Pinostrobin targets the PI3K/AKT/CCL2 axis in intestinal epithelial cells to inhibit intestinal macrophage infiltration and alleviate dextran sulfate sodium-induced colitis in mice

doi:10.12122/j.issn.1673-4254.2025.10.16

Abstract

Abstract:

Objective To investigate the mechanism through which pinostrobin (PSB) alleviates dextran sulfate sodium (DSS)-induced colitis in mice. Methods C57BL/6 mice were randomized into control group, DSS model group, and PSB intervention (30, 60, and 120 mg/kg) groups. Colitis severity of the mice was assessed by examining body weight changes, disease activity index (DAI), colon length, and histopathology. The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining, and macrophage infiltration and polarization were analyzed with flow cytometry. ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors (TNF‑α and IL-6) and chemokines (CCL₂, CXCL₁₀, and CX₃CL₁) in the colon tissues, and PI3K/AKT phosphorylation levels were analyzed with Western blotting. In cultured Caco-2 and RAW264.7 cells, the effect of PSB on CCL₂-mediated macrophage migration was assessed using Transwell assay. Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB. Results In DSS-induced mouse models, PSB at 60 mg/kg optimally alleviated colitis, shown by reduced weight loss and DAI scores and increased colon length. PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues, inhibited colonic macrophage infiltration, and promoted the shift of macrophage polarization from M1 to M2 type. In cultured intestinal epithelial cells, PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL₂ expression. PSB treatment obviously blocked CCL₂-mediated macrophage migration of RAW264.7 cells, which could be reversed by exogenous CCL₂. Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL₂ signaling as the core targets of PSB. Conclusion PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling, reducing CCL₂ secretion, and blocking macrophage chemotaxis and migration, highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.

Key words: pinostrobin, dextran sulfate sodium, colitis, infiltration of macrophages, PI3K/AKT signaling pathway, CCL₂

Keni¹ ZHANG, Tong¹ QIAO, Lin¹ YIN, Ju HUANG, Zhijun GENG, Lugen³ ZUO, Jianguo HU, Jing LI. Pinostrobin targets the PI3K/AKT/CCL₂ axis in intestinal epithelial cells to inhibit intestinal macrophage infiltration and alleviate dextran sulfate sodium-induced colitis in mice[J]. Journal of Southern Medical University, 2025, 45(10): 2199-2209.

Figures/Tables 8

Tab.1 Primer sequences for RT-qPCR

Gene name	Gene ID	Primer sequences (5'-3')
IL-6	16193	F:TCTATACCACTTCACAAGTCGGA
		R:GAATTGCCATTGCACAACTCTTT
TNF-α	21926	F:CAGGCGGTGCCTATGTCTC
		R:CGATCACCCCGAAGTTCAGTAG
CCL₂	20296	F:TAAAAACCTGGATCGGAACCAAA
		R:GCATTAGCTTCAGATTTACGGGT
CXCL₁₀	15945	F:CCAAGTGCTGCCGTCATTTTC
		R:TCCCTATGGCCCTCATTCTCA
CX₃CL₁	20312	F:CTGCCCTCACTAAAAATGGTGG
		R:GAATTGCCATTGCACAACTCTTT
GAPDH	14433	F:TGGCCTTCCGTGTTCCTAC
		R:GAGTTGCTGTTGAAGTCGCA

Fig.1 Effect of pinostrobin (PSB) on symptoms of dextran sodium sulfate (DSS)-induced colitis in mice. A: Daily body weight changes in wild-type (WT), DSS, PSB-30 mg/kg, PSB-60 mg/kg and PSB-120 mg/kg groups. B: Disease activity index (DAI) scores in the 5 groups. C: Representative images of the mouse colon. D: Colon lengths of the mice. E: Representative images of mouse spleens. F: Spleen weight. G: Spleen index. *P<0.05 vs WT; #P<0.05 vs DSS.

Fig.2 Effect of PSB on intestinal tissue injury and inflammation in the mouse models of colitis. A: HE staining of mouse colon tissues. B: Inflammation scores of mouse colon tissue. C: Levels of inflammatory factors (TNF-α and IL-6) in the colonic mucosa detected by ELISA. D: Relative mRNA expressions of TNF-α and IL-6 in the colonic mucosa detected by RT-qPCR. *P<0.05 vs WT; #P<0.05 vs DSS.

Fig.3 Effect of PSB on intestinal barrier damage in mice with DSS-induced colitis. A: Immunofluorescence staining of ZO-1 and Claudin-1 in the colon of the mice. B: Fluorescence intensity of ZO-1. C: Fluorescence intensity of claudin-1. *P<0.05 vs WT; #P<0.05 vs DSS.

Fig.4 Effect of PSB on macrophage infiltration and polarization in the mouse models of colitis. A: Expression of F4/80 analyzed by immunohistochemical staining. B: Average optical density (AOD) of F4/80. C: Flow cytometry gating strategy. D, E: Assessment of macrophage percentage (CD45+F4/80+CD11b+ population) in mouse intestinal tissue by flow cytometry. F,G: Assessment of M1 macrophage percentage (F4/80+ CD11b+iNOS+ population) in mouse intestinal tissue by flow cytometry. H,I: Assessment of M2 Macrophage percentage (F4/80+ CD11b+CD206+ population) in mouse intestinal tissue by flow cytometry. *P<0.05 vs WT; #P<0.05 vs DSS.

Fig.5 PSB network pharmacology. A: Venn diagram. B: PPI network diagram. C: KEGG analysis. D: GO analysis (BP, CC, MF).

Fig.6 Effects of PSB on macrophage chemokines and the PI3K/AKT signaling pathway. A: Levels of chemokines (CCL2, CXCL10 and CX3CL1) in the colonic mucosa detected by ELISA. B: Relative mRNA expressions of chemokines (CCL2, CXCL10 and CX3CL1) in the colonic mucosa detected by RT-qPCR. C, D: Western blot analysis of p-PI3K and p-AKT, the key proteins of the PI3K/AKT pathway, in mouse intestinal mucosal tissues. *P<0.05 vs WT; #P<0.05 vs DSS.

Fig.7 Effect of PSB on the chemotaxis of macrophages by regulating the PI3K/AKT/CCL2 signaling axis in intestinal epithelial cells. A, B: Determination of the cytotoxicity of PSB in Caco-2 and RAW264.7 cells by CCK-8 assay. C: Concentration-dependent inhibition of DSS-induced CCL2 mRNA expression by PSB (RT-qPCR). D: Western blot bands and quantitative analysis (p-PI3K and p-AKT). E, F: Expression levels of CCL2 mRNA (RT-qPCR) and protein (ELISA). G,H: Transwell migration images (crystal violet staining) and statistics of the number of migrated cells. *P<0.05 vs Con; #P<0.05 vs DSS; △P<0.05 vs DSS+PSB.

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Pinostrobin targets the PI3K/AKT/CCL₂ axis in intestinal epithelial cells to inhibit intestinal macrophage infiltration and alleviate dextran sulfate sodium-induced colitis in mice

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