ABSTRACT
Notoginsenoside R1 (NGR1) is a neoteric phytoestrogen extracted from Panax notoginseng, and possesses comprehensive pharmacological functions in multitudinous ailments. But, whether NGR1 is utilized in neonatal pneumonia is not clear. This research study aspired to disclose the protective activity of NGR1 in neonatal pneumonia. WI-38 cells were co-stimulated with NGR1 and lipopolysaccharide (LPS, 10 ng/mL), CCK-8 and flow cytometry assays were implemented for cell viability and apoptosis assessment. Real-time quantitative plymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot analysis were executed for inflammatory cytokine determination. MicroRNA-181a (miR-181a) expression was evaluated through RT-qPCR, simultaneously, the impact of miR-181a was estimated in NGR1 and LPS co-managed cells. Dual luciferase report assay was performed to disclose the relation between miR-181a and Toll-like receptor 4 (TLR4). The nuclear factor-κB (NF-κB) and TAK1/JNK pathways were ultimately appraised. We found that NGR1 decreased cell viability, evoked apoptosis and impeded interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) expression and secretions in LPS-managed WI-38 cells. MiR-181a expression was enhanced by NGR1, and miR-181a inhibition inverted the impacts of NGR1 in LPS-managed WI-38 cells. Besides, TLR4 was predicted to be a firsthand direct target of miR-181a. Furthermore, NGR1 hindered NF-κB and TAK1/JNK pathways through modulating TLR4. These discoveries disclosed the fact that NGR1 protected WI-38 cells against LPS-triggered injury via adjusting the miR-181a/TLR4 and NF-κB and TAK1/JNK pathways.
Subject(s)
Ginsenosides/pharmacology , Lung/cytology , MicroRNAs/metabolism , Toll-Like Receptor 4/metabolism , Cell Line , Cell Proliferation/drug effects , Cytokines/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Lipopolysaccharides/toxicity , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/drug effects , NF-kappa B/metabolism , Pneumonia/metabolism , Pneumonia/pathology , Protective Agents/pharmacology , Toll-Like Receptor 4/geneticsABSTRACT
A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6.
