CYPs x Prostaglandin-endoperoxide synthase family x lipoxygenase

Megakaryocytes, a type of myeloid cell primarily produced in the bone marrow, are essential for releasing platelets into the bloodstream. Through platelet production, megakaryocytes transfer their own biological content to these cells, including inflammatory enzymes. Amongst these enzymes, 12-lipoxygenase (12-LO) has been shown to modulate platelet activation and is involved in several chronic inflammatory conditions. However, the role of 12-LO in megakaryocyte maturation and the subsequent release of platelets remains uninvestigated. This study demonstrates the importance of 12-LO expression in megakaryocyte maturation and functions. Flow cytometry and fluorescence microscopy were utilized to analyze DAMI cell differentiation. Inflammatory enzyme profiles were assessed through Western blot analysis and LC-MS/MS. To evaluate megakaryocyte functionality, platelet-type 12-LO knockout mice were employed. Differentiation of DAMI cells into mature megakaryocytes resulted in increased surface marker expression, enhanced platelet-like particle production, reduced cell proliferation, and elevated cell death. The arachidonic acid release from cell membranes was increased in differentiated cells as well as the expression of several inflammatory enzymes such as 12-LO, cyclooxygenase-1 and thromboxane synthase. The production of 12(S)-hydroxyeicosatetraenoic acid and thromboxane was also increased in these cells. In DAMI cells deficient in 12-LO expression (ALOX12^-/-), we found no difference in CD41 expression following differentiation compared to ALOX12^+/+ cells. However, 12-LO-deficient cells produced fewer proplatelets and, consequently, fewer platelet-like particles. In vivo, platelet counts in 12-LO-deficient mice were comparable to those in wild-type mice, and the number of bone marrow megakaryocytes remained consistent between the groups. Ex vivo analysis revealed that megakaryocytes lacking 12-LO produced fewer platelets. These findings indicate that 12-LO may play a regulatory role in megakaryocyte function, particularly in proplatelet formation and platelet release.

The most effective lipid mediators in inflammation are eicosanoids, which involve numerous enzymes such as cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP). Arctoscopus japonicus lipids (AJL) have been shown to inhibit inflammation, but their effects on cellular mechanisms via lipid mediators remain unclear. In this study, we examined the eicosanoid synthesis pathways for the anti-inflammatory effects of AJL in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. AJL significantly reduced LPS-activated macrophages production of pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), as well as the expression of interferon-γ (IFN-γ) and IL-12. It also increased the expression of anti-inflammatory cytokines transforming growth factor (TGF)-β and IL-10. AJL inhibited LPS-activated COX, LOX, and CYP metabolites from producing thromboxane A₂ (TXA₂), prostaglandins (PG) H₂, PGI₂, PGE₂, and PGF_2α, leukotriene B₄ (LTB₄), as well as epoxyeicosatrienoic acids (EETs). Furthermore, AJL decreased LPS-induced the expression of both of COXs (COX-1 and COX-2) and 5-LOX mRNAs and proteins in the COX and LOX pathways, respectively. AJL also suppressed the expression of CYP4A11 proteins in the CYP pathway. Furthermore, CD86 expression was down regulated by AJL in LPS-stimulated cells. These findings conclusively reveal that AJL modulates eicosanoid synthesis pathways, contributing to its anti-inflammatory properties.

Ershen Wan (ESW), a classic traditional Chinese medicine (TCM) prescription composed of Psoralea corylifolia Linn. and Myristica fragrans Houtt., has been applied to treat gastrointestinal disorders in clinical practices for thousands of years. However, its potential molecular mechanism in alleviating ulcerative colitis (UC) remains to be elusive.

The purpose of the study is to explore the underlying mechanism of ESW in treating UC.

The protective effect of ESW on dextran sodium sulfate (DSS)-induced UC mice was assessed by body weight, disease activity index (DAI), colon length, colon tissue pathology, and colonic inflammatory factors. Furthermore, network pharmacology was applied to dissect the possible targets and biological pathways regulated by ESW. The plasma and fecal metabolomics were comprehensively analyzed by UPLC-Q-TOF/MS. Subsequently, an efficient and feasible approach integrating network pharmacology, metabolomics, and molecular docking was used to explore the key targets obtained from the metabolite-reaction-enzyme-gene network. And the effect of ESW on the MAPK signaling mediated intestinal epithelial cell apoptosis was further investigated by in vitro and in vivo experiments.

ESW could notably alleviate colon injury and inflammation of UC mice. Network pharmacology suggested that the bioactive components of ESW could mainly modulate signaling pathways associated with inflammation and metabolism. Consistently, plasma and fecal metabolomics further indicated that ESW could regulate the metabolic pathways of arachidonic acid, linoleic acid, sphingolipid, tryptophan, and glycerophospholipid. And the combined analysis of network pharmacology and metabolomics revealed that 14 pivotal targets were modulated by ESW, including PTGS1, PTGS2, CYP1A1, FADS1, CBR1, ALOX5, EPHX1, EPHX2, HPGD, PLA2G1B, PLA2G7, MGLL, ACHE, and SPHK1. Additionally, molecular docking suggested that bioactive components of ESW could bind well to these potential targets. And in vitro and in vivo experiments further verified that ESW could markedly ameliorate pathological symptoms of UC mice through inhibiting MAPK signaling mediated colonic epithelial cell apoptosis.

Collectively, these findings indicated that ESW could effectively alleviate the pathological symptoms of UC mice, mainly involving in the modulation of lipid and amino acid metabolism pathways, and the suppression of MAPK signaling-mediated apoptosis. In this study, the potential mechanism of ESW for the treatment of UC was first clarified, which provided a solid scientific foundation for its clinical application. Notably, the proposed strategy facilitated a comprehensive prediction and validation of the efficacy and molecular mechanism of TCMs, and also provided a novel approach for revealing the intricate biological pathogenesis of diseases.