Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics

Abstract

Background: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC.

Aim: To investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology.

Methods: Male Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs.

Results: The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01).

Conclusion: EA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

Keywords: Differential proteins; Electroacupuncture; Moxibustion; Proteomics; Ulcerative colitis.

Figure 1

Schematic diagram of herb-partitioned moxibustion at the rat Tianshu (ST25, bilateral) and Qihai (CV6) acupoints.

Figure 2

Flow chart of proteomics detection and analysis. A: Sprague-Dawley rat; B: Protein extraction of colon tissue; C: Protein enzymatic digestion, peptide labelling, separation, and high-performance liquid chromatography analysis; D: Bioinformatics analysis.

Figure 3

Colon tissue injuries in each group. A: Gross structure; B: Histopathological observation (hematoxylin and eosin, × 200); C: Macroscopic scores; D: Histopathological scores; E: Colonic length. ^aP < 0.01 vs N group; ^bP < 0.01 vs M group. N: Normal group; M: Dextran sulfate sodium-induced ulcerative colitis model group; HM: Herb-partitioned moxibustion group; EA: Electroacupuncture group.

Figure 4

Volcano plot of differentially expressed proteins between groups. A: Volcano plot of differentially expressed proteins (DEPs) between the normal and dextran sulfate sodium-induced ulcerative colitis model group; B: Volcano plot of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and herb-partitioned moxibustion group; C: Volcano plot of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and electroacupuncture group.

Figure 5

Venn diagram of differentially expressed proteins among groups. A: Venn diagram of differentially expressed proteins (DEPs) in dextran sulfate sodium-induced ulcerative colitis model group that can be regulated by herb-partitioned moxibustion; B: Venn diagram of DEPs in dextran sulfate sodium-induced ulcerative colitis model group that can be regulated by electroacupuncture. N: Normal group; M: Dextran sulfate sodium-induced ulcerative colitis model group; HM: Herb-partitioned moxibustion group; EA: Electroacupuncture group.

Figure 6

Biological function annotation of differentially expressed proteins between groups. A: Biological function annotation of differentially expressed proteins (DEPs) between the normal and dextran sulfate sodium-induced ulcerative colitis model group; B: Biological function annotation of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and herb-partitioned moxibustion group; C: biological function annotation of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and electroacupuncture group.

Figure 7

Kyoto Encyclopedia of Genes and Genomes pathway enrichment of differentially expressed proteins between groups. A: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed proteins (DEPs) between the normal and dextran sulfate sodium-induced ulcerative colitis model group; B: KEGG pathway enrichment of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and herb-partitioned moxibustion group; C: KEGG pathway enrichment of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and electroacupuncture group. Each point in the KEGG pathway enrichment plot represents a KEGG pathway, with the left axis showing the pathway name and the abscissa showing the enrichment factor, which represents the ratio of the number of DEPs annotated to that pathway to the number of proteins annotated to that pathway for that species’ protein. A larger enrichment factor indicates more reliable enrichment of DEPs in the pathway.

Figure 8

Protein-protein interaction network of differentially expressed proteins between groups. A: Protein-protein interaction (PPI) network of differentially expressed proteins (DEPs) between the normal and dextran sulfate sodium-induced ulcerative colitis model group; B: PPI network of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and herb-partitioned moxibustion group; C: PPI network of DEPs between the dextran sulfate sodium-induced ulcerative colitis model and electroacupuncture group. Circle colours indicate changes in protein expression, with red indicating up-regulation and green indicating down-regulation, and line thickness indicates interaction intensity.

Figure 9

Verification of differentially expressed proteins expressions by western blot. A and B: Band diagrams of protein expressions. All data are expressed as mean ± SD; C: Synaptic vesicle glycoprotein 2A; D: Nuclear cap binding protein subunit 1; E: Carbamoyl phosphate synthetase 1; F: Cytochrome c oxidase subunit 4 isoform 1; G: ATP synthase beta subunit precursor 1; H: Doublecortin like kinase 3. ^aP < 0.01 vs normal group; ^bP < 0.01 vs dextran sulfate sodium-induced ulcerative colitis model group. N: Normal group; M: Dextran sulfate sodium-induced ulcerative colitis model group; HM: Herb-partitioned moxibustion group; EA: Electroacupuncture group; Sv2a: Synaptic vesicle glycoprotein 2A; Ncbp1: Nuclear cap binding protein subunit 1; Cps1: Carbamoyl phosphate synthetase 1; Cox4i1: Cytochrome c oxidase subunit 4 isoform 1; Atp5f1: ATP synthase beta subunit precursor; Dclk3: Doublecortin like kinase 3.