Cottonseed-derived gossypol and ethanol extracts differentially regulate cell viability and VEGF gene expression in mouse macrophages

Authors: Cao, Heping; Sethumadhavan, Kandan; WU, XIAOYU - Jiangxi Agricultural University; ZENG, XIAOCHUN - Yichun University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2021
Publication Date: 8/3/2021
Citation: Cao, H., Sethumadhavan, K., Wu, X., Zeng, X. 2021. Cottonseed-derived gossypol and ethanol extracts differentially regulate cell viability and VEGF gene expression in mouse macrophages. Scientific Reports. 11:15700. https://doi.org/10.1038/s41598-021-95248-4.
DOI: https://doi.org/10.1038/s41598-021-95248-4

Interpretive Summary: Cottonseed is the secondary product of cotton plant because it accounts for only 20% of the crop value. One way to increase cottonseed value is to isolate bioactive extracts and compounds from cottonseed because these value-added products possess health promotion and disease prevention potentials. Vascular endothelial growth factor (VEGF) is a key mitogenic and angiogenic factor involved in inflammation, tumor progression, collateral vessel formation, and diabetic retinopathy. Plant extracts and chemical compounds that can regulate VEGF gene expression may have positive effect on nutrition and health. We recently isolated bioactive extracts essentially free of gossypol from cottonseed. These bioactive cottonseed extracts are shown to affect human cancer cell growth and regulate diacylglycerol acyltransferase gene expression in mouse macrophages. The objective of this study was to assess the value of cottonseed extracts free of gossypol as a safe source of plant polyphenols by investigating the regulation of VEGF gene expression along with toxic gossypol and endotoxin LPS in cultured mouse RAW264.7 macrophages. This study demonstrated that cottonseed extracts exhibited modest effects on VEGF gene expression in the macrophages. Gossypol and LPS had stronger stimulation on VEGF gene expression, but gossypol increased much more VEGF mRNA and protein than LPS and the effect was sustained in longer treatment. These results suggest that gossypol and cottonseed coat extracts may have health and nutritional benefits for VEGF-related diseases.

Technical Abstract: Vascular endothelial growth factor (VEGF) plays an important role in chronic inflammation. Many plant extracts have nutritional and healthy benefits by down-regulating VEGF expression, but there was no report on VEGF regulation by cottonseed extracts in any biological system. The objective was to investigate cell viability and VEGF expression regulated by gossypol and ethanol extracts using lipopolysaccharides (LPS) as a control. MTT, qPCR and immunoblotting techniques were used to monitor cell viability, VEGF mRNA and protein levels in mouse RAW264.7 macrophages. Gossypol dramatically reduced macrophage viability but cottonseed extracts and LPS exhibited minor effect on cell viability. VEGFb mRNA levels were approximately 40 fold of VEGFa in the macrophages. Gossypol increased VEGFa and VEGFb mRNA levels up to 27 and 4 fold, respectively, and increased VEGF protein. LPS increased VEGFa mRNA by 6 fold but decreased VEGFb mRNA. LPS increased VEGF protein in 2-4 h but decreased in 8-24 h. Glanded seed extracts showed some stimulating effects on VEGF mRNA levels. Glandless seed coat extract showed increased VEGFb mRNA levels but its kernel extract reduced VEGF mRNA levels. This study demonstrated that gossypol and ethanol extracts differentially regulated cell viability and VEGF expression in mouse macrophages.