Isolation and Purification of Musk Peptides and Study on Their Anti inflammatory Mechanisms Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS) is a serious and complex respiratory disease characterized by damage to alveolar epithelial cells and vascular endothelial cells, hypotension, and alveolar edema, ultimately leading to acute hypoxic respiratory failure. The pathogenesis of ALI/ARDS is complex, with dysregulation of inflammatory response, increased permeability of pulmonary endothelial cells and epithelium leading to disruption of the pulmonary microvascular barrier being the core of the pathophysiological disorders of ALI/ARDS. Wu et al. reported that macrophage pyroptosis is involved in the pathogenesis of lipopolysaccharide induced ALI/ARDS, and the Caspase-1 specific inhibitor Ac YVADCMK can alleviate lung tissue damage and reduce the production of pro-inflammatory cytokines.

Musk (Moschus) is a dry secretion from the mature male sachet of the deer family animals Moschus berezovskii Flerov, Moschus sifanicus Przewalski, or Moschus moschiferus Linnaeus. Musk is warm in nature, pungent in taste, and belongs to the heart and spleen meridians. It has the effects of opening the orifices, awakening the mind, promoting blood circulation, unblocking collaterals, reducing swelling, and relieving pain. Modern medical research has found that musk has complex chemical components and pharmacological effects such as anti-inflammatory, anti dementia, anti-tumor, anti cerebral ischemia, immune regulation, and hormone regulation. Zhu et al. have demonstrated strong anti-inflammatory activity of musk peptides in various in vivo inflammatory models such as croton oil ear inflammation in mice, yeast and agar arthritis in rats, and scald inflammation in rats, but the anti-inflammatory mechanism is still unclear. This study aims to extract and purify musk peptides from natural musk, investigate the anti-inflammatory activity of musk peptides in lipopolysaccharide induced THP-1 in vitro cell models and acute lung injury mouse models, and explore their potential molecular mechanisms of action, in order to provide reference for further research, development, and clinical application of the precious Chinese medicinal herb musk.

 

 

Bioactive peptides, due to their high selectivity, predictable metabolism in vivo, low toxicity, and ease of synthesis, have attracted widespread attention from researchers both domestically and internationally as potential therapeutic drugs for various diseases. According to reports, musk peptides are one of the anti-inflammatory active ingredients in natural musk. Japanese scholars Masayasu Kimura and others have isolated for the first time a peptide with a molecular weight of about 1000 from musk, which has strong anti-inflammatory activity and can significantly inhibit the migration of guinea pig white blood cells. Its strength of action is about 40 times that of hydrocortisone. Zhu et al. confirmed in a mouse ear swelling model induced by croton oil that the anti-inflammatory effect of musk 21 is three times that of hydrocortisone, the anti-inflammatory effect of musk 65 is about six times that of hydrocortisone, and the anti-inflammatory strength of musk 1 is 36 times that of hydrocortisone. Liu et al. separated Mua-1 with a molecular weight of about 200000 through gel filtration method, which has a significant inhibitory effect on ear inflammation of croton oil mice, with an inflammation inhibition rate of 72.3%. THP-1 monocytes can differentiate into macrophages induced by PMA, exhibiting a phenotype and function similar to that of human primary macrophages. The THP-1 cell line has been widely used as an in vitro model for human monocytes and macrophages for studying the mechanisms of inflammatory diseases. LPS is the active ingredient in the cell wall of Gram negative bacteria, which can cause strong inflammatory reactions and is often used as an inducer for in vivo and in vitro inflammation models. In this study, the author used an ion exchange chromatography column to separate and purify five fractions from natural musk extract, namely SXP1 SXP2、SXP3、 SXP4、SXP5。 In the LPS induced THP-1 macrophage model, SXP4 was found to significantly inhibit the production of TNF – α and IL-1 β, demonstrating strong anti-inflammatory activity. In addition, SDS-PAGE analysis revealed that the molecular weight of peptide proteins in SXP4 mainly ranges from 10 to 26 kDa.

Acute lung injury and more severe acute respiratory distress syndrome have a high mortality rate, characterized by pulmonary inflammation caused by infection, trauma, lung tissue contusion, or aspiration of gastric contents. This study found that SXP4 can significantly improve the pathological damage of mouse lung tissue, reduce the expression of TNF – α, IL-6 in serum and IL-1 β, IL-18 in lung tissue of mice with lipopolysaccharide induced acute lung injury. Pyroptosis is a programmed cell death form that relies on the enzymatic activity of Caspase family members to mediate the formation of transmembrane pores on the cell membrane by Gasdermin family proteins, causing cell swelling and rupture, as well as the release of inflammatory mediators such as IL-1 β, IL-18, HMGB1, and promoting inflammatory response. Research has shown that alveolar macrophage pyroptosis plays an important role in acute lung injury. Therefore, inhibiting cell pyroptosis is a potential therapeutic target for acute lung injury.

There are two activation pathways for cell pyroptosis, namely the classical pathway dependent on Caspase-1 and the non classical pathway dependent on Caspase-4, -5/-11, both of which are accompanied by Gasdermin D (GSDMD) lysis and release of IL-1 β and IL-18. The classic pathway of cell pyroptosis is mediated by inflammasomes. Inflammatory bodies are large molecular complexes within cells, composed of sensor NOD like receptors (NLRs), adaptor protein apoptosis related spot like protein (ASC), and effector Caspase-1 precursor (ProCaspase-1). Among them, NLRP3 is the most extensively studied member in the NLRs family. Grailer et al. found that NLRP3 inflammasome and Caspase-1 play a crucial role in the pathogenesis of acute lung injury. In LPS induced acute lung injury mice, lung tissue damage and inflammatory response were significantly reduced in NLRP3-/- mice and Caspase-1-/- mice. Activated NLRP3 recruits ASC and ProCaspase-1 to assemble into NLRP3 inflammasome, and Pro-Caspase-1 is cleaved into the active form Caspase-1. Caspase-1 cleaves GSDMD, exposing its amino acid end domain and inducing cell membrane perforation, leading to cell swelling and lysis. At the same time, Caspase-1 also processes IL-1 β and IL-18 precursors (Pro-IL-1 β, Pro-IL-18), releasing mature IL-1 β and IL-18 through pores on the cell membrane. The immunohistochemical results of this study showed that SXP4 can significantly reduce the expression levels of NLRP3, ASC, Caspase-1, and GSDMD in lung tissue of mice with acute lung injury (see Figure 13).

In summary, musk peptide SXP4 may reduce the production of pro-inflammatory cytokines, alleviate the inflammatory response in mice with acute lung injury, and alleviate lung tissue pathological damage by inhibiting the NLRP3/Caspase-1-mediated cell apoptosis pathway.