Coelonin protects against PM2 .5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1ß. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

Inhibition of inflammation-induced injury and cell migration by coelonin and militarine in PM2.5-exposed human lung alveolar epithelial A549 cells.

Accumulating studies suggest that fine particulate matter (PM2.5) pollutants in the air are easily enter into alveoli and even the bloodstream, resulting in an inflammatory response that not only triggers respiratory disorders but also causes permanent damage to various organs. Recent findings suggest that coelonin and militarine enriched in orchids can inhibit inflammation-induced injury against respiratory diseases. Here, we evaluated the anti-inflammatory properties of coelonin and militarine and examined their underlying molecular mechanisms in A549 cells exposed to PM2.5. PM2.5 induced significant intracellular reactive oxidative stress accumulation at a concentration of 250 µg/ml, as determined using the dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay. Cell viability was assessed via the MTS assay to determine the concentrations of compounds appropriate for use in subsequent experiments. Data from the enzyme-linked immunosorbent assay (ELISA) showed that both coelonin (10 and 20 µg/ml) and militarine (5 and 10 µg/ml) mitigated PM2.5-induced inflammation by reducing the generation of inflammatory factors, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Quantitative real-time PCR (qRT-PCR) analysis revealed a remarkable decrease in IL-6, TNF-α, cyclooxygenase-2 (COX-2) and interleukin-1ß (IL-1ß) mRNA levels in the coelonin and militarine-pretreated groups. In Western blot analysis, expression of inhibitor of NF-κB (IκBα) protein in the coelonin and militarine pretreatment groups was significantly increased compared with the PM2.5 (only) treatment group (P < 0.05), concomitant with a significant decrease in phospho-IκB kinase ß/IκB kinase ß (p-IKKß/IKKß), phospho-nuclear factor of kappa B p65/nuclear factor of kappa B p65 (p-NF-κBp65/NF-κBp65) and COX-2 proteins (P < 0.05). Both coelonin and militarine inhibited migration and inflammation by suppressing PM2.5-induced IKK phosphorylation, and followed by IκBα protein degradation and NF-κB activation. Our collective data strongly supported the utility of coelonin and militarine as novel sources for development of treatments for PM2.5-induced lung diseases.

Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential Mechanism.

Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 µg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1ß, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

[Comparison of contents of three active ingredients in Bletilla striata from different sources].

In order to understand the difference of contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 µm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 ℃ and the injection volume was 4µL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin Ⅲ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.

Comparison of contents of three active ingredients in Bletilla striata from different sources / 中国中药杂志

In order to understand the difference of contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 μm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 ℃ and the injection volume was 4μL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin Ⅲ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.