羟氯喹减轻氟尿嘧啶诱导的小鼠肠炎及机制

目的探讨羟氯喹(HCQ)在化疗药物诱导的肠炎中的治疗作用及其可能的分子机制。方法 30只C57B6/J小鼠随机分为0、 1、 3、 5、 7 d组,第1~3天腹腔注射氟尿嘧啶(5-FU)200μL/d(50 mg/kg),分别于第0、 1、 3、 5、 7天处死,双链DNA(dsDNA)定量试剂盒检测血清及小肠液dsDNA水平,腹泻评分评估小鼠肠炎病情。体外培养HCT-116细胞,分别以(0、 0.01、 0.1、 0.5、 1、 10)μmol/L 5-FU处理72 h或1μmol/L 5-FU处理24、 48、 72 h,dsDNA定量试剂盒检测各组细胞培养上清dsDNA水平。24只C57B6/J小鼠随机分为:对照组(腹腔注射并灌胃生理盐水200μL/d)、模型组(腹腔注射50 mg/kg 5-FU 200μL/d、灌胃生理盐水200μL/d)、 HCQ治疗组(腹腔注射50 mg/kg 5-FU 200μL/d前一天起灌胃60 mg/kg HCQ溶液200μL/d)、 HCQ单药组(灌胃60 mg/kg HCQ溶液200μL/d),处理6 d处死小鼠。HE染色观察小肠病变,原位末端转移酶标记技术(TUNEL)检测小鼠小肠细胞凋亡情况,免疫荧光组织化学染色法检测小肠组织CD11c、 Toll样受体9(TLR9)、核因子κB (NF-κB)的表达水平, ELISA检测血清IL-1β水平。体外培养小鼠骨髓来源的树突状细胞(BMDC), LipofectamineTM 3000转染dsDNA以刺激BMDC,蛋白印迹法检测BMDC中TLR9表达,免疫荧光细胞化学染色法检测NF-κB表达, ELISA检测细胞上清中白细胞介素1β(IL-1β)水平。结果 5-FU处理的小鼠肠上皮细胞明显凋亡,血清及小肠灌洗液中dsDNA水平动态变化与腹泻评分变化一致;5-FU处理的HCT-116细胞上清中dsDNA水平随5-FU浓度和时间的增加而增加。HCQ处理可减轻小鼠小肠上皮的破坏,降低小肠组织TLR9、 NF-κB表达及血清IL-1β水平。模型小鼠小肠存在大量DC的浸润,用dsDNA刺激BMDC后,其TLR9、 NF-κB、 IL-1β表达均明显增加,而HCQ预处理后均显著降低。结论 HCQ减轻5-FU诱导的小鼠肠道炎症,抑制DC中TLR9、 NF-κB依赖的DNA感受通路和IL-1β的释放。

Hydroxychloroquine alleviates 5-fluorouracil-induced enteritis in mice and its mechanism

Objective To investigate the effect of hydroxychloroquine(HCQ) on 5-fluorouracil(5-FU)-induced enteritis in mice and its mechanism. Methods Thirty C57B6/J mice were randomly divided into 0-, 1-, 3-, 5-and 7-day groups and sacrificed separately at day 0, 1, 3, 5, and 7 after intraperitoneal administration of 5-FU 200 μL/d(50 mg/kg) at day 1-3. The double-stranded DNA(dsDNA) levels in serum and small intestinal fluid were detected by dsDNA quantification kit. Severity of enteritis was evaluated by diarrhea score. HCT-116 cells were cultured in vitro and treated with 5-FU at different concentrations(0, 0.01, 0.1, 0.5, 1, 10 μmol/L) for 72 hours or 5-FU at 1 μmol/L for different time(24, 48, 72 hours). The dsDNA concentration in the cell culture supernatant of each group was detected by dsDNA quantification kit. Twenty-four C57B6/J mice were randomly divided into 4 groups: control group(intraperitoneal injection and intragastric administration of 200 μL/d saline), model group(intraperitoneal injection of 50 mg/kg 5-FU of 200 μL/d, intragastric administration of saline 200 μL/d), HCQ treatment group(intragastric administration of 60 mg/kg HCQ of 200 μL/d, starting at 1 day before the first intraperitoneal injection of 50 mg/kg 5-FU of 200 μL/d) and HCQ group(intragastric administration of 60 mg/kg HCQ solution of 200 μL/d). And they were sacrificed after 6 days. Small intestine lesions were observed by HE staining. Apoptotic cells in the small intestine were detected by TUNEL staining. The expression levels of CD11c, Toll-like receptor 9(TLR9) and nuclear factor κB(NF-κB) in the small intestine were assessed by immunofluorescence staining. Interleukin-1β(IL-1β) levels in the serum were detected by ELISA. Mouse bone marrow-derived dendritic cells(BMDCs) were cultured in vitro and stimulated by dsDNA using LipofectamineTM 3000. The expression of TLR9 and NF-κB in BMDCs were detected by Western blotting and immunofluorescent staining, respectively. IL-1β level in the cell supernatant was detected by ELISA. Results Large amounts of apoptotic cells were observed in the small intestine of 5-FU-treated mice and the dynamic changes of dsDNA levels in the serum and small intestinal lavage fluid were consistent with that of diarrhea score. 5-FU triggered dsDNA release from HCT-116 cells in a dose-and time-dependent manner. HCQ alleviated the destruction in small intestinal epithelium and inhibited the expression levels of TLR9, NF-κB and IL-1β in the serum. The infiltration of a large number of dendritic cells in the small intestine of model mice was observed. After BMDCs were stimulated with dsDNA, the expression of TLR9, NF-κB, and IL-1β all significantly increased and HCQ significantly decreased. Conclusion HCQ alleviates 5-FU-induced enteritis in mice and inhibit TLR9 and NF-κB-dependent DNA sensing pathways and the secretion of IL-1β in dendritic cells.