Regulation of cell viability and anti-inflammatory tristetraprolin family gene expression in mouse macrophages by cottonseed extracts

Authors: Cao, Heping; Sethumadhavan, Kandan

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2019
Publication Date: 1/21/2020
Citation: Cao, H., Sethumadhavan, K. 2020. Regulation of cell viability and anti-inflammatory tristetraprolin family gene expression in mouse macrophages by cottonseed extracts. Scientific Reports. 10:775. https://doi.org/10.1038/s41598-020-57584-9.
DOI: https://doi.org/10.1038/s41598-020-57584-9

Interpretive Summary: Cotton is an important plant because it produces fiber and cottonseed. Cottonseed accounts for approximately 20% of the crop value. However, cottonseed usage is limited to feeding ruminant animals and not for human or other animal consumption due to toxic gossypol in glanded cottonseed, the commonly cultivated cotton. Glandless cottonseed does not have pigment glands and accumulates only trace amounts of gossypol; which has potential being used as a food ingredient or as a feed for non-ruminant animals. However, diets containing glandless cottonseed kernels produced significant number of hepatocellular carcinomas in rainbow trout. Because glandless cottonseed kernels are available for purchase and consumption by the general public, it is therefore important to investigate the cytotoxicity of glandless cottonseeds. Here we report our findings on the cytotoxicity and gene expression regulation of ethanol extracts from glandless and glanded cottonseed along with pure gossypol and lipopolysaccharides using cultured mouse macrophages. This study showed that no cytotoxicity effect was observed in cells treated with extracts from glandless cottonseed coat or kernels, nor with glanded cottonseed coat, but with glanded cottonseed kernel extract. Lipopolysaccharides did not show toxic activity but gossypol at high concentration or long treatment inhibited cell viability. Cottonseed extracts and gossypol increased both proinflammatory tumor necrosis factor and anti-inflammatory tristetraprolin gene expression in the macrophages, to a much less extent than those of lipopolysaccharides. The effects of ethanol extracts from cottonseeds and gossypol on stimulating anti-inflammatory tristetraprolin gene expression are similar to those of cinnamon extract and green tea extract. These results suggest that glandless cottonseed extract is harmless towards the cultured macrophages but glanded cottonseed extract from its kernel, like gossypol, is harmful to the immunological cells. Our results further suggest that ethanol extracts from cottonseed, especially glandless cottonseed may have potential health/nutritional benefits for inflammation-related diseases such as arthritis and diabetes.

Technical Abstract: Cotton plant provides two economically important products: fiber and cottonseed. Cotton produces more seed than fiber in quantity. Cottonseed typically accounts for 20% of the crop value. Due to presence of toxic gossypol in the glanded cotton, the use of cottonseed is limited to feeding cows and not for human or other animal consumption. To explore the potential use of glandless cottonseed which has only trace amount of gossypol, we investigated the cytotoxicity and gene expression modulation of ethanol extracts from glandless cottonseed along with those from glanded cottonseed and compared directly with gossypol and lipopolysaccharide (LPS). Cottonseeds were separated into coat and kernel fractions. Both fractions were homogenized in acetic acid, defatted with chloroform and hexane and extracted with ethanol. The ethanol extracts were reconstituted in DMSO before being added to mouse RAW264.7 macrophages using gossypol and LPS as controls. Cell cytotoxicity and gene expression were determined with MTT and qPCR assays. No cytotoxicity effect was observed in cells treated with extracts from glandless cottonseed coat or kernel, or glanded cottonseed coat, but glanded cottonseed kernel extract inhibited mitochondrial activity by 40%. LPS did not show toxic activity but gossypol inhibited cell viability. Cottonseed extracts and gossypol increased both proinflammatory TNF and anti-inflammatory TTP/ZFP36 gene expression in the macrophages, to a much less extent than those of LPS. The effects of ethanol extracts from cottonseeds and gossypol on stimulating anti-inflammatory TTP gene expression were similar to those of cinnamon extract and green tea extract. These results suggest that glandless cottonseed extract is harmless towards the cultured macrophages but glanded cottonseed extract from its kernel, like gossypol, is harmful to the immunological cells, and that ethanol extracts from cottonseed, especially glandless cottonseed may have health/nutritional benefits for inflammation-related diseases.