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Effect of Lactobacillus johnsonii Strain SQ0048 on the TLRs-MyD88/NF-κB Signaling Pathway in Bovine Vaginal Epithelial Cells.

ABSTRACT: Vaginal inflammation is a common disease of the dairy cows' reproductive tract. Lactic acid bacteria can combat purulent inflammation caused by pathogenic bacteria and regulate the NF-κB signaling pathway mediated by toll-like receptors (TLRs) in the inflammatory response. We studied the effect of Lactobacillus johnsonii SQ0048, an isolate with antibacterial activity, on the NF-κB signaling pathway in cow vaginal epithelial cells. The expression levels of serial effectors related to the TLRs-MyD88/NF-κB signaling pathway (TLR2, TLR4, MyD88, IKK, NF-κB, IL-1β, IL-6, TNF-α, and IL-10) were measured with real-time polymerase chain reaction (RT-PCR), ELISA, and Western blot analyses. TLR2 and TLR4 were activated by SQ0048 cells, as noted by increased mRNA expression levels of TLR2 and TLR4 in SQ0048-treated bovine vaginal epithelial cells relative to control cells (P <0.01). SQ0048 treatment also significantly increased MyD88 and IKK expression, and activated NF-κB in vaginal epithelial cells (P <0.01). In addition, SQ0048 treatment also significantly increased mRNA expression levels of IL-1β, IL-6, and TNF-α, but decreased IL-10 mRNA expression levels (P <0.01). These data indicate that strain SQ0048 presence can improve the immune functions of cow vaginal epithelial cells by activating TLRs-MyD88/NF-κB signaling pathways. However, further in vivo studies are required to confirm these findings.

SUBMITTER: Cheng C

PROVIDER: S-EPMC8383737 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lactobacillus strain SQ0048 isolated from bovine vagina has been shown to exhibit specific adherence to the epithelium and to produce inhibitory substances; however, the underlying mechanisms remain unclear. We cultured and identified primary bovine vaginal epithelial cells treated with SQ0048 to investigate the pathways involved in host cell responses using transcriptome sequencing (RNA-seq). Transcription profiling showed 296 significantly altered differentially expressed genes (DEGs), of which 170 were upregulated and 126 downregulated. Gene Ontology (GO) enrichment analysis of the DEGs revealed significant enrichment of 424 GO terms throughout the differentiation process (P < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were successfully annotated as members of 171 pathways, with 23 significantly enriched KEGG pathways (P < 0.05). A relatively high number of genes were enriched for the endoplasmic reticulum protein processing and interleukin-17 (IL-17) signaling pathways and for antigen processing and presentation. DEGs were verified by quantitative reverse transcription-PCR (RT-qPCR) and determination of which were most enriched for endoplasmic reticulum protein processing pathways, the activation of which might be a major factor underlying Lactobacillus adhesion to cells and pathogenic inhibition.IMPORTANCE Bovine bacterial vaginitis causes infertility, abortion, and postpartum uterine diseases, causing serious economic loss to the dairy industry. The large-scale use of antibiotics destroys normal genital tract flora and hinders the defense mechanisms of the host. Recent research suggests that lactobacilli present in the vaginal microflora of healthy cows constitute the primary microbiological barrier to infection by genital pathogens, exerting a protective role on the reproductive tract via specific adherence to the epithelium and the production of inhibitory substances. Our research identified the mechanisms for Lactobacillus adhesion and pathogenic inhibition, providing valuable information for the development of new probiotics and the discovery of novel therapeutic targets for the prevention of infections in dairy cows.

Project description:Purpose: The goals of this study is to investigate host cell responses treated with lactobacillus in various molecular pathways by RNA-Seq and real-time quantitative PCR (RT-qPCR) methods to and evaluate protocols for optimal high-throughput data analysis. Methods: mRNA profiles of bovine vaginal epithelial cells treated with lactobacillus and control cells were generated by sequencing, in triplicate, utilizing Illumina HiSeq 4000 platform. The raw RNA-seq data was filtered to remove adaptor contamination, low quality bases, and undetermined bases utilizing Cutadapt software.The filtered reads were aligned and mapped to the reference genome using Hisat 2.0. RT-qPCR validation was performed using TaqMan and SYBR Green assays. Results: The transcription profiling results showed that 296 differentially expressed genes (DEGs) were significantly altered, of which 170 were upregulated and 126 downregulated. Gene ontology (GO) enrichment analysis of the DEGs revealed a significant enrichment of 424 GO terms throughout the differentiation process (p< 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were mainly involved in signal transduction, immune endocrine systems, transport and catabolism, as well as metabolism and genetic information processing. We found a relatively high number of genes enriched in protein processing in the endoplasmic reticulum and IL-17 signaling pathway, as well as antigen processing and presentation. DEGs that enriched into pathways were verified by RT-qPCR. The highest number of DEGs participated in protein processing in pathways involving the endoplasmic reticulum of which might be a major factor causing the adhesion of lactobacillus to cells and pathogenic inhibition. Conclusions:Our study provides new insights on the interaction between lactobacillus and host cells, emphasizing the importance of novel candidate pathways involving the protein processing in endoplasmic reticulum and analyzing the possible anti-pathogenic mechanisms of the SQ0048 strain that inhibit pathogenic colonization.

Project description:BackgroundContrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI.MethodsOn the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells.ResultsMEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1β, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65).ConclusionAtorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.

Project description:Dairy farming is the most important economic activity in animal husbandry. Mastitis is the most common disease in dairy cattle and has a significant impact on milk quality and yield. The natural extract allicin, which is the main active ingredient of the sulfur-containing organic compounds in garlic, has anti-inflammatory, anticancer, antioxidant, and antibacterial properties; however, the specific mechanism underlying its effect on mastitis in dairy cows needs to be determined. Therefore, in this study, whether allicin can reduce lipopolysaccharide (LPS)-induced inflammation in the mammary epithelium of dairy cows was investigated. A cellular model of mammary inflammation was established by pretreating bovine mammary epithelial cells (MAC-T) with 10 µg/mL LPS, and the cultures were then treated with varying concentrations of allicin (0, 1, 2.5, 5, and 7.5 µM) added to the culture medium. MAC-T cells were examined using RT-qPCR and Western blotting to determine the effect of allicin. Subsequently, the level of phosphorylated nuclear factor kappa-B (NF-κB) was measured to further explore the mechanism underlying the effect of allicin on bovine mammary epithelial cell inflammation. Treatment with 2.5 µM allicin considerably decreased the LPS-induced increase in the levels of the inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and inhibited activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in cow mammary epithelial cells. Further research revealed that allicin also inhibited the phosphorylation of inhibitors of nuclear factor kappa-B-α (IκB-α) and NF-κB p65. In mice, LPS-induced mastitis was also ameliorated by allicin. Therefore, we hypothesize that allicin alleviated LPS-induced inflammation in the mammary epithelial cells of cows probably by affecting the TLR4/NF-κB signaling pathway. Allicin will likely become an alternative to antibiotics for the treatment of mastitis in cows.

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Effect of Lactobacillus johnsonii Strain SQ0048 on the TLRs-MyD88/NF-κB Signaling Pathway in Bovine Vaginal Epithelial Cells.

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