海藻糖对小鼠放射性肠损伤的防护作用及机制研究

Protective effect and mechanism of trehalose on ionizing radiation-induced intestinal damage in mice

目的探讨海藻糖对小鼠放射性肠损伤的防护作用及分子机制。方法采用简单随机抽样法将8周龄的雄性C57 BL/6 J小鼠分为4组:对照组(不给予任何处理)、海藻糖组(饮用2 g/100 ml海藻糖水)、照射组(以13 Gy剂量对小鼠腹部照射)、海藻糖+照射组(饮用2 g/100 ml海藻糖水+以13 Gy剂量对小鼠腹部照射),每组6只。经13 Gy照射和2 g/100 ml海藻糖饮水处理后,每天记录小鼠体重,14 d后处死小鼠并采集小鼠的血液、小肠和结肠,测量结肠长度。采用苏木精-伊红(HE)染色法和过碘酸-雪夫(PAS)染色法评估小鼠肠道损伤程度;酶联免疫吸附实验(ELISA)检测血清和小肠组织中肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)的水平;小鼠小肠上皮MODE-K细胞增殖实验、细胞克隆形成实验、细胞凋亡检测实验、活性氧测定实验和DNA损伤检测实验分别检测细胞活力、细胞克隆形成能力、细胞凋亡率、活性氧水平和DNA双链断裂数量;实时定量聚合酶链式反应(qRT-PCR)法和Western blot法分别检测自噬相关基因mRNA和蛋白水平的表达情况。组间比较采用两独立样本t检验。结果动物实验结果显示,与照射组比较,海藻糖+照射组可显著缓解电离辐射(IR)后小鼠体重的下降,并可促进其恢复(t=4.064,P<0.05),且小鼠结肠缩短程度较轻,2组间的差异有统计学意义(t=4.044,P<0.05)。HE和PAS染色法评估结果显示,海藻糖可缓解IR后小鼠小肠黏膜损伤,与照射组比较,海藻糖+照射组小鼠绒毛较长、隐窝较深、杯状细胞数量较多,2组间的差异有统计学意义(t=4.373、8.414、6.515,均P<0.05)。ELISA检测结果显示,与照射组比较,海藻糖+照射组可显著降低IR后小鼠血清及小肠组织中TNF-α和IL-6的水平,且差异均有统计学意义(t=4.475、8.686、3.993、6.007,均P<0.05)。细胞增殖和克隆实验结果显示,与照射组比较,海藻糖+照射组可促进IR后小鼠小肠上皮MODE-K细胞活性和克隆形成能力[(0.885±0.127)对(0.644±0.151)、(97.330±5.937)对(64.000±7.324)],且差异均有统计学意义(t=2.566、4.411,均P<0.05);细胞凋亡检测实验结果显示,与照射组比较,海藻糖+照射组可抑制IR后小鼠小肠MODE-K细胞凋亡率[(15.270±0.647)%对(9.334±0.854)%],且差异有统计学意义(t=8.315,P<0.05);活性氧测定实验和DNA损伤检测实验结果显示,与照射组比较,海藻糖+照射组可降低IR后小鼠细胞内ROS水平和DNA双链断裂数量(t=8.884、4.802,均P<0.05)。qRT-PCR法和Western blot法检测结果显示,与照射组比较,海藻糖+照射组小鼠小肠组织和MODE-K细胞中自噬相关基因TFEB、MAP1LC3B的表达水平显著增加[(208.210±26.143)对(8.986±5.362)、(13.970±4.587)对(5.815±1.873)],且差异均有统计学意义(t=8.875,8.461,均P<0.05)。结论海藻糖通过调控转录因子EB介导的自噬反应激活缓解小鼠放射性肠损伤。

Objective To investigate the protective effect and molecular mechanism of trehalose in ionizing radiation(IR)-induced small intestinal damage.Methods Eight-week-old male C57 BL/6 J mice were randomly divided into four groups,with each group containing six mice:control(no treatment given),trehalose-treated(consumption of 2 g/100 ml seaweed sugar water),IR(whole abdominal irradiation group treated with 13 Gy IR),and trehalose+IR groups(trehalose+total abdominal irradiation treatment with 13 Gy IR).Daily monitoring of body weight was performed after 13 Gy irradiation and 2 g/100 ml trehalose drinking water treatment.After 14 days,the mice were euthanized,and samples from the blood,small intestine,and colon were collected.Intestinal injury was assessed via hematoxylin-eosin(HE)and periodic acid-schiff(PAS)staining.The levels of tumor necrosis factor(TNF)-αand interleukin(IL)-6 in the serum and small intestine tissues were quantified through enzyme-linked immunosorbent assay(ELISA).Experiments on mouse small-intestine epithelial cell proliferation,cell clone formation,cell apoptosis rate,reactive oxygen species(ROS)determination,and DNA double-strand breaks(DSBs)detection were conducted to detect cell viability,clone formation ability,apoptosis,ROS levels,and the number of DSBs,respectively.The expression levels of autophagy-related genes and proteins were determined via quantitative real-time polymerase chain reaction(qRT-PCR)method and Western blot method.Inter group comparison was achieved through two independent sample t-tests.Results The animal study results demonstrated that compared with the irradiation group,trehalose significantly alleviated the weight loss,facilitated the recovery of irradiated mice,and promoted its recovery(t=4.064,P<0.05),the trehalose+IR group exhibited milder colon shortening degree of mice,and statistically significant difference(t=4.044,P<0.05).In addition,the HE and PAS staining evaluation results indicated that trehalose can alleviate intestinal mucosal damage in mice after IR.Compared with the irradiation group,the trehalose+irradiation group presented longer villi,deeper crypts,and more goblet cells.The two groups exhibited a statistically significant difference(t=4.373,8.414,6.515,all P<0.05).ELISA demonstrated that trehalose substantially decreased the TNF-αand IL-6 levels in mouse serum and small intestinal tissues after IR,and the differences were statistically significant(t=4.475,8.686,3.993,6.007,all P<0.05).In vitro,the results of cell proliferation and cloning experiments revealed that trehalose promoted the activity and clone formation ability of mouse small-intestine MODE-K cells after IR[(0.885±0.127)vs.(0.644±0.151),(97.330±5.937)vs.(64.000±7.324)],with the differences showing statistical significance(t=2.566,4.411,all P<0.05).The findings of the apoptosis detection experiment indicated that trehalose inhibited the apoptosis rate of mouse small intestine MODE-K cells after IR treatment[(15.270±0.647)%vs.(9.334±0.854)%],and the difference was statistically significant(t=8.315,P<0.05).According to ROS assay and DSB detection experiments,trehalose reduced intracellular ROS levels and DSBs after IR(t=8.884,4.802,both P<0.05).The findings of qRT-PCR and Western blot analysis showed that compared with the IR group,the trehalose+IR group showed significantly increased expressions of autophagy-related genes and proteins transcription factor EB(TFEB)and microtubule-associated proteins 1 light chain 3 beta in the small-intestine tissue and MODE-K mouse cells[(208.210±26.143)vs.(8.986±5.362),(13.970±4.587)vs.(5.815±1.873)],and the differences were statistically significant(t=8.875,8.461,all P<0.05).Conclusion Trehalose alleviates IR-induced intestinal damage in mice through the regulation of TFEB-mediated autophagy activation.