Ji-Chuan decoction ameliorates slow transit constipation via regulation of intestinal glial cell apoptosis

Figure 1 Results of the constipation-related index. A: Particle counts of stool; B: Intestinal propulsive rate; C: The expression levels of colonic acetylcholine. ACH: Acetylcholine; HC: Healthy control; STC: Slow transit constipation; JCD: Ji-Chuan decoction; JCDL: Low-dose Ji-Chuan decoction treatment; JCDM: Middle-dose Ji-Chuan decoction treatment; JCDH: High-dose Ji-Chuan decoction treatment; MSP: Mosapride treatment. Compared with the healthy control group: ^aP < 0.05, ^bP < 0.01. Compared with the slow transit constipation group: ^cP < 0.05, ^dP < 0.01.

Figure 2 Histological changes in the distal colon. A: Distal colon of the healthy control group; B: In the distal colons of mice in the slow transit constipation group, more severe inflammatory infiltration was observed; C: In the distal colons of mice in the low-dose Ji-Chuan decoction (JCD) treatment group, more severe inflammatory infiltration was observed; D: In the distal colon of mice in the middle-dose JCD group, there was reduced inflammation; E: High-dose JCD treatment restored the normal tissue morphology of the distal colon of constipated mice; F: Mosapride treatment restored the normal tissue morphology of the distal colon of constipated mice. Sections were observed at 400 × using a light microscope.

Figure 3 Different intestinal metabolites detected by ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry. A: Principal component analysis; B: Orthogonal partial least squares discrimination analysis score plots of intestinal metabolic profiling in the healthy control (HC) vs slow transit constipation (STC) groups; C: STC vs high-dose Ji-Chuan decoction (JCDH) treatment groups under negative ion mode; D: Permutation test in negative ion mode of the HC vs STC groups; E: Permutation test in negative ion modes of the STC vs JCDH groups; F: Volcano plot of the different metabolites between the STC and HC groups; G: Volcano plot of the different metabolites between the JCDH and STC groups; H: Critical metabolic pathways of the STC vs JCDH groups. HC: Healthy control; STC: Slow transit constipation; JCD: Ji-Chuan decoction; JCDL: Low-dose Ji-Chuan decoction treatment; JCDM: Middle-dose Ji-Chuan decoction treatment; JCDH: High-dose Ji-Chuan decoction treatment; MSP: Mosapride treatment; MAG: 1-(6Z,9Z,12Z,15Z-Octadecatetraenoyl)-sn-glycerol; 11.12-EET: (5Z,8Z,14Z)-11,12-Epoxyeicosa-5,8,14-trienoic acid; LPE: 2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine; QC: Quality control.

Figure 4 Network construction, screening and validation of the core targets for Ji-Chuan decoction treatment of slow transit constipation. A: Protein-protein interaction (PPI) network; B: Top 30 protein targets with the largest betweenness in the PPI network; C: Betweenness analysis of the top 30 protein targets; D and E: The relative expression of AKT as assessed by immunofluorescent staining. AKT: Serine/threonine-protein kinase; HC: Healthy control; STC: Slow transit constipation; JCD: Ji-Chuan decoction; JCDL: Low-dose Ji-Chuan decoction treatment; JCDM: Middle-dose Ji-Chuan decoction treatment; JCDH: High-dose Ji-Chuan decoction treatment; MSP: Mosapride treatment; SLC6A4: Sodium-dependent serotonin transporter; AKT1: RAC-alpha serine/threonine-protein kinase; IL: Interleukin; NQO1: NAD(P)H dehydrogenase (quinone)1; GABRB2: Gammaaminobutyric acid receptor subunit beta-2; CASP3: Caspase-3; CAT: Catalase; ESR1: Estrogen receptor; MAPK3: Mitogen-activated protein kinase 3; FOS: Proto-oncogene c-Fos; VEGFA: Vascular endothelial growth factor A; EGFR: Epidermal growth factor receptor; EGF: Pro-epidermal growth factor; CAV1: Caveolin-1; JUN: Transcription factor AP-1; PTGS2: Prostaglandin G/H synthase 2; MAPK8: Mitogen-activated protein kinase 8; MYC: Myc proto-oncogene protein; MAPK1: Mitogen-activated protein kinase 1; CCND1: G1/S-specific cyclin-D1; CREB1: Cyclic AMP-responsive element-binding protein 1; TH: Thyroid hormone; CYCS: Cytochrome c; HMOX1: Heme oxygenase 1; MMP9: Matrix metalloproteinase-9; ERBB2: Receptor tyrosine-protein kinase erbB-2; FN1: Fibronectin. Data are expressed as the mean ± SD. Compared with the healthy control group: ^bP < 0.01. Compared with the slow transit constipation group: ^cP < 0.05, ^dP < 0.01.

Figure 5 Integrative analysis and experimental validation of improved network pharmacology and metabolites. A: Integration analysis between the modified network pharmacology and metabolites. The ordinate stands for pathways, the primary abscissa stands for minus false discovery rates, and the secondary abscissa stands for impact; B and C: The relative expression of Caspase3+ Glial fibrillary acidic protein was assessed by immunofluorescent staining. GFAP: Glial fibrillary acidic protein; HC: Healthy control; STC: Slow transit constipation; JCD: Ji-Chuan decoction; JCDL: Low-dose Ji-Chuan decoction treatment; JCDM: Middle-dose Ji-Chuan decoction treatment; JCDH: High-dose Ji-Chuan decoction treatment; MSP: Mosapride treatment; FDR: False discovery rates; KEGG: Kyoto Encyclopedia of Genes and Genomes; Th: Thyroid hormone; EGFR: Epidermal growth factor receptor; MAPK: Mitogen-activated protein kinase; IL: Interleukin; TNF: Tumor necrosis factor; HIF: Hypoxia-inducible factor. Data are expressed as the mean ± SD. Compared with the healthy control group: ^aP < 0.05, ^bP < 0.01. Compared with the slow transit constipation group: ^cP < 0.05, ^dP < 0.01.