOS signaling pathway

Abstract

Endothelial progenitor cells (EPCs) dysfunction is closely correlated with the coronary artery injury induced by Kawasaki disease (KD). The level of tumor necrosis factor-α (TNF-α) elevated significantly in acute phase of KD which can damage the functions of EPCs. The aim of this study was to investigate whether berberine (BBR) can protect EPCs from the inhibition caused by TNF-α via the PI3K (Phosphatidyl Inositol 3-kinase) /AKT (Serine/threonine protein kinase B) /eNOS (endothelial Nitric Oxide synthase) signaling pathway. The cell proliferative ability of EPCs was determined by MTT (methyl thiazolyl tetrazolium) assays. Nitric oxide (NO) level was determined in supernatants. The mRNA level of eNOS, PI3K and AKT were measured by Real Time-Polymerase Chain Reaction (RT-PCR), and the protein levels of eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt) and PI3K were analyzed using Western-blot. The results demonstrated that TNF-α inhibits the proliferative ability of EPCs. However, BBR improves the proliferative activity of EPCs inhibited by TNF-α. Blockade of PI3K by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) and blockade of eNOS by l-NAME (NG-Nitroarginine Methyl Ester) attenuates the effect of BBR. BBR can increase the level of PI3K/Akt/eNOS mRNA and the protein level of PI3K, p-Akt, eNOS and p-eNOS, which can be blocked by PI3K inhibitor (LY294002) and eNOS inhibitor (l-NAME). Therefore, we concluded that impaired EPCs proliferation could be reversed by BBR via the PI3K/AKT/eNOS signaling pathway.

Keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (PubChem CID: 64966); AKT (Serine/threonine protein kinase B); Berberine; Berberine (PubChem CID: C12456); Dimethylsulfoxide (PubChem CID: 679); Endothelial progenitor cells; FITC (Fluorescein isothiocyanate) (PubChem CID: 18730); LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) (PubChem CID: 3973); PI3K (Phosphatidyl Inositol 3-kinase); Paraformaldehyde (PubChem CID: 712) Rosiglitazone (PubChem CID 77999); TNF-α (Tumor Necrosis Factor-α); agarose (PubChem CID: 11966311); eNOS (endothelial Nitric Oxide Synthase); ethidium bromide (PubChem CID: 14710); l-NAME (NG-Nitroarginine Methyl Ester) (PubChem CID: 39836).