透明化三维成像研究艾灸对结肠炎大鼠肠道血管生成的作用

Study of the Effect of Moxibustion on Intestinal Angiogenesis in Colitis Rats Using Three-dimensional Imaging

目的应用组织透明化技术观察隔药灸对2-4-6三硝基苯磺酸(TNBS)诱导的实验性结肠炎大鼠肠道血管生成的影响,从三维水平探讨隔药灸治疗结肠炎的潜在机制。方法采用TNBS灌肠法制备结肠炎大鼠模型,将大鼠随机分为正常组、模型组、隔药灸组和抑制剂组,每组8只。隔药灸组采用隔药灸天枢、气海穴治疗;抑制剂组采用10 mg/mL的血管内皮细胞生长因子受体(VEGFR)抑制剂溶液按1 mL/kg体质量进行腹腔注射治疗。采用苏木素-伊红(HE)染色法观察各组结肠组织病理学改变;采用CUBIC组织透明化技术对各组大鼠结肠组织进行透明化处理,通过血小板内皮细胞黏附分子1(PECAM-1/CD31)染色标记血小板内皮细胞,采用Imaris三维图像处理软件对结肠血管网络进行三维测量和分析。结果 HE染色结果显示,模型组大鼠结肠形态结构紊乱,黏膜固有层有明显裂隙样溃疡,并有大量炎性细胞浸润;隔药灸组和抑制剂组上述病理改变均减轻,可见新生的黏膜上皮修复。结肠血管网络三维测量和分析结果显示,与正常组比较,模型组结肠组织病理学评分显著升高,结肠平均血管密度增加,分支等级增加,血管直径降低;与模型组比较,隔药灸组和抑制剂组结肠组织病理学评分均显著降低,结肠平均血管密度降低,分支等级降低,血管直径增加,而隔药灸组和抑制剂组无明显差异。结论隔药灸能有效减轻结肠炎大鼠结肠炎症和组织损伤,抑制结肠炎大鼠结肠血管生成,从而发挥对结肠炎的治疗作用。

Objective To observe the effect of herb-partitioned moxibustion on intestinal angiogenesis in rats with experimental colitis induced by TNBS using tissue clearing technology, and to explore the potential mechanism of herb-partitioned moxibustion treatment of colitis at the three dimensional level. Method The colitis rat model was established by TNBS via enema, and the rats were randomly divided into a normal group, a model group, a herb-partitioned moxibustion group and an inhibitor group, with 8 rats in each group. The herb-partitioned moxibustion group was treated with herb-partitioned moxibustion at Tianshu(ST25) and Qihai(CV6);the inhibitor group received intraperitoneal injection of 10 mg/mL vascular endothelial growth factor receptor(VEGFR)inhibitor at 1 mL/kg. HE staining was used to observe the histopathological changes of colon tissues in each group;the CUBIC tissue clearing technique was used to clear rat colon tissues, the platelet endothelial cells were stained and labeled with PECAM-1/CD31, and Imaris three-dimensional image processing software was used to perform three-dimensional measurement and analysis of the colonic vascular network. Result HE staining result showed that the morphological structure of colon in the model group was disordered, the lamina propria of mucosa presented with obvious slit-like ulcers, and a large number of inflammatory cell infiltration;the above-mentioned pathological changes were alleviated in the herb-partitioned moxibustion group and the inhibitor group, along with mucosal epithelial repair.The three-dimensional measurement and analysis of colonic vascular network demonstrated that compared with the normal group, the colonic histopathology score increased significantly in the model group, the average colonic blood vessel density increased, the blood vessel branch grade increased, and the blood vessel diameter decreased;compared with the model group, the colonic histopathological score dropped significantly in the herbpartitioned moxibustion group and the inhibitor group, the average colonic blood vessel density decreased, the blood vessel branch grade decreased, and the blood vessel diameter increased;there were no significant differences between the herb-partitioned moxibustion group and the inhibitor group. Conclusion Herb-partitioned moxibustion can effectively reduce intestinal inflammation and tissue damage, and inhibit colonic angiogenesis in colitis rats, thus achieving therapeutic effect for colitis.