Anthocyanins Acts on Megakaryocytopoiesis and Proplatelet Formation in Vitro: Involving the Signaling Pathways of MAPKs and PI3K/Akt/NF‐κB
Authors: Yan Yang , Li Ya Fu , Jing Ren , hua Ling Wen 
Published: November 2023

BackgroundCardiovascular diseases (CVDs) have become a major public health problem nowadays. Platelets are small anucleated cells derived from the megakaryocytes(MKs), which play a vital role in hemostasis and thrombosis, and contribute to inflammation and atherosclerosis. Excessive platelet activation and aggregation have been shown to be involved in a variety of cardiovascular diseases (CVDs), particularly atherothrombosis. An increasing number of people believe that natural foods that contain bioactive constituents could possibly diminish the risk of CVDs. There are increasing evidences suggesting that dietary intake of phytochemicals can exert protective potential for CVDs. Anthocyanins, a category of phenolic flavonoids, are abundant in various fruits, vegetables and beverages, and are commonly consumed in human diet. Numerous of epidemiological and medical anthropological investigations have indicated that anthocyanins are believed to play a protective role against atherosclerosis. Our previous reports have demonstrated that anthocyanins significantly inhibited platelet activation, aggregation and thrombus formation in vitro, and significantly inhibited platelet granule secretion in hypercholesterolemia subjects. Furthermore, anthocyanins did not cause the tail bleeding in mice. However, the effects of cyanidin‐3‐O‐β‐glucoside (Cy‐3‐g), one of the most abundant compounds of anthocyanins, on the megakaryocyte, which is the precursor of platelet, have not yet been investigated before.ObjectivesTo investigate the effects of Cy‐3‐g on megakaryocytopoiesis, megakaryocytic proplatelet formation and its possible mechanisms in vitro.MethodsHuman megakaryocytic cell line Meg01were incubated with different concentrations of Cy‐3‐g (0.05μM, 5μM, 50μM and 100μM) or solvent control for indicated times. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay and colony forming unit‐megakaryocyte (CFU‐MK) assay. Flow cytometry and Giemsa staining assay were used to examine DNA ploidy. The positive expression level of CD41 in megakaryocytes was measured by flow cytometry. The effect of Cy‐3‐g on proplatelet formation was further examined. The expression and phosphorylation levels of proteins involved in the signaling pathways of MAPKs, PI3K/Akt and NF‐κB in MKs were determined by western blotting analysis.ResultsCy‐3‐g significantly increased cell viability and CFU‐MK numbers comparing with the solvent control in Meg01 cells. DNA ploidy and CD41 expression on MKs were also significantly increased. Cy‐3‐g significantly increased the number of proplatelet forming‐megakaryocytes in a dose‐dependent manner after 48 h and 72 h treatment. Western blotting analysis showed that Cy‐3‐g dose‐dependently increased the phosphorylation of Erk1/2 and p38 MAPK in MKs after 48 h treatment. However, Cy‐3‐g treatment showed no significant increase in the phosphorylation of JNK. Furthermore, Cy‐3‐g, like the pan‐PI3K inhibitor LY294002, potently inhibited phosphorylation of the PI3K downstream effector Akt. However, it should be noted that LY294002 demonstrated more potent inhibition of AktSer473 phosphorylation than Cy‐3‐g. Moreover, Cy‐3‐g significantly decreased IκB‐α phosphorylation and degradation dose dependently and blocked the NF‐κB p65 subunit into the nucleus after 48 h treatment. We also showed that treatment cells with LY294002 could significantly inhibit IκB‐α phosphorylation and degradation and then block the NF‐κB p65 subunit into the nucleus, indicating that NF‐κB was the downstream‐regulated protein of the PI3K‐AKT axis. Furthermore, Cy‐3‐g appeared to inhibit NF‐κB p65 more potently than LY294002. The combination of Cy‐3‐g and LY294002 showed an additional inhibitory efficacy.ConclusionsThe study collectively demonstrated that Cy‐3‐g enhanced megakaryocyte proliferation, differentiation and proplatelet formation in the human megakaryocytic cell line in vitro, involving the signaling pathways of the Erk1/2 and p38 MAPK activation, and PI3K‐Akt‐NF‐κB inhibition.Support or Funding InformationThis work was supported by National Natural Science Foundation of China Research Grants (No. 81573145 and No. 81372978), Science and Technology Grants of Guangdong province (No. 2015A0303131 and No.2014A020212111)