血根碱通过调控Nrf2/NF-κB通路缓解小鼠溃疡性结肠炎

doi:10.12122/j.issn.1673-4254.2024.08.05

摘要/Abstract

摘要：

目的探讨血根碱（SA）对葡聚糖硫酸钠（DSS）诱导的溃疡性结肠炎（UC）的作用机制。方法随机将56只雄性C57BL/6小鼠分为空白对照组、模型组（DSS组）、SA低剂量组（DSS+SA-L，SA 1 mg/kg）、SA中剂量组（DSS+SA-M，SA 5 mg/kg）、SA高剂量组（DSS+SA-H，SA 10 mg/kg）、柳氮磺吡啶组（DSS+SASP，SASP 400 mg/kg）、SA高剂量组+Nrf2抑制剂ML385组（DSS+SA-H+ML385，SA 10 mg/kg+ML385 30 mg/kg），8只/组。除空白对照组外，均采用3.5%DSS诱导建立UC模型，SA干预组和柳氮磺吡啶组给与对应药物灌胃治疗，通路抑制剂组SA 10 mg/kg灌胃同时腹腔注射ML385 30 mg/kg。通过监测小鼠体质量变化、疾病活动指数（DAI）评分、结肠长度测量、HE染色评估小鼠炎症；检测结肠组织中活性氧（ROS）水平；比色法检测结肠组织中丙二醛含量（MDA）； RT-qPCR检测结肠组织中炎性因子；Western blotting法检测结肠组织中核因子E2相关因子2（Nrf2）、血红素加氧酶1（HO-1）、Kelch样环氧氯丙烷相关蛋白1（Keap-1）、磷酸化p65蛋白（p-p65）、p65、紧密连接蛋白（occludin、ZO-1）蛋白表达。结果与DSS组相比，SA治疗可缓解DSS组小鼠体质量下降、结肠长度缩短、DAI评分升高（P<0.05）。HE染色显示，DSS组结肠腺体结构破坏，SA可明显改善结肠隐窝结构。RT-qPCR显示，SA干预组结肠组织中TNF-α、IL-1β和IL-6水平下降（P<0.05），ROS、MDA下降（P<0.05）。Western blotting结果显示，结肠组织中Nrf2、HO-1、occludin、ZO-1蛋白表达上升（P<0.05），Keap-1、p-p65蛋白表达下降（P<0.05），p65无明显变化（P>0.05），且DSS+SA-L、DSS+SA-M、DSS+SA-H组各指标变化呈剂量依赖性。Nrf2通路抑制剂ML385降低高剂量组SA对UC小鼠结肠黏膜的改善作用。结论 SA可改善UC样小鼠肠炎，作用机制可能与激活Nrf2通路，抑制Nrf2介导的NF-κB通路有关。

关键词: 溃疡性结肠炎, 血根碱, Nrf2, NF-κB

Abstract:

Objective To investigate the mechanism of sanguinarine (SA) for alleviating ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) in mice. Methods Male C57BL/6 mouse models of 3.5% DSS-induced UC were randomized for treatment with 1, 5 and 10 mg/kg SA by gavage, 400 mg/kg sulfasalazine by gavage, or 10 mg/kg SA combined with intraperitoneal injection of 30 mg/kg ML385 (a Nrf2 inhibitor). The changes in intestinal inflammation was assessed by monitoring weight changes, disease activity index (DAI) score, colon length measurement, and HE staining. After the treatments, the colon tissues were collected for detection of malondialdehyde (MDA) content using colorimetry, mRNA expressions of inflammatory factors using RT-qPCR, and the expressions of Nrf2, HO-1, Keap-1, p-p65, p65, occludin, and ZO-1 proteins were detected using Western blotting. Results SA treatment obviously alleviated weight loss, colon length shortening and DAI score increase and ameliorated structural destruction of the colon glands and colonic crypts in mice with DSS-induced UC. SA intervention significantly decreased the levels of TNF-α, IL-1β and IL-6 mRNA and lowered ROS and MDA levels in the colon tissue of UC mice. The mouse models receiving SA treatment showed significantly increased expressions of Nrf2, HO-1, occludin and ZO-1 and lowered expressions of Keap-1 and P-P65 in the colon tissue without significant changes of p65 expression, and these changes were SA dose-dependent. Treatment with ML385 obviously attenuated the effect of high-dose SA for improving UC in the mouse models. Conclusion SA can improve UC-like enteritis in mice possibly by activating the Nrf2 pathway and inhibiting the NF-κB pathway in the colon tissue.

Key words: ulcerative colitis, sanguinarine, Nrf2, nuclear factor-κB

赵娜, 沈梦迪, 赵睿, 奥迪, 骆泽谭, 张银亮, 徐志东, 范方田, 郑海伦. 血根碱通过调控Nrf2/NF-κB通路缓解小鼠溃疡性结肠炎[J]. 南方医科大学学报, 2024, 44(8): 1467-1475.

Na ZHAO, Mengdi SHEN, Rui ZHAO, Di AO, Zetan LUO, Yinliang ZHANG, Zhidong XU, Fangtian FAN, Hailun ZHENG. column:Sanguinarine alleviates ulcerative colitis in mice by regulating the Nrf2/NF-κB pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1467-1475.

图/表 7

表1 引物序列

Tab.1 Primer sequence for RT-qPCR

Gene	Forward	Reverse
TNF-α	CGTCGTAGCAAACCACCAA	GGGCAGCCTTGTCCCTTGA
IL- 1β	CTCAACTGTGAAATGCCACC	GAGTGATACTGCCTGCCTGA
IL-6	ATGGCAATTCTGATTGTATG	GACTCTGGCTTTGTCTTTCT
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA

图1 SA干预对UC模型小鼠的保护作用

Fig.1 Protective effect of SA against DSS-induced UC in mice. A: Changes of body weight. B: Changes of DAI scores. C, D: Comparison of colon length among the groups. *P<0.05 vs DSS group. #P<0.05 vs control.

图2 HE染色观察各组小鼠结肠组织病理学变化

Fig.2 HE staining of mouse colon tissues in different groups.

图3 SA干预对DSS诱导的UC小鼠肠道屏障功能影响

Fig.3 Effect of SA intervention on intestinal barrier function in DSS-induced UC mice. A: Western blotting results. B, C:Comparison of protein expression levels among the groups. *P<0.05 vs DSS group. #P<0.05 vs control.

图4 SA对UC小鼠的肠道炎症和氧化应激的影响

Fig.4 Effect of SA intervention on intestinal inflammation and oxidative stress in UC mice. A-C: Changes in intestinal inflammatory factors in different groups. D-F: Changes in intestinal oxidative stress in different groups. *P<0.05 vs DSS group, #P<0.05 vs control.

图5 各组小鼠肠组织中NF-κBp65、p-p65、Keap-1、Nrf2、HO-1蛋白表达

Fig.5 Expression levels of NF-κBp65, p-p65, Keap-1, Nrf2 and HO-1 in intestinal tissues of mice in each group. A: Western blotting results. B-F: Changes in the protein expression levels.*P<0.05 vs DSS group. #P<0.05 vs control.

图7 ML385对UC小鼠的炎症因子、ROS、MDA的影响。

Fig.7 Effect of ML385 on inflammatory factors, ROS, and MDA in UC mice. A-C: Changes in inflammatory factors in colonic tissues. D-F: Changes in indicators of oxidative stress in the colon tissue. *P<0.05 vs DSS group. #P<0.05 vs control. $P<0.05 vs DSS+SA-H.

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