目的 考察药根碱对葡聚糖硫酸钠（dextran sulfate sodium，DSS）诱导的溃疡性结肠炎（ulcerative colitis，UC）小鼠的治疗作用及机制，并评估其安全性。方法 将50只C57BL/6J小鼠随机分为对照组、模型组、药根碱组（160 mg/kg）和DSS＋药根碱低、高剂量（80、160 mg/kg）组，除对照组和药根碱组外，其余组给予小鼠含3% DSS的饮用水构建小鼠UC模型。对小鼠体质量、结肠长度、疾病活动指数（disease activity index，DAI）评分、结肠组织病理学进行观察，检测血清中谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）活性和总抗氧化能力（total antioxidant capacity，T-AOC），以及结肠组织中超氧化物歧化酶（superoxide dismutase，SOD）活性和丙二醛（malondialdehyde，MDA）水平，考察药根碱对UC小鼠的治疗作用及抗氧化作用；采用免疫荧光法探究药根碱对UC小鼠结肠黏膜炎症反应、肠道黏蛋白（mucin 2，MUC2）、肠道屏障功能相关蛋白闭合蛋白（occludin）和闭锁小带蛋白-1（zonula occludens-1，ZO-1）的表达以及F4/80⁺和CD11b⁺细胞浸润的影响；采用分子对接考察药根碱与凋亡、抗氧化蛋白的结合能力，采用TUNEL染色评估UC小鼠结肠上皮细胞的凋亡情况，Western blotting法检测凋亡和抗氧化应激相关蛋白B淋巴细胞2（B-cell lymphoma 2，Bcl-2）、B淋巴细胞瘤-2相关X蛋白（B-cell lymphoma-2 associated X protein，Bax）、核因子E相关因子2（nuclear factor erythroid-2 related factor 2，Nrf2）及血红素加氧酶-1（heme oxygenase-1，HO-1）的表达。通过检测UC小鼠血清丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）活性和主要器官组织病理学变化评估药根碱的安全性。结果 药根碱可显著缓解DSS诱导的UC小鼠症状，降低DAI评分，恢复结肠长度（P＜0.01），缓解结肠组织损伤；药根碱升高了UC小鼠血清中T-AOC、GSH-Px水平（P＜0.05、0.01）以及结肠组织中SOD活性（P＜0.05），并降低了结肠组织中MDA水平（P＜0.05）；免疫荧光结果显示，药根碱组的F4/80⁺和CD11b⁺表达降低，MUC2、ZO-1和occludin表达升高；TUNEL染色结果显示，药根碱可改善UC小鼠结肠上皮细胞凋亡。分子对接结果表明，药根碱与凋亡、抗氧化蛋白均有良好的结合能力。Western blotting结果显示，药根碱治疗后Bax表达显著降低（P＜0.05、0.01），Bcl-2、Nrf2和HO-1蛋白表达显著升高（P＜0.05、0.01）。此外，药根碱组血清ALT和AST活性及肝、心、脾、肺和肾组织病理结果与对照组无差异。结论 药根碱可能通过抑制肠上皮细胞凋亡，激活Nrf2/HO-1信号通路减轻DSS诱导的UC小鼠结肠黏膜损伤，并表现出较好的安全性。

Objective To investigate the therapeutic effect and mechanism of jatrorrhizine on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice and to assess its safety. Methods Fifty C57BL/6J mice were randomly divided into control group, DSS group, DSS + jatrorrhizine (80 mg/kg) group, DSS + jatrorrhizine (160 mg/kg) group, and jatrorrhizine (160 mg/kg) group. The UC mice model was replicated by giving mice drinking water containing 3% DSS. Body weight, colon length, disease activity index (DAI) score, and colon histopathology were observed, and glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC) in serum were measured, as well as superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in colon tissue to examine the therapeutic and antioxidant effects of jatrorrhizine on UC mice. Immunofluorescence method was used to investigate the effects of drug radicals on colonic mucosal inflammation, expression of mucin 2 (MUC2), occludin and zonula occludens-1 (ZO-1), and cell infiltration of F4/80⁺ and CD11b⁺ in mice. Molecular docking was used to validate the docking of jatrorrhizine with apoptotic and antioxidant proteins. TUNEL staining was used to evaluate apoptosis of colonic epithelial cells in mice. Western blotting (WB) was used to detect the expression of intestinal barrier function proteins occludin and ZO-1, and expressions of oxidative stress and apoptosis-related proteins B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-2 associated X protein (Bax), nuclear factor erythroid-2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Results Jatrorrhizine significantly alleviated the symptoms of DSS-induced UC mice, reduced DAI scores, restored the length of the colon (P < 0.05, 0.01), slowed down the colon tissue damage, and activities of GSH-Px and T-AOC in serum and SOD in colon tissue were increased (P < 0.05, 0.01), and the level of MDA in colon tissue was decreased (P < 0.05). Immunofluorescence results suggested that expressions of F4/80⁺ and CD11b were reduced, and that the expressions of MUC2, ZO-1, and occludin were increased in jatrorrhizine-treated groups. TUNEL staining results showed that apoptosis of mouse colon epithelial cells was improved in jatrorrhizine group. Molecular docking results showed that jatrorrhizine had a good binding ability to apoptotic and antioxidant proteins, and WB results showed that the expression of Bax was significantly reduced, and the expressions of Bcl-2, Nrf2 and HO-1 were significantly increased after jatrorrhizine treatment (P < 0.05, 0.01). In addition, the expressions of ALT and AST and histopathological results of liver, heart, spleen, lung and kidney in the group administered with jatrorrhizine alone did not differ from those in the control group. Conclusion Jatrorrhizine may attenuate DSS-induced colonic mucosal injury in UC mice by regulating apoptosis and activating the Nrf2/HO-1 signaling pathway, and the results of toxicological study have shown that jatrorrhizine has good safety.

R285.5

国家自然科学基金重大项目(82192915);国家重点研究与发展计划项目(2018YFC1704500)