Lycium barbarum polysaccharides alleviate LPS-induced inflammatory responses through PPARγ/MAPK/NF-κB pathway in bovine mammary epithelial cells

As the main component of the Gram-negative bacterial cell wall, lipopolysaccharide (LPS) is well documented as an inducer of inflammation in bovine mammary cells. Lycium barbarum (goji) polysaccharides (LBP) have been used in nonruminants as prebiotics to improve growth performance, immune ability, and antioxidant capacity. We aimed to investigate the underlying effects of LBPs on proinflammatory responses in LPS-stimulated primary bovine mammary epithelial cells (bMECs). Cells were isolated from mammary tissue of three lactating Holstein cows without clinical disease (30.26 +/- 3.1 kg/d of milk yield; 175 +/- 6 DIM). For the pre-experimental treatment, bMECs were precultured with serum-free medium for 12 h. Treatments were as follows: pretreatment with culture medium devoid of LPS or LBP for 30 h (CON); CON for 24 h followed by challenge with 2 mu g/mL LPS for 6 h (LPS); pretreatment with 100 or 300 mu g/mL LBP for 24 h followed by LPS challenge (2 mu g/mL) for 6 h (LBP(100)+LPS; LBP(300)+LPS). To further determine if the effect of LBP on immuneregulation is peroxisome proliferator-activated receptor-gamma (PPAR gamma) activation dependent, an inhibitor of PPAR gamma, GW9662, at a concentration of 1 mu M was used. Cells treated with LBP at 100, 300, and 500 mu g/mL had upregulated protein abundance of PPAR gamma, while PGC1 alpha had a higher expression only at 300 mu g/mL of LBP treatment. Compared with CON, cells pretreated with LBP at 100 and 300 mu g/mL had greater protein abundance of SCD1 and SREBP1. 5-Ethynyl-2'-deoxyuridine (EdU) staining and cell wound healing assays showed that the negative effect of LPS alone on cell proliferation was reversed by pretreatment with LBP at both 100 and 300 mu g/mL. Upregulation of gene and protein abundance of proinflammatory factors and cytokines (COX-2, NLRP3, TNF-alpha, IL-1 beta, and IL-6) induced by LPS stimulation were alleviated by LBP pretreatment at 300 mu g/mL (more than 2-fold decrease). Compared with LPS challenge alone, phosphorylation of proteins involved in NF-kappa B (I kappa B alpha and p65) and MAPK (p38, JNK, and ERK) pathways was downregulated following LBP treatment. Additionally, inhibition of PPAR gamma by GW9662 weakened the protective effect of LBP on LPS-induced protein abundance of phosphorylated p65, COX-2, IL-1 beta, and TNF-alpha. These results indicated that the protective effect of LBP on LPS-induced bMECs inflammatory responses is PPAR gamma activation-dependent. As such, this knowledge might help design strategies for intervening against the detrimental effects of bovine mastitis. Interpretive summary: Current research examined Lycium barbarum polysaccharides (LBP) for combating LPS-induced inflammatory responses in primary bovine mammary epithelial cells. We uncovered a preventive role of LBP in reducing detrimental effects induced by LPS including inhibition of NF-kappa B and MAPK along with peroxisome proliferator-activated receptor-gamma (PPAR gamma) activation. The decrease in cell proliferation due to LPS was curtailed by pretreatment with LBP. Moreover, the effect of LBP on regulation of inflammatory responses in bovine mammary epithelial cell was PPAR gamma dependent. Collectively, data suggest that LBP reverses LPS-induced inflammatory response via MAPK/NF-kappa B signaling in a PPAR gamma- activation-dependent manner. Thus, the study provides new insights into therapeutic strategies for combating mastitis using LBP and highlighted the link between PPAR gamma and regulation of mammary cell inflammation.

Automated summary

This study investigated the effects of Lycium barbarum polysaccharides (LBP) on proinflammatory responses in lipopolysaccharide (LPS)-stimulated primary bovine mammary epithelial cells. The results showed that LBP reduced the expression of proinflammatory factors and cytokines induced by LPS stimulation, inhibited the NF-kappa B and MAPK pathways, and activated peroxisome proliferator-activated receptor-gamma (PPAR gamma). LBP also reversed the negative effect of LPS on cell proliferation. The protective effect of LBP on LPS-induced inflammation was found to be dependent on PPAR gamma activation.