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

Abstract

Vascular endothelial growth factor (VEGF) plays an important role in chronic inflammation associated with several diseases. 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 sixfold 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.

Thymus musilii Velen. Methanolic Extract: In Vitro and In Silico Screening of Its Antimicrobial, Antioxidant, Anti-Quorum Sensing, Antibiofilm, and Anticancer Activities

“…Idebenone metabolites (Benzenebutanoic acid and 2,5-dihydroxy-3,4-dimethoxy-6-methyl-) were previously reported in the methanolic and ethanolic extracts of Monotheca buxifolia [ 55 ] and from the butanolic fraction of Delphinium brunonianum together [ 56 ]. Gossypol, firstly isolated in 1889 from the seeds, roots, and stems of Gossypium members [ 57 , 58 ], was also reported in our study in the methanolic extract of T. musilii . Similarly, tripeptides and dipeptides were successfully identified by using the HR-LCMS technique in the organic extracts from T. polium [ 24 ], D. brunonianum [ 55 ], and A. subhirsutum [ 59 , 60 ].…”

Plant Polyphenol Gossypol Induced Cell Death and Its Association with Gene Expression in Mouse Macrophages

“…The results from this study and several previous studies suggest that gossypol is a strong stimulator of gene expression in mouse macrophages for the following reasons: (1) gossypol increases mRNA-destabilizing anti-inflammatory TTP family gene expression (TTP/ZFP36L1, ZFP36L2, ZFP36L2 and ZFP36L3) (this study); (2) gossypol increases proinflammatory cytokine gene expression (this study); (3) gossypol increases mRNA-stabilizing human antigen R (HuR) gene expression [ 52 ]; (4) gossypol increases vascular endothelial growth factor (VEGF) gene expression [ 53 ]; (5) gossypol increases glucose transporter gene expression (this study); (6) gossypol increases insulin signaling pathway gene expression (INSR, AKT1 and PIK3R1) (this study); (7) gossypol increases diacylglycerol acyltransferase (DGAT) gene expression [ 48 ]. However, since gossypol induced cell death at the same time, it is still to be determined if the gene expression effect was due to the direct or indirect effect of gossypol in the cells.…”

Plant Polyphenol Gossypol Induced Cell Death and Its Associ-ation with Gene Expression in Mouse Macrophages

“…This study demonstrated that gossypol induced macrophage death and reduced protein content that was accompanied with elevated levels of anti-inflammatory TTP family and proinflammatory cytokine gene expression as well as glucose transporter and insulin signaling pathway gene expression in mouse macrophages. We recently showed that gossypol strongly stimulated DGAT, HuR and VEGF gene expression in mouse macrophages [48,52,53]. Taking together, these studies indicate that gossypol derived from cottonseed may be a powerful stimulator of gene expression involved in inflammatory responses, glucose transport, insulin signaling and lipid biosynthesis in mouse macrophages, regardless its direct or indirect effects.…”

“…The results from this study and several previous studies suggest that gossypol is a strong stimulator of gene expression in mouse macrophages for the following reasons: 1) Gossypol increases mRNA destabilizing anti-inflammatory TTP family gene expression (TTP/ZFP36L1, ZFP36L2, ZFP36L2 and ZFP36L3) (this study); 2) Gossypol increases proinflammatory cytokine gene expression (this study); 3) Gossypol increases mRNA stabilizing human antigen R (HuR) gene expression [52]; 4) Gossypol increases vascular endothelial growth factor (VEGF) gene expression [53]; 5) Gossypol increases glucose transporter gene expression (this study); 6) Gossypol increases insulin signaling pathway gene expression (INSR, AKT1 and PIK3R1) (this study); 7) Gossypol increases diacylglycerol acyltransferase (DGAT) gene expression [48]. However, since gossypol induced cell death at the same time, it is still to be determined if gene expression effect was due to direct or indirect effect of gossypol in the cells.…”