Lactobacillus rhamnosus GR-1 Alleviates Escherichia coli-Induced Inflammation via NF-κB and MAPKs Signaling in Bovine Endometrial Epithelial Cells

Abstract

Escherichia coli counts as a major endometritis-causing pathogen among dairy cows, which lowered the economic benefits of dairy farming seriously. Probiotic consumption has been reported to impart beneficial effects on immunomodulation. However, the inflammatory regulation mechanism of probiotics on endometritis in dairy cows remains unexplored. The current work aimed to clarify the mechanism whereby Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) resists bovine endometrial epithelial cells (BEECs) inflammatory injury induced by E. coli. The model of cellular inflammatory injury was established in the BEECs, which comes from the uterus of healthy dairy cows using E. coli. The outcome of L. rhamnosus GR-1 addition on inflammation was evaluated in BEECs with E. coli-induced endometritis. The underlying mechanisms of anti-inflammation by L. rhamnosus GR-1 were further explored in E. coli-stimulated BEECs. In accordance with the obtained results, the use L. rhamnosus GR-1 alone could not cause the change of inflammatory factors, while L. rhamnosus GR-1 could significantly alleviate the expression of E. coli-induced inflammatory factors. Based on further study, L. rhamnosus GR-1 significantly hindered the TLR4 and MyD88 expression stimulated by E. coli. Moreover, we observed that in BEECs, L. rhamnosus GR-1 could inhibit the E. coli-elicited expressions of pathway proteins that are associated with NF-κB and MAPKs. Briefly, L. rhamnosus GR-1 can effectively protect against E. coli-induced inflammatory response that may be closely related to the inhibition of TLR4 and MyD88 stimulating NF-κB and MAPKs.

Keywords: Escherichia coli; Lactobacillus rhamnosus; MAPKs; NF-κB; bovine endometrial epithelial cell; inflammatory response.

Figure 1

Cell viability in BEECs. Cell viability was detected by MTT at different periods. Data are presented as the mean ± SEM of three independent experiments.

Figure 2

Effect of L. rhamnosus GR-1 on the mRNA expression of inflammatory factors in BEECs induced by E. coli. IL-1β, IL-8, IL-6, and TNF-α mRNA were quantified using the qPCR assay. (A) The expression of IL-1β mRNA, (B) IL-8 mRNA, (C) IL-6 mRNA, and (D) TNF-α mRNA. Data represented are means ± SEMs from triplicate experiments. *P <0.05, **P <0.01.

Figure 3

L. rhamnosus GR-1 affects the TLR4 and MyD88 expression in E. coli-induced BEECs. (A) Expression of TLR4 mRNA and (B) MyD88 mRNA. Data are presented as the mean ± SEM of three independent experiments. *P <0.05, **P <0.01.

Figure 4

L. rhamnosus GR-1 affects the NF-κB pathway protein expression in E. coli-induced BEECs. The p-p65 and p-IκBα expressions in the L. rhamnosus GR-1- and E. coli-elicited BEECs were explored by Western blotting. (A) p-p65, p-IκBα. The resultant phosphorylated protein as percentages of total protein is averaged and plotted, (B) p-p65/p65 and (C) p-IκBα/β-actin. Data represented are means ± SEMs from triplicate experiments. **P <0.01.

Figure 5

L. rhamnosus GR-1 affects the p-JNK1, p-p38, and p-ERK1/2 protein expression in E. coli-induced BEECs. The protein levels of p-JNK1, p-p38, and p-ERK1/2 in BEECs stimulated with E. coli and L. rhamnosus GR-1 was investigated by Western blotting. (A) p-JNK1, p-p38, p-ERK1/2. The resultant mean ratio of phosphorylated protein to total protein is depicted, (B) p-JNK1/GAPDH, (C) p-38/GAPDH and (D) p-ERK1/2/GAPDH. Data are presented as the mean ± SEM of three independent experiments. *P <0.05, **P <0.01.