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Study of the mechanism of acupuncture and moxibustion in protecting the intestinal mucosal barrier in DSS-induced UC rats based on the IL-9/IL-9R pathway

基于IL-9/IL-9R途径研究针灸保护DSS诱导的UC大鼠肠黏膜屏障的作用机制

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Abstract

Objective

To observe the effects of acupuncture and moxibustion on interleukin (IL)-9/IL-9 receptor (IL-9R) in the colon tissue of rats with ulcerative colitis (UC) and investigate the protective mechanism of acupuncture and moxibustion on the intestinal mucosal barrier in UC rats.

Methods

Male Sprague-Dawley rats were randomly divided into a normal control (NC) group and a modeling group. UC models were prepared by giving 4% dextran sulfate sodium (DSS) water for 7 d. After the successful construction of the UC rat model, the modeling group was randomly divided into a UC group, a herb-partitioned moxibustion (HM) group, and an electroacupuncture (EA) group. HM and EA interventions at bilateral Tianshu (ST25) were performed once a day for 7 d. Hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the colon. The serum concentrations of IL-9, IL-6, IL-1β, and hemoglobin-H (HbH) were determined by enzyme-linked immunosorbent assay. The protein expression levels of IL-9, IL-9R, Claudin-2, zonula occludens-1 (ZO-1), and occludin in the colon tissue were measured by Western blotting or immunohistochemistry. Immunofluorescence was used to detect the co-expression of PU.1 and CD4 with the IL-9 protein.

Results

Compared with the NC group, the colon tissue of UC rats was severely damaged and ulcerated with congestion and edema, and the colonic histopathological score increased significantly (P<0.01). The serum HbH concentration decreased significantly (P<0.01), while the serum concentrations of IL-9, IL-6, and IL-1β increased (P<0.01). The protein expression of colonic ZO-1 and occludin decreased significantly (P<0.01), while the protein expression of colonic IL-9 and IL-9R increased (P<0.05). The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 increased in the colon tissue (P<0.05). Compared with the UC group, the colonic mucosal structures were gradually repaired in both HM group and EA group, and healed ulcers could be observed, the colonic histopathological score decreased significantly (P<0.05). The serum concentration of HbH increased (P<0.01), while the serum concentrations of IL-9, IL-6, and IL-1β decreased (P<0.05). The protein expression levels of ZO-1 and occludin increased (P<0.05), while the protein expression levels of IL-9 and IL-9R decreased (P<0.01). The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 decreased in the colon tissue (P<0.05).

Conclusion

Both HM and EA can inhibit the protein expression levels of IL-9 and IL-9R in the UC colon by regulating the transcription factor PU.1, promote the repair of intestinal mucosal barrier, and down-regulate protein contents of proinflammatory factors IL-9, IL-6, and IL-1β in the serum, which may be one of the key mechanisms of acupuncture and moxibustion in reducing the inflammation of UC colonic mucosa and protecting the intestinal mucosal barrier.

摘要

目的

观察针灸对溃疡性结肠炎(UC)大鼠结肠组织肠黏膜白细胞介素(IL)-9/IL-9 受体(IL-9R)的调节作 用, 探讨针灸对UC 大鼠肠黏膜屏障的保护机制。

方法

将雄性Sprague-Dawley 大鼠随机分为正常组和造 模组。造模组先予以4%葡聚糖硫酸钠(DSS)水溶液连续自由饮用7 d。大鼠UC 模型构建成功后, 将造模组 大鼠随机分为模型组、隔药灸组和电针组。隔药灸组和电针组分别接受隔药灸和电针双侧天枢穴干预, 每 日1 次, 连续干预7 d。采用苏木素-伊红(HE)染色法观察大鼠结肠组织病理学变化并评分; 采用酶联免疫吸 附测定法测定血清中IL-9、IL-6、IL-1β 和血红蛋白H(HbH)的蛋白含量; 采用免疫印迹法或免疫组化法检测 大鼠结肠组织IL-9、IL-9R、Claudin-2、紧密连接蛋白1(ZO-1)和Occludin 的蛋白表达; 采用免疫荧光法检测 PU.1、CD4 与IL-9 蛋白共表达。

结果

与正常组比较, 模型组大鼠结肠组织损伤严重, 出现大面积的溃疡, 伴有充血水肿, 结肠组织病理学评分显著升高(P<0.01), 血清HbH 浓度降低(P<0.01), 血清中IL-9、IL-6 和IL-1β 蛋白含量均升高 (P<0.01), 结肠组织中ZO-1 和Occludin 蛋白表达降低(P<0.01), 结肠组织中IL-9、IL-9R 蛋白表达均增高(P<0.05), 结肠组织中IL-9/PU.1 和IL-9/CD4 阳性共表达增加(P<0.05)。与模型组相比, 隔药 灸组和电针组大鼠结肠黏膜结构逐渐修复, 可见愈合性溃疡, 结肠组织病理学评分降低(P<0.05), 血清HbH 浓度增加(P<0.01), 血清IL-9、IL-6 和IL-1β 蛋白含量均降低(P<0.05), 结肠组织中ZO-1 和Occludin 蛋白表达 升高 (P<0.05), 结肠组织中IL-9、IL-9R 蛋白表达降低(P<0.01), 结肠组织中IL-9/PU.1 和IL-9/CD4 阳性共表达 降低(P<0.05)。

结论

隔药灸和电针均能通过调节转录因子PU.1 抑制UC 结肠组织中IL-9 与IL-9R 的蛋白表 达, 促进肠黏膜屏障修复, 降低血清中促炎因子IL-9、IL-6 和IL-1β 的蛋白含量, 这可能是针灸减轻UC 结肠 黏膜炎症, 保护肠黏膜屏障的关键机制之一。

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Acknowledgments

This work was supported by the Projects of National Natural Science Foundation of China (国家自然科学基金 项目, No. 82174501, No. 82105012, No. 82004475, No. 81873372); Shanghai Municipal Natural Science Foundation (上海市自然科学基金项目, No. 21ZR1460200, No. 22ZR1458400); Shanghai Talent Development Fund Project (上海市人才发展基金项目, No. 2021058); One Hundred Person Project of Xinglin of Shanghai University of Traditional Chinese Medicine (上海中医药大学“杏林 百人计划”项目, No. [2020] 23).

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Authors and Affiliations

  1. Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 200437, China

    Yan Huang  (黄艳), Kexin Sun  (孙可鑫), Jing Xu  (徐静), Zhe Ma  (马喆), Jimeng Zhao  (赵继梦), Chen Zhao  (赵琛), Yue Zhang  (张玥), Luyi Wu  (吴璐一) & Rude Huang  (黄儒德)

  2. Shanghai Research Institute of Acupuncture and Meridian, Shanghai, 200030, China

    Yan Huang  (黄艳), Zhe Ma  (马喆), Jimeng Zhao  (赵继梦), Lingjie Li  (李灵杰), Ling Yang  (杨玲), Feng Li  (李峰), Yanan Liu  (刘雅楠) & Rude Huang  (黄儒德)

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  1. Yan Huang  (黄艳)
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Contributions

HUANG Yan, HUANG Rude, and WU Luyi designed the study; HUANG Yan and SUN Kexin finished experiments and wrote the manuscript; MA Zhe, ZHAO Jimeng, ZHAO Chen, and YANG Ling performed partial research; LI Feng, LIU Yanan, and ZHANG Yue analyzed the data; XU Jing and LI Lingjie helped revise the manuscript.

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Correspondence to Luyi Wu  (吴璐一) or Rude Huang  (黄儒德).

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The authors declare that there is no potential conflict of interest in this article.

Statement of Human and Animal Rights

All experimental operations were conducted under the guidance of the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. The treatment of animals in this experiment conformed to the ethical criteria.

Additional information

Co-first Authors: HUANG Yan, M.D., associate researcher; SUN Kexin, master degree candidate

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Huang, Y., Sun, K., Xu, J. et al. Study of the mechanism of acupuncture and moxibustion in protecting the intestinal mucosal barrier in DSS-induced UC rats based on the IL-9/IL-9R pathway. J. Acupunct. Tuina. Sci. 22, 91–103 (2024). https://doi.org/10.1007/s11726-024-1424-6

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