Systems pharmacology approach reveals protective mechanisms of Jian-Pi Qing-Chang decoction on ulcerative colitis

Figure 1 Candidate compound screening and corresponding target matching for Jian-Pi Qing-Chang. A: Compound-targets networks were constructed for the candidate compounds of each Chinese herb of Jian-Pi Qing-Chang (JPQC) and their corresponding targets; B: An integrated compound-targets network was constructed for all the putative compounds of JPQC and their corresponding targets. JPQC: Jian-Pi Qing-Chang; AM: Astragali mongholicus; CP: Codonopsis pilosula; POL: Portulaca oleracea L; RS: Radix sanguisorbae; PN: Panax notoginseng; BR: Bletillae rhizoma; RA: Radix aucklandiae; CC: Coptis chinensis; RG: Radix glycyrrhizae.

Figure 2 Protein-protein interaction network constitution and putative target identification for Jian-Pi Qing-Chang against ulcerative colitis. A: Protein-protein interaction networks were constituted for the candidate targets of Jian-Pi Qing-Chang (JPQC) and ulcerative colitis (UC), respectively, and an intersection was performed to identify the putative targets of JPQC on UC based on the degree centrality value; B and C: Enrichment analyses of biological processes and signalling pathways of JPQC were performed by ClueGO. JPQC: Jian-Pi Qing-Chang; UC: Ulcerative colitis. DC: Degree centrality.

Figure 3 Jian-Pi Qing-Chang inhibits the HIF-1α signalling pathway in dextran sulfate sodium-induced colitis mice. A: The animal experimental flow. An experimental colitis model was induced by administration of 3.5% dextran sulfate sodium (DSS) in drinking water for 7 d in male C57BL/6 mice. Normal saline or Jian-Pi Qing-Chang (JPQC) (15 g/kg/d) was given intragastrically daily for the control/DSS groups and JPQC group, respectively. n = 7-10; B and C: The body weight and disease activity index scores were gauged every day; D: The length of colons and statistical graph; E: Haematoxylin and eosin staining and immunohistochemical staining for TNF-α; F and G: Serum concentrations of TNF-α and IL-1β were measured by ELISA; H: The relative mRNA expression in colon tissues was measured by PCR; I-K: The relative protein expression in colon tissues was measured by Western blot; L: Immunofluorescence staining for HIF-1α in colon tissues. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. JPQC: Jian-Pi Qing-Chang; DSS: Dextran sulfate sodium.

Figure 4 Jian-Pi Qing-Chang restrains the HIF-1α signalling pathway in bone marrow-derived macrophages. A: The isolating method and culturing conditions of bone marrow-derived macrophages (BMDMs) from wild-type C57BL/6 mice. Mature BMDMs were treated with TNF-α (20 ng/mL) in the presence or absence of either Jian-Pi Qing-Chang (100 μg/mL) or rapamycin (2 μM) for 24 h, or pre-treated with pyrrolidinedithiocarbamate (50 μM) for 30 min; B: The relative mRNA expression in BMDMs was measured by PCR; C-E: The relative protein expression in BMDMs was measured by Western blot; F: Immunofluorescence staining for HIF-1α in BMDMs. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. BMDMs: Bone marrow-derived macrophages; JPQC: Jian-Pi Qing-Chang; PDTC: Pyrrolidinedithiocarbamate; RAP: Rapamycin.

Figure 5 Jian-Pi Qing-Chang depresses the mTOR and NF-κB signalling pathways in bone marrow-derived macrophages. A-D: The relative protein expression in bone marrow-derived macrophages (BMDMs) was measured by Western blot; E: Immunofluorescence staining for NF-κB in BMDMs. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. BMDMs: Bone marrow-derived macrophages; JPQC: Jian-Pi Qing-Chang; PDTC: Pyrrolidinedithiocarbamate; RAP: Rapamycin.

Figure 6 Jian-Pi Qing-Chang regulates tight junctions via the NF-κB/HIF-1α signalling pathway in Caco2 cells. A: The cell co-culture assay. Bone marrow-derived macrophages (BMDMs) were co-cultured in the upper chamber of a trans-well plate, with Caco2 cells placed in the lower chamber on day 5. Mature BMDMs were treated with TNF-α (20 ng/ml) in the presence or absence of either Jian-Pi Qing-Chang (100 μg/mL) or RAP (2 μM) for 24 h, or pre-treated with pyrrolidinedithiocarbamate (50 μM) for 30 min; B: The relative mRNA expression in Caco2 cells was measured by PCR; C-F: The relative protein expression in Caco2 cells was measured by Western blot; G and H: Double immunofluorescence staining for HIF-1α/Tublin and NF-κB/Claudin-1 in Caco2 cells. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. BMDMs: Bone marrow-derived macrophages; JPQC: Jian-Pi Qing-Chang; PDTC: Pyrrolidinedithiocarbamate; RAP: Rapamycin.

Figure 7 Jian-Pi Qing-Chang improves intestinal epithelial barrier function via the NF-κB/HIF-1α signalling pathway in dextran sulfate sodium-induced colitis mice. A-F: The relative protein expression in colon tissues was measured by Western blot; G: Immunofluorescence staining for Claudin-1 in colon tissues; H: The schematic diagram of therapeutic mechanism by which Jian-Pi Qing-Chang improves the mucosal inflammatory response and intestinal epithelial barrier function during the development of ulcerative colitis. Data are presented as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. JPQC: Jian-Pi Qing-Chang; DSS: Dextran sulfate sodium.