ISGylation Inhibits an LPS-Induced Inflammatory Response via the TLR4/NF-κB Signaling Pathway in Goat Endometrial Epithelial Cells.
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ABSTRACT:
SUBMITTER: Xiao J
PROVIDER: S-EPMC8470639 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Inflammation plays a crucial role in the progression of Subacute Ruminal Acidosis (SARA). The experiment was designed to investigate anti-inflammatory effects of glycyrrhizin on goats ruminal epithelial cells (GREC) which were induced SARA by Lipopolysaccharide (LPS) in vitro. The GREC were induced SARA by adding LPS at the concentration of 5 μm and glycyrrhizin was added at different concentration of 0, 60, 90, 120, 150 μm. The structural integrity of LPS-induced GREC with the treatment of glycyrrhizin were observed by electron microscope; The levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured by ELISA; The number of Zo-1 and Occludin were measured, the expression of tight junction protein Occludin were measured by Western blot, and the mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12 were measured in vitro. The results showed that higher concentration treatment of glycyrrhizin led to better morphology in LPS-induced GREC. Glycyrrhizin inhibited the growth of inflammatory factors TNF-α, IL-1β, IL-6, IL-8 and IL-12 in a dose-dependent manner. The number of ZO-1 and Occludin increased with the increase of adding of glycyrrhizin. Western blot analysis showed that the expression of tight junction protein Occludin in LPS-induced GREC increased with the adding of glycyrrhizin in a dose-dependent manner. Furthermore, the mRNA expression of NF-κB, TNF-α, IL-1β, IL-6, IL-8 and IL-12 decreased significantly with the increase treatment of glycyrrhizin. Glycyrrhizin significantly inhibits LPS-induced inflammatory mediators in GREC and the effects are better with the increase treatment of glycyrrhizin in vitro.
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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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Project description:ObjectiveThe aim of this study was to investigate the anti-inflammatory effect of Rosiglitazone (RGZ) on lipopolysaccharide (LPS) -induced Endometritis and explore its possible mechanism.MethodsThe preventive and therapeutic effects of RGZ on Endometritis were studied in vivo and in vitro. A total of 40 female C57BL/6 mice were randomly divided into the following 4 groups: RGZ+LPS, RGZ control, LPS and DMSO control. The mice uterine tissue sections were performed with HE and immunohistochemical staining. Human endometrial stromal cells (HESCs) were cultured, and different concentrations of LPS stimulation groups and RGZ and/or a TLR4 signaling inhibitor TAK-242 pretreatment +LPS groups were established to further elucidate the underlying mechanisms of this protective effect of RGZ.ResultsThe HE results in mice showed that RGZ+LPS group had less tissue loss than LPS group. Immunohistochemical staining (IHC) results showed that the expression of TLR4 after RGZ treatment was significantly lower than that in LPS group. These findings suggested that RGZ effectively improves the pathological changes associated with LPS-induced endometritis by inhibiting TLR4. Reverse transcription-polymerase chain reaction and western blot analysis demonstrated that RGZ pretreatment suppresses the expression of Toll-like receptor 4 (TLR4) and its downstream activation of nuclear factor-κB (NF-κB). In vitro, RGZ inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner and also downregulated LPS induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein.ConclusionsThese results suggest that RGZ may inhibit LPS-induced endometritis through the TLR4-mediated NF-κB pathway.
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Project description:Catalpol is the main active ingredient of an extract from Radix rehmanniae, which in a previous study showed a protective effect against various types of tissue injury. However, a protective effect of catalpol on uterine inflammation has not been reported. In this study, to investigate the protective mechanism of catalpol on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and mouse endometritis, in vitro and in vivo inflammation models were established. The Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB), and immunofluorescence techniques. The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) and CXCL5, both in bEECs and in uterine tissue. From the experimental results of WB, qRT-PCR, and immunofluorescence, the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group. The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase (MPO) activity. The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.
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Project description:Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment.
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