Polypeptide from Moschus Suppresses Lipopolysaccharide-Induced Inflammation by Inhibiting NF-B-ROS/NLRP3 Pathway

Chinese journal of integrative medicine(2023)

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Abstract
Objective: To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus (PPM) in lipopolysaccharide (LPS)-induced THP-1 macrophages and BALB/c mice. Methods: The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice. In LPS-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays; the proinflammatory cytokines and reactive oxygen species (ROS) were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively; and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR), respectively. In LPS-induced BALB/c mice, the proinflammatory cytokines were measured, and lung histology and cytokines were observed by hematoxylin and eosin (HE) and immunohistochemical (IHC) staining, respectively. Results: The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10-26 kD. In vitro, PPM reduced the production of interleukin 1 beta (IL-1 beta), IL-18, tumor necrosis factor alpha (TNF-alpha), IL-6 and ROS in LPS-induced THP-1 macrophages ( P< 0.01). Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factor kappa B (NF-kappa B) pathway and thioredoxin interacting protein (TXNIP)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome pathway by reducing protein expression of phospho-NF-kappa B p65, phosphoinhibitors of NF-kappa B (I kappa Bs) kinase alpha/beta (IKK alpha/beta), TXNIP, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1 ( P< 0.05 or P< 0.01). In addition, qRT-PCR revealed the inhibitory effects off PPM on the mRNA levels of TXNIP, NLRP3, ASC, and caspase-1 ( P< 0.05 or P< 0.01). Furthermore, in LPS-induced BALB/c mice, PPM reduced TNF-alpha and IL-6 levels in serum ( P< 0.05 or P< 0.01), decreased IL-1 beta and IL-18 levels in the lungs ( P< 0.01) and alleviated pathological injury to the lungs. Conclusion: PPM could attenuate LPS-induced inflammation by inhibiting the NF-kappa B-ROS/NLRP3 pathway, and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.
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Key words
Moschus, polypeptide, inflflammation, NLRP3 inflammasome, thioredoxin interacting rotein, nuclear factor kappa B, Chinese medicine
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