炎调方调控Rho/ROCK信号通路对脓毒症急性胃肠损伤小鼠肠上皮细胞凋亡的研究
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1.河南省中医院;2.黄河科技学院

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国家自然科学基金青年项目(82104833)、全国名老中医药专家传承工作室(国中医药人教函【2022】75号)、河南省中医药科学研究专项课题(20-21ZT2114、2022ZY1086)、河南省科技攻关项目(212102310339)


The mechanism of Yantiao fang regulating Rho/ROCK signaling pathway on apoptosis of intestinal epithelial cells in mice with acute gastrointestinal injury caused by sepsis
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1.Henan Provincial Hospital of Traditional Chinese Medicine;2.Yellow River Institute of Science and Technology

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    摘要:

    目的 探讨炎调方通过调控Rho/ROCK信号通路对脓毒症急性胃肠损伤小鼠肠上皮细胞凋亡的影响。 方法 取70只BALB/c小鼠随机分为空白组(10只)、假手术组(10只)和造模小鼠(50只)。空白组小鼠正常饲养,不予处理;假手术组小鼠仅开腹游离盲肠不进行穿孔结扎;通过盲肠结扎穿孔术(CLP)构建脓毒症急性胃肠损伤小鼠模型,将造模成功的小鼠随机分为模型组,炎调方低、中、高剂量组,ROCK抑制剂组。采用苏木素-伊红(HE)染色观察小鼠回肠组织病理学改变;ELISA 法检测各组小鼠血清白细胞介素-1β( IL-1β) 、白细胞介素-6( IL-6)、肿瘤坏死因子-α( TNF-α) 、白细胞介素-10( IL-10) 水平;免疫组织化学检测观察小鼠回肠组织细胞的增殖程度PCNA、Ki-67的表达;蛋白印迹法检测回肠组织凋亡蛋白Caspase 3 蛋白、Bax蛋白的表达;RT-qPCR检测小鼠回肠组织Rho/ROCK 通路上ROCK mRNA、MLC mRNA表达。结果 脓毒症急性胃肠损伤模型小鼠回肠组织肠黏膜萎缩明显、绒毛排列杂乱,可见断裂、脱落,上皮细胞细胞坏死脱落,炎症细胞浸润明显以及Chiu’s病理评分升高;较空白组和假手术组相比,模型组小鼠外周血血清中促炎介质水平(IL-1β、IL-6、TNF-α)和Caspase 3、Bax、ROCK mRNA、MLC mRNA水平升高,而抑炎介质水平(IL-10)和回肠组织中PCNA、Ki-67表达降低。与模型组比较,炎调方各组随着剂量的增加,回肠组织病理学改变均得到不同程度的改善:Chiu’s病理评分降低、血清中IL-1β、IL-6、TNF-α水平降低、IL-10水平升高;且回肠组织中PCNA、Ki-67表达升高,Caspase 3、Bax、ROCK mRNA、MLC mRNA的表达降低。结论 炎调方通过调控Rho/ROCK信号通路抑制脓毒症急性胃肠损伤小鼠的肠上皮细胞凋亡,从而具有减轻肠道组织炎症反应,最终达到防止肠黏膜组织损伤的作用。

    Abstract:

    Objective To investigate the effect of Yantiao Formula on apoptosis of intestinal epithelial cells in mice with acute gastrointestinal injury caused by sepsis by regulating Rho/ROCK signaling pathway. Methods 70 BALB/c mice were randomly divided into blank group (10 mice), sham operation group (10 mice) and model group (50 mice).Mice in blank group were fed normally without treatment.Sham operation group mice only open free cecum without perforation ligation;A mouse model of acute gastrointestinal injury caused by sepsis was established by cecal ligation and perforation (CLP). The mice were randomly divided into model group, Yantiao formula low-dose, medium-dose and high-dose groups, and ROCK inhibitor group.Hematoxylin-eosin (HE) staining was used to observe the histopathological changes of mice ileum.Serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were determined by ELISA.The expression of PCNA and Ki-67 in mouse ileal cells was detected by immunohistochemistry.The expressions of Caspase 3 and Bax were detected by western blotting.The expression of ROCK mRNA and MLC mRNA in the Rho/ROCK pathway of mouse ileum were detected by RT-qPCR. Results The intestinal mucosa of the ileum tissue of mice with acute gastrointestinal injury caused by sepsis was atrophied obviously, villi were disordered, rupture and shedding were observed, epithelial cells were necrotic and shedding, inflammatory cell infiltration was obvious, and Chiu's pathological score was increased.Compared with blank group and sham group, the serum levels of pro-inflammatory mediators (IL-1β, IL-6, TNF-α) and mRNA levels of Caspase 3, Bax, ROCK and MLC in peripheral blood of mice in model group were increased, while the levels of anti-inflammatory mediators (IL-10) and expressions of PCNA and Ki-67 in ileum were decreased.Compared with model group, the histopathological changes of ileum in all Yantiao fang groups were improved to varying degrees with the increase of dose: the pathological score of Chiu's was decreased, the levels of IL-1β, IL-6 and TNF-α in serum were decreased, and the levels of IL-10 were increased.In addition, the expressions of PCNA and Ki-67 were increased, while the expressions of Caspase 3, Bax, ROCK mRNA and MLC mRNA were decreased. Conclusion Yantiao fang can inhibit the apoptosis of intestinal epithelial cells in mice with acute gastrointestinal injury caused by sepsis by regulating Rho/ROCK signaling pathway, so as to reduce the inflammatory reaction of intestinal tissue and ultimately prevent the damage of intestinal mucosal tissue.

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  • 收稿日期:2023-04-10
  • 最后修改日期:2023-11-29
  • 录用日期:2024-01-17
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