Multi-omics Analysis at Serum Proteomic and Metabolomic Levels Reveals Mechanisms of Moxibustion for a Mouse Model of Ankylosing Spondylitis

Abstract

Background: Moxibustion is widely used in ameliorating symptoms of ankylosing spondylitis (AS). The aim of this study was to identify the potential molecular profiles of moxibustion on relieving AS mice through incorporating ultra-high-performance liquid chromatography quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF/MS) based metabolomics approach and data independent acquisition-mass spectrometry (DIA-MS) based proteomic. Methods: In this research, mice with proteoglycan-induced spondylitis (PGISp) were intervened with moxibustion for four weeks at specific acupuncture points or at non-acupuncture points. Arthritis severity, histopathological examinations, cytokines and osteoblast (OB) related genes were assessed. Protein profiling and metabolite profiling of serum of mice were examined by UHPLC-Q-TOF/MS based metabolomics technology and DIA-MS based proteomic approach. A multi-omic analysis was implemented for integrating the results of the single proteomic and metabolomic. The levels of some novel proteins in significantly enriched integrated pathways were validated by the enzyme-linked immunosorbent assay (ELISA) method. Results: Our findings clearly indicate that acupuncture points’ moxibustion can significantly decrease the arthritis index (AI) score, improve the histopathological examination and reduce the serum levels of C-reactive protein (CRP), Interferon-γ (IFN-γ), Interleukin-17 (IL-17), Alkaline phosphatase (ALP) and Osteocalcin (OCN) for AS mice. A total of 21 differentially expressed metabolites and 21 differentially expressed proteins (DEPs) were identified as potential moxibustion-intervened biomarkers for AS mice. These molecules were mainly involved in six integrative canonical pathways: mineral absorption, lipid metabolism, purine metabolism, glycolysis/gluconeogenesis, glycine, serine and threonine metabolism and phenylalanine metabolism pathways. Furthermore, The ELISA analysis of four key proteins was consistent with the results of the proteomic analysis. Conclusions: Combing UHPLC-Q-TOF/MS based metabolomics approach and DIA-MS based proteomic, as a promising tool, can advance our understanding of potential mechanisms of action of moxibustion for AS from a holistic perspective.

Research Progress on Molecular Mechanism of Moxibustion in the Treatment of Ankylosing Spondylitis

“…37 At present, many original pieces of research have explored the bone metabolism regulation mechanism of moxibustion intervention in AS, which are summarized below. Xu et al 38 revealed that moxibustion at acupoints of Shenshu (BL 23), Zusanli (ST36), and Mingmen (GV4) of AS mice could effectively inhibit the overexpression of ALP and OCN, reduce the new bone formation, and improve the mobility of the spine in AS mice. Lin and Wu 39 explored the effect of medicineseparated moxibustion on the pain and bone metabolism of AS.…”

“…The results showed that moxibustion could achieve the balance of intestinal flora by down-regulating the number of sulfate-reducing bacteria and Bacteroides fragilis in intestinal microorganisms. Xu et al 38 found that moxibustion could reduce the permeability of the intestinal tract and improve the mechanical barrier function of colon by down-regulating the L-phenylalanine of intestinal flora and up-regulating the expression of short-chain fatty acid and intestinal mucosal synthetic protein L-threonine. Moreover, moxibustion could prohibit the inflammatory signaling pathway, up-regulate the protein expression of Foxp3 and IL-10, and finally improve the intestinal defense against inflammation.…”