芍药苷调节RIP1/RIP3/MLKL信号通路对慢性结肠炎大鼠坏死性凋亡的影响

doi:10.3969/j.issn.1006-6233.2024.10.05

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摘要 目的:探究芍药苷(PAE)调节受体相互作用蛋白1(RIP1)/受体相互作用蛋白3(RIP3)/混合系激酶区域样蛋白(MLKL)信号通路对慢性结肠炎大鼠坏死性凋亡的影响。方法:取健康SD大鼠随机分组,每组6只,包括对照组(Control组)、模型组(Model组)、药物低、中、高剂量灌胃7d处理组(1.25g·kg^-1·d^-1 PAE-L组、2.5g·kg^-1·d^-1 PAE-M组、5g·kg^-1·d^-1 PAE-H组)和高剂量芍药苷灌胃7d+尾静脉注射7d RIP1激活剂组(5g·kg^-1·d^-1 PAE-H+1.5mg·kg^-1·d^-1 Z-VAD-fmk组)。建立慢性结肠炎模型,造模成功后,大鼠眼眶采血,取其结肠黏膜组织,用HE染色观察结肠组织形态;分析结肠黏膜组织损伤指数(CMDI)评分;用TUNEL检测试剂盒检测结肠组织细胞凋亡;用免疫组化检测结肠组织中基质金属蛋白酶(MMP-1、MMP-2)表达;用ELISA法检测大鼠血清炎症因子(IL-6、TNF-α)水平;用免疫印迹法检测结肠组织中RIP1/RIP3/MLKL通路蛋白表达。结果:与Control组相比,Model组结肠黏膜组织糜烂、水肿,出现炎性细胞浸润,CMDI评分、细胞凋亡率、IL-6、TNF-α水平上升,MMP-1、MMP-2、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL蛋白表达上调(P<0.05);与Model组比较,PAE-L、PAE-M、PAE-H组结肠黏膜组织炎症细胞浸润逐渐减少,CMDI评分、细胞凋亡率、IL-6、TNF-α水平降低,MMP-1、MMP-2、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL蛋白表达下调(P<0.05);与PAE-H组相比,PAE-H+Z-VAD-fmk组结肠黏膜组织炎症加重,CMDI评分、细胞凋亡率、IL-6、TNF-α水平升高,MMP-1、MMP-2、p-RIP1/RIP1、p-RIP3/RIP3、p-MLKL/MLKL蛋白表达上调(P<0.05)。结论:芍药苷能够抑制RIP1/RIP3/MLKL信号通路缓解慢性结肠炎大鼠坏死性凋亡。

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	牟忠颜
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关键词 ：慢性结肠炎, 芍药苷, 受体相互作用蛋白1/受体相互作用蛋白3/混合系激酶区域样蛋白, 坏死性凋亡

Abstract：Objective: To explore the effect of paeoniflorin (PAE) on necroptosis in chronic colitis rats by regulating the receptor-interacting protein 1 (RIP1)/receptor-interacting protein 3 (RIP3)/mixed lineage kinase domain-like protein (MLKL) signaling pathway. Methods: Healthy SD rats were randomly divided into six groups, with six rats per group: Control group, Model group, and three treatment groups with low, medium, and high doses of PAE administered via gavage for 7 days (1.25g·kg^-1·d^-1 PAE-L group, 2.5g·kg^-1·d^-1 PAE-M group, 5g·kg^-1·d^-1 PAE-H group), and a combination group with high-dose PAE gavage for 7 days plus tail vein injection of RIP1 activator for 7 days (5g·kg^-1·d^-1 PAE-H + 1.5mg·kg^-1·d^-1 Z-VAD-fmk group). A chronic colitis model was established, and after successful modeling, blood was collected from the rats via the orbital vein. Colonic mucosa tissues were collected for HE staining to observe the morphology of colon tissues; colonic mucosal damage index (CMDI) scores were analyzed; TUNEL assay was used to detect apoptosis in colon tissues; immunohistochemistry was used to detect the expression of matrix metalloproteinases (MMP-1, MMP-2) in colon tissues; ELISA was used to detect serum levels of inflammatory cytokines (IL-6, TNF-α); and western blotting was used to detect the expression of RIP1/RIP3/MLKL signaling pathway proteins in colon tissues. Results: Compared with the Control group, the Model group exhibited colonic mucosal erosion, edema, and inflammatory cell infiltration, with increased CMDI scores, apoptosis rate, IL-6, TNF-α levels, and upregulated expression of MMP-1, MMP-2, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL proteins (P<0.05). Compared with the Model group, the PAE-L, PAE-M, and PAE-H groups showed a gradual reduction in inflammatory cell infiltration in colonic mucosa tissues, decreased CMDI scores, apoptosis rate, IL-6, TNF-α levels, and downregulated expression of MMP-1, MMP-2, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL proteins (P<0.05). Compared with the PAE-H group, the PAE-H + Z-VAD-fmk group showed exacerbated inflammation in colonic mucosa tissues, with increased CMDI scores, apoptosis rate, IL-6, TNF-α levels, and upregulated expression of MMP-1, MMP-2, p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL proteins (P<0.05). Conclusion: Paeoniflorin can alleviate necroptosis in chronic colitis rats by inhibiting the RIP1/RIP3/MLKL signaling pathway.

Key words： Chronic colitis Paeoniflorin Receptor-interacting protein 1/receptor-interacting protein 3/mixed lineage kinase domain-like protein Necroptosis

基金资助:山东省烟台市科技计划项目,(编号:2021YD076)

引用本文:

牟忠颜, 张建平, 肖强. 芍药苷调节RIP1/RIP3/MLKL信号通路对慢性结肠炎大鼠坏死性凋亡的影响[J]. 河北医学, 2024, 30(10): 1609-1614.
MU Zhongyan, ZHANG Jianping, et al. The Effect of Paeoniflorin on Necroptosis in Chronic Colitis Rats by Regulating the RIP1/RIP3/MLKL Signaling Pathway. HeBei Med, 2024, 30(10): 1609-1614.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2024.10.05 或 http://www.hbyxzzs.cn/CN/Y2024/V30/I10/1609

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