Esculetin inhibits the pyroptosis of microvascular endothelial cells through NF-κB /NLRP3 signaling pathway.

The effect of Esculetin on pyroptosis and its possible mechanism in endothelium were explored. 10 µg/mL LPS and 0.5 mM ATP were used to stimulate the rat intestinal microvascular endothelial cells. Then add different concentrations of Esculetin (20µM, 40 µM) to the culture medium containing LPS and ATP culturing for 24 h. The expression of p-NF-κB p65, NF-κB p65, I-κB, p-I-κB, NLRP3, ASC, caspase-1, and gasdermin-D were detected by Western blot, and the release level of IL-18 and IL-1ß were measured by ELISA. The NLRP3 inhibitor MCC950 was used at the concentration of 10 µM for 4 h to disentangle the potential mechanism of the influence of Esculetin on pyroptosis. In our experiments, the expression of gasdermin-d and important proteins of NF-κB and NLRP3 signaling pathways were inhibited by Esculetin. Besides, Esculetin also attenuated the morphological changes like swelling rupture and pores on the membrane caused by pyroptosis thereby protecting cells from being damaged by pyroptosis. Combining with the effect of Esculetin on proteins above and its protective effect on cell morphology, we believe that Esculetin has an anti-pyroptosis effect. The inhibiting pyroptosis effects mentioned above are similar to MCC950, which means the anti-pyroptosis effects of Esculetin are associated with the NLRP3 signaling pathway. In conclusion, Esculetin inhibits the pyroptosis of microvascular endothelial cells through the NF-κB/NLFP3 signaling pathway and is expected to be conducive in treating pyroptosis-related diseases.

Coixol Suppresses NF-κB, MAPK Pathways and NLRP3 Inflammasome Activation in Lipopolysaccharide-Induced RAW 264.7 Cells.

Coixol, a plant polyphenol extracted from coix (Coix lachryma-jobi L.var.ma-yuen Stapf), has not been investigated for its anti-inflammatory effect. In this study, using a lipopolysaccharide (LPS)-induced macrophage cell model, we observed that coixol can effectively reduce the expression of interleukin (IL)-1ß, IL-6, IL-18, tumor necrosis factor (TNF)-α, nitric oxide (NO), inducible nitric oxide synthases (iNOS), and cyclooxygenase (COX)-2, but had no effect on the expression of the anti-inflammatory mediator IL-10. Furthermore, we found that coixol inhibits mitogen-activated protein kinases (MAPKs), nuclear transcription factor κ B (NF-κB) pathways, and NOD-like receptor protein (NLRP) 3 inflammasome activation. In conclusion, the present study demonstrates that coixol exerts certain anti-inflammatory effects by inhibiting the expression of pro-inflammatory mediators in vitro. The mechanism of this effect was in part related to its ability to inhibit the activation of NF-κB, MAPKs pathways, and NLRP3 inflammasome.

Kaempferol prevents angiogenesis of rat intestinal microvascular endothelial cells induced by LPS and TNF-α via inhibiting VEGF/Akt/p38 signaling pathways and maintaining gut-vascular barrier integrity.

Kaempferol is a major flavonoid found in natural plant extracts; it shows great potential in anti-inflammatory and anti-cancer medicine. However, the underlying mechanism of the protective action of kaempferol on the gut-vascular barrier (GVB) and the active sites preventing intestinal micro-angiogenesis has not been reported. The purpose of our study is to investigate the protective effect of kaempferol on the barrier damage induced by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α), and its mechanism of protective action on intestinal micro-angiogenesis. Our data showed that the combination of LPS and TNF-α activates the inflammatory response of the rat intestinal microvascular endothelial cells (RIMVECs), leading to overexpression of vascular endothelial growth factors (VEGFs). Also, the permeability of GVB and transepithelial electrical resistance (TEER) constructed by Transwell and the tubular structure of RIMVEC were significantly affected. Kaempferol (25, 50, and 100 µM) decreased the inflammatory factor secretion and GVB permeability, down-regulated the expression of VEGFs, p-Akt, and hypoxia-inducible factor-1alpha (HIF-1α). It also alleviated the abnormal expression of tight junction proteins (TJs). Moreover, kaempferol may prevents intestinal angiogenesis in the presence of Akt inhibitor (MK-2206 2HCl) by regulating tube formation and downstream signaling of the VEGF/Akt pathways. In addition, the wound healing test showed that kaempferol had a similar effect in the presence of p38 inhibitor (SB203580), which intuitively restrained the migration of RIMVECs and reduced the p38 MAPK signaling. Our results demonstrated that kaempferol exhibits significant anti-inflammatory effects in LPS and TNF-α induced inflammatory environments. Kaempferol prevents intestinal angiogenesis by impeding the tube formation and migration of RIMVECs. It also suppresses the expression of angiogenesis-related signals, thereby protecting the GVB.

Naringenin prevents TNF-α-induced gut-vascular barrier disruption associated with inhibiting the NF-κB-mediated MLCK/p-MLC and NLRP3 pathways.

The microvasculature endothelium accurately regulates the passage of molecules across the gut-vascular barrier (GVB), which plays an essential role in intestinal immunity. Naringenin is reported to have therapeutic potential against several intestinal disorders. However, the effect of naringenin on GVB disruption has been rarely studied. This study aims to investigate the effect of naringenin on GVB function and the potential mechanism. In the present study, the in vitro GVB disruption of rat intestinal microvascular endothelial cells (RIMVEC) was induced by 50 ng mL-1 of tumor necrosis factor-α (TNF-α). The integrity of the in vitro GVB was determined by Evans blue (EB)-albumin efflux assay and trans-endothelial electrical resistance (TER). Meanwhile, the expression of tight junction proteins and the related NF-κB, MLCK/p-MLC and NLRP3 pathways were determined using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunofluorescence. The results show that naringenin (100 µM) inhibits TNF-α-induced interleukin (IL)-6 hypersecretion, alleviates GVB disruption and mitigates the change in the tight junction protein expression pattern. Naringenin inhibits the GVB-disruption-associated activation of the MLCK/p-MLC system and TLR4/NF-κB/NLRP3 pathways. Furthermore, naringenin shows a similar effect to that of NF-κB inhibitor Bay 11-7082 in reducing the TNF-α-induced activation of NLRP3, p-MLC and secondary GVB disruption. The results suggest that naringenin evidently alleviates TNF-α-induced in vitro GVB disruption via the maintenance of a tight junction protein pattern, partly with the inhibition of the NF-κB-mediated MLCK/p-MLC and NLRP3 pathway activation.

Coix Seed Diet Ameliorates Immune Function Disorders in Experimental Colitis Mice.

Coix seed is a functional food in the Chinese diet that possesses the ability to alleviate ulcerative colitis clinically. However, the underlying mechanisms remain ambiguous. In this study, we investigated the protective effect of the Coix seed diet on experimental colitis mice. The mice were randomly divided into four groups: control group, model group, Coix seed feed group, and positive control group. The maintenance feed of the mice was replaced with Coix seed feed 10 days before orally administering the mice 5% (w/v) dextran sulfate sodium drink. As a result, the Coix seed feed alleviated colitis symptoms, maintained the complete blood count at a normal level, reduced the pathological score, relieved inflammatory cytokine secretion, and alleviated oxidative stress. Network pharmacology analysis was used for further exploration of the targets of Coix seed feed. The results showed that T-cell regulation is one of the targets of Coix seed feed, and the analysis of the T-lymphocyte subset and innate immune cell distribution of the colon tissue supported the network pharmacology results. In conclusion, Coix seed, as a staple food, can alleviate experimental colitis, and the mechanism may be related to the immune regulation effect of Coix seeds.