Background & Aims

Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying.

Methods

Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45⁺, CD11b⁺, F4/80⁺ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow–derived macrophages from wild-type mice.

Results

Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45⁺, CD11b⁺, and F4/80⁺ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying.

Conclusions

In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.

中文翻译：

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巨噬细胞种群的变化促进了小鼠胃排空延迟的发展

背景与目标

Muscularis propria 巨噬细胞靠近调节胃肠运动功能的细胞，包括 Cajal 间质细胞 (ICC) 和肌间神经元。在糖尿病性胃轻瘫动物模型中，胃排空延迟的发展与表达细胞保护标志物的巨噬细胞丢失和 ICC 网络减少有关。患有长期糖尿病和胃排空正常的小鼠具有表达抗炎标志物的巨噬细胞，并且胃 ICC 正常。集落刺激因子 1 基因 (Csf1op/op) 自发突变的纯合子小鼠没有巨噬细胞；当他们被给予链脲佐菌素诱发糖尿病时，他们不会出现胃排空延迟。

方法

给予野生型和 Csf1op/op 小鼠链脲佐菌素诱导糖尿病。一些 Csf1op/op 小鼠每天腹腔注射 CSF1，持续 7 周；收集胃组织并通过免疫组织化学分析细胞分布。CD45 ⁺、CD11b ⁺、F4/80 ⁺巨噬细胞从胃固有肌层中分离出来，通过流式细胞仪分离，并通过定量实时聚合酶链反应进行分析。将来自 Csf1op/op 小鼠的培养的胃固有肌层暴露于培养基，该培养基通过来自野生型小鼠的骨髓来源的巨噬细胞进行培养。

结果

给予含有巨噬细胞的 CSF1 的 Csf1op/op 小鼠的胃固有肌层；给予 CSF1 的 15 只糖尿病小鼠中有 11 只出现胃排空延迟并且 ICC 受损。在非糖尿病 Csf1op/op 小鼠中，给予 CSF1 减少了胃肌间神经元的数量，但不影响氮能神经元或 ICC 的比例。在给予 CSF1 的糖尿病 Csf1op/op 小鼠胃排空延迟时，氮能神经元的比例与非糖尿病野生型对照相同。先前暴露于氧化损伤的巨噬细胞条件培养基对 Csf1op/op 小鼠培养的胃固有肌层中的 ICC 造成损害；针对 IL6R 或 TNF 的中和抗体防止了对 ICC 的这种损害。CD45 ⁺、CD11b ⁺和 F4/80 ⁺从胃排空延迟的糖尿病野生型小鼠中分离出的巨噬细胞表达较高水平的编码炎症标志物（IL6 和诱导型一氧化氮合酶）的信使 RNA 和较低水平的编码抗炎细胞标志物（血红素加氧酶 1、精氨酸酶 1、和 FIZZ1）比从胃排空正常的糖尿病小鼠中分离的巨噬细胞。

结论

在对患有糖尿病的 Csf1op/op 和野生型小鼠的研究中，我们发现胃排空延迟与巨噬细胞产生的炎症因子增加和抗炎因子的产生减少有关，从而导致 ICC 丢失。