南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (11): 2456-2465.doi: 10.12122/j.issn.1673-4254.2025.11.18
黄晴晴1,4(
), 杨晶晶4, 姜雪凝4, 张文静1, 汪煜4, 左芦根2,4, 王炼2,4, 王月月1,4, 张小凤3,4, 宋雪3,4, 胡建国1,4(
)
收稿日期:2025-04-15
出版日期:2025-11-20
发布日期:2025-11-28
通讯作者:
胡建国
E-mail:hqq10100@163.com;jghu9200@bbmc.edu.cn
作者简介:黄晴晴,在读硕士研究生,E-mail: hqq10100@163.com
基金资助:
Qingqing HUANG1,4(
), Jingjing YANG4, Xuening JIANG4, Wenjing ZHANG1, Yu WANG4, Lugen ZUO2,4, Lian WANG2,4, Yueyue WANG1,4, Xiaofeng ZHANG3,4, Xue SONG3,4, Jianguo HU1,4(
)
Received:2025-04-15
Online:2025-11-20
Published:2025-11-28
Contact:
Jianguo HU
E-mail:hqq10100@163.com;jghu9200@bbmc.edu.cn
摘要:
目的 探讨刺桐碱(HYP)对小鼠克罗恩病(CD)样结肠炎的作用及其分子机制。 方法 本研究采用2,4,6-三硝基苯磺酸(TNBS)诱导构建小鼠CD样结肠炎模型。将30只C57BL/6J雄性小鼠随机分为3组:WT组、TNBS组和HYP组,10只/组。TNBS组和HYP组采用TNBS诱导结肠炎,HYP组每日灌胃15 mg/kg HYP,其余2组给予等量生理盐水。通过疾病活动指数(DAI)评分、体质量变化、结肠长度及组织病理学评分等指标,评估HYP对小鼠CD样结肠炎的治疗效果。在体外实验中,采用LPS刺激的Caco-2细胞建立肠上皮炎症模型,分为Control组、LPS组和LPS+HYP组。采用qRT-PCR、免疫荧光等技术检测HYP对肠上皮炎症反应及屏障功能的影响。进一步通过GO和KEGG富集分析预测HYP的作用机制,并利用Western blotting验证关键信号通路的调控。 结果 体内研究结果显示,HYP干预可改善TNBS诱导的小鼠结肠炎症状,具体表现为:体质量下降趋势减缓、结肠长度缩短程度改善、DAI评分及组织炎症评分降低,同时结肠黏膜中促炎因子表达水平下调(P<0.05)。在肠屏障功能方面,HYP干预后TNBS模型小鼠结肠组织TEER值升高,细菌移位率(肠系膜淋巴结、肝脏、脾脏)降低,血清中I-FABP和FITC-Dextran的浓度下降(P<0.05)。此外,HYP干预还可增加结肠组织杯状细胞数量,并上调MUC2和紧密连接蛋白(Claudin-1、ZO-1)表达(P<0.05)。体外研究结果显示,与LPS组相比,HYP处理可抑制Caco-2细胞促炎因子表达并恢复紧密连接蛋白水平(P<0.05)。Western blotting分析显示,HYP在体内外模型中均能下调TLR4/MyD88信号通路关键蛋白的表达(P<0.05)。 结论 HYP可能通过抑制肠上皮炎症反应并改善肠屏障功能,从而缓解小鼠CD样结肠炎。
黄晴晴, 杨晶晶, 姜雪凝, 张文静, 汪煜, 左芦根, 王炼, 王月月, 张小凤, 宋雪, 胡建国. 刺桐碱通过抑制肠上皮炎症反应并改善肠屏障功能缓解小鼠克罗恩病样结肠炎[J]. 南方医科大学学报, 2025, 45(11): 2456-2465.
Qingqing HUANG, Jingjing YANG, Xuening JIANG, Wenjing ZHANG, Yu WANG, Lugen ZUO, Lian WANG, Yueyue WANG, Xiaofeng ZHANG, Xue SONG, Jianguo HU. Hypaphorine alleviates Crohn's disease-like colitis in mice by inhibiting intestinal epithelial inflammatory response and protecting intestinal barrier function[J]. Journal of Southern Medical University, 2025, 45(11): 2456-2465.
| Gene name | Primer sequences |
|---|---|
| TNF-α | Forward:5'-CAGGCGGTGCCTATGTCTC-3' |
| Reverse:5'-CGATCACCCCGAAGTTCAGTAG-3' | |
| IL-6 | Forward:5'-TCTATACCACTTCACAAGTCGGA-3' |
| Reverse:5'-GAATTGCCATTGCACAACTCTTT-3' | |
| IL-1β | Forward:5'-GAAATGCCACCTTTTGACAGTG-3' |
| Reverse:5'-TGGATGCTCTCATCAGGACAG-3' | |
| GAPDH | Forward:5'-AGGTCGGTGTGAACGGATTTG-3' |
| Reverse:5'-GGGGTCGTTGATGGCAACA-3' |
表1 引物序列
Tab.1 Primer sequences for qRT-PCR in this study
| Gene name | Primer sequences |
|---|---|
| TNF-α | Forward:5'-CAGGCGGTGCCTATGTCTC-3' |
| Reverse:5'-CGATCACCCCGAAGTTCAGTAG-3' | |
| IL-6 | Forward:5'-TCTATACCACTTCACAAGTCGGA-3' |
| Reverse:5'-GAATTGCCATTGCACAACTCTTT-3' | |
| IL-1β | Forward:5'-GAAATGCCACCTTTTGACAGTG-3' |
| Reverse:5'-TGGATGCTCTCATCAGGACAG-3' | |
| GAPDH | Forward:5'-AGGTCGGTGTGAACGGATTTG-3' |
| Reverse:5'-GGGGTCGTTGATGGCAACA-3' |
图1 HYP给药可缓解TNBS模型小鼠CD样结肠炎
Fig.1 HYP treatment alleviates CD-like colitis in TNBS-treated mice. A: Body weight changes. B: DAI score. C: Gross observation of the dissected colon from each groups. D: Colon length in each group. E: HE staining of colonic sections of the mice from each group (scale bar=1 mm or 100 μm). F: Colonic inflammation scores in each group. *P<0.05 vs WT, #P<0.05 vs TNBS.
图2 HYP给药降低TNBS模型小鼠结肠黏膜中炎症介质水平
Fig.2 HYP treatment reduces the levels of inflammatory mediators in the colonic mucosa of TNBS-treated mice. A-C: Expression levels of TNF-α, IL-6 and IL-1β mRNAs in mouse colonic mucosal tissue. D-F: Expression levels of TNF-α, IL-6 and IL-1β proteins in mouse colonic mucosal tissue. *P<0.05 vs WT, #P<0.05 vs TNBS.
图3 HYP给药改善TNBS模型小鼠肠屏障损伤
Fig.3 HYP ameliorates intestinal barrier damage in TNBS-treated mice. A: TEER values of mouse colon tissues in each group. B-D: Bacterial translocation rate in the mesenteric lymph nodes, liver, and spleen. E, F: Serum levels of I-FABP and FD4 in each group. G: AB-PAS and immunohistochemical staining of colonic sections (scale bar=100 μm). H, I: Number of goblet cells per crypt and MUC2 expression level in mouse colon tissue in the 3 groups. J, L: Immunofluorescence staining of colonic sections from each group (scale bar=100 μm). K, M: Expression levels of claudin-1 and ZO-1 in mouse colon tissues in each group. *P<0.05 vs WT, #P<0.05 vs TNBS.
图4 HYP干预降低LPS诱导的Caco-2细胞中炎症介质水平
Fig.4 HYP treatment reduces inflammatory mediator levels in LPS-induced Caco-2 cells. A-C: Expression levels of TNF-α, IL-6 and IL-1β mRNAs in Caco-2 cells with different treatments. D-F: Protein levels of TNF-α, IL-6 and IL-1β in Caco-2 cells with different treatments. *P<0.05 vs Control, #P<0.05 vs LPS.
图5 HYP干预改善LPS诱导的Caco-2细胞屏障损伤
Fig.5 HYP treatment ameliorates LPS-induced barrier damage in Caco-2 cells. A: TEER value of Caco-2 cells. B: Immunofluorescence staining of Caco-2 cells with different treatments (scale bar=100 μm). C-E: Expression levels of claudin-1 and ZO-1 in Caco-2 cells with different treatments. *P<0.05 vs Control, #P<0.05 vs LPS.
图6 HYP缓解CD样肠炎可能与炎症反应和TLR信号通路有关
Fig.6 Therapeutic mechanism of HYP for CD may involve inflammatory response and TLR signaling pathway. A, B: GO and KEGG enrichment analyses.
图7 HYP可下调TLR4/MyD88信号通路
Fig.7 HYP downregulates the TLR4/MyD88 signaling pathway in both the mouse and cell models. A-C: Protein levels of TLR4 and MyD88 in mouse colonic mucosa tissues. D-F: Protein levels of TLR4 and MyD88 in Caco-2 cells with different treatments. *P<0.05 vs WT or Control, #P<0.05 vs TNBS or LPS.
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