球松素靶向肠上皮细胞PI3K/AKT/CCL2轴抑制巨噬细胞肠道浸润缓解葡聚糖硫酸钠诱导的小鼠结肠炎

doi:10.12122/j.issn.1673-4254.2025.10.16

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (10): 2199-2209.doi: 10.12122/j.issn.1673-4254.2025.10.16

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球松素靶向肠上皮细胞PI3K/AKT/CCL₂轴抑制巨噬细胞肠道浸润缓解葡聚糖硫酸钠诱导的小鼠结肠炎

张可妮¹(), 乔通¹, 尹林¹, 黄菊²^,³, 耿志军²^,³, 左芦根³, 胡建国¹^,³, 李静¹^,³()

^1.蚌埠医科大学第一附属医院，检验科，安徽蚌埠 233000
^2.蚌埠医科大学第一附属医院，中心实验室，安徽蚌埠 233000
^3.蚌埠医科大学第一附属医院，炎症相关性疾病基础与转化研究安徽省重点实验室，安徽蚌埠 233000

收稿日期:2025-05-14 出版日期:2025-10-20 发布日期:2025-10-24
通讯作者: 李静 E-mail:kenizhang0906@163.com;lijingbyfy@bbmu.edu.cn
作者简介:张可妮，在读硕士研究生，E-mail: kenizhang0906@163.com
基金资助:
安徽省临床医学研究转化项目(202427b10020088);炎症相关性疾病基础与转化研究安徽省重点实验室开放课题(YZ2024D10);安徽省高校优秀青年基金项目(2022AH030138);安徽省教育厅优秀青年教师培育项目(YQYB2023019)

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

Keni¹ ZHANG¹(), Tong¹ QIAO¹, Lin¹ YIN¹, Ju HUANG²^,³, Zhijun GENG²^,³, Lugen³ ZUO³, Jianguo HU¹^,³, Jing LI¹^,³()

^1.Department of Clinical Laboratory, First Affiliated Hospital of Bengbu Medical University, Bengbu 233000, China
^2.Central Laboratory, First Affiliated Hospital of Bengbu Medical University, Bengbu 233000, China
^3.Anhui Provincial Key Laboratory of Basic and Translational Research on Inflammatory Diseases, First Affiliated Hospital of Bengbu Medical University, Bengbu 233000, China

Received:2025-05-14 Online:2025-10-20 Published:2025-10-24
Contact: Jing LI E-mail:kenizhang0906@163.com;lijingbyfy@bbmu.edu.cn

摘要/Abstract

摘要：

目的探讨天然黄酮类化合物球松素（PSB）调控巨噬细胞肠道浸润缓解葡聚糖硫酸钠（DSS）诱导的小鼠结肠炎的作用机制。方法将30只C57BL/6雄性小鼠随机分为对照组（WT）、DSS模型组（DSS）及PSB干预组（30、60、120 mg/kg），6只/组。通过体质量变化、疾病活动指数（DAI）、结肠长度及组织病理学评分评估结肠炎表型；免疫荧光检测紧密连接蛋白ZO-1/Claudin-1表达；流式细胞术定量分析结肠巨噬细胞（CD45⁺F4/80⁺CD11b⁺）浸润及极化表型（M1：iNOS⁺；M2：CD206⁺）；ELISA及RT-qPCR检测炎症因子（TNF-α、IL-6）及趋化因子（CCL₂、CXCL₁₀、CX₃CL₁）表达；Western blotting分析PI3K/AKT磷酸化水平；体外浓度梯度实验明确PSB抑制CCL₂的最佳剂量；Transwell实验验证CCL₂介导的巨噬细胞迁移；结合网络药理学预测关键靶点通路。结果预实验确定PSB 60 mg/kg为最佳治疗剂量，缓解DSS诱导的结肠炎症状：与DSS组相比，PSB组体质量下降减少（P<0.05），DAI评分降低（P<0.05），结肠长度恢复（P<0.05）。PSB上调紧密连接蛋白ZO-1/Claudin-1表达（P<0.05），抑制结肠固有层巨噬细胞浸润（F4/80阳性细胞显著减少，P<0.05）并调控其极化（抑制M1促炎亚群，激活M2修复亚群，P<0.05）。分子机制分析结果显示，PSB抑制肠上皮细胞PI3K/AKT磷酸化水平（P<0.05），抑制趋化因子CCL₂表达（P<0.05），并阻断CCL₂介导的RAW264.7细胞迁移，该效应可被外源性CCL₂逆转。网络药理学富集分析及体外补救实现证明PI3K/AKT及CCL₂趋化因子信号通路为核心调控靶点。结论球松素通过靶向抑制肠上皮细胞PI3K/AKT通路活化，减少CCL₂分泌，进而阻断巨噬细胞趋化迁移并调控其极化表型，缓解DSS诱导的小鼠结肠炎，为炎症性肠病的天然化合物干预提供新策略。

关键词: 球松素, 葡聚糖硫酸钠, 结肠炎, 巨噬细胞浸润, PI3K/AKT信号通路, CCL₂

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

张可妮, 乔通, 尹林, 黄菊, 耿志军, 左芦根, 胡建国, 李静. 球松素靶向肠上皮细胞PI3K/AKT/CCL₂轴抑制巨噬细胞肠道浸润缓解葡聚糖硫酸钠诱导的小鼠结肠炎[J]. 南方医科大学学报, 2025, 45(10): 2199-2209.

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.

图/表 8

表1 RT-qPCR的引物序列

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

图1 PSB对小鼠DSS诱导的结肠炎症状的影响

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.

图2 PSB对小鼠实验性结肠炎肠组织损伤及炎症的影响

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.

图3 PSB对小鼠实验性结肠炎肠屏障损伤的影响

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.

图4 PSB抑制对小鼠实验性结肠炎巨噬细胞浸润和极化的影响

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.

图5 PSB网络药理学分析

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

图6 PSB对巨噬细胞趋化因子和PI3K/AKT信号通路的影响

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.

图7 PSB调控肠上皮细胞PI3K/AKT/CCL2信号轴对巨噬细胞趋化的影响

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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球松素靶向肠上皮细胞PI3K/AKT/CCL₂轴抑制巨噬细胞肠道浸润缓解葡聚糖硫酸钠诱导的小鼠结肠炎

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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