雾化吸入花椒精油对炎症相关性结直肠癌小鼠炎癌转化的影响及作用机制

Effect of Atomization Inhalation of Huajiao(Zanthoxylum bungeanum) Essential Oil on Inflammatory and Cancer Transformation in CAC Mice and Its Mechanism

目的 观察花椒精油雾化吸入对炎症相关性结直肠癌(colitis associated cancer, CAC)小鼠模型肠道炎癌转化的影响及作用机制。方法 将72只C57BL/6小鼠随机均分为正常组、模型组、花椒高中低(8%、5%、2%)浓度组以及溶媒对照组(2%Tween-80)。除空白组外,其余各组均用氧化偶氮甲烷(azoxymethane, AOM)/葡聚糖硫酸钠(dextran sulfate sodium salt, DSS)联合诱导构建CAC模型,分别用不同浓度花椒精油雾化吸入给药进行干预。观察并记录小鼠一般情况,监测体质量、疾病活动指数(disease activity index, DAI)变化;利用苏木素-伊红(hematoxylin-eosin, HE)染色观察结肠组织病理,筛选最佳药物干预浓度;通过实时荧光定量PCR法(real-time quantitative PCR detecting system, RT-qPCR)检测结肠组织中白细胞介素-6(interleukin 6,IL-6)的mRNA相对表达;比色法检测结肠中乙酰胆碱酯酶(acetylcholinesterase, AChE)、乙酰胆碱转移酶(choline acetyltransferase, ChAT)活性;蛋白免疫印迹法(Western blot, WB)检测结肠中α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α-7nAChR)蛋白表达水平。结果 (1)HE染色示:与空白组相比,模型组小鼠结肠组织病理损伤显著,上皮细胞结构紊乱,出现不典型增生及大量炎性细胞浸润;与模型组相比,花椒高、低浓度组小鼠结肠黏膜损伤、结构紊乱、不典型增生、炎性浸润等较轻;花椒中浓度组小鼠结肠黏膜轻微损伤,未出现高分化腺癌样改变,无明显增生现象。据此确定花椒精油最佳干预浓度为中浓度。(2)与空白组相比,模型组AChE活性升高(P<0.01)、ChAT活性降低(P<0.05),α-7nAChR蛋白表达降低(P<0.01),IL-6的mRNA相对表达上调(P<0.01)。与模型组相比,花椒中浓度组AChE活性降低(P<0.01)、ChAT活性升高(P<0.05),α-7nAChR蛋白表达升高(P<0.05),花椒中浓度组IL-6的mRNA相对表达下调(P<0.05)。结论 雾化吸入花椒精油可延缓CAC小鼠炎癌转化进程,其作用机制可能与胆碱能抗炎通路激活、IL-6表达下调有关。

Objective To observe the effect and mechanism of atomized inhalation of Huajiao(Zanthoxylum bungeanum) essential oil on intestinal inflammation and cancer transformation in a mouse model of inflammation related colorectal cancer(CAC). Methods Seventy-two C57 BL/6 mice were randomly divided into normal group, model group, Huajiao(Zanthoxylum bungeanum) high, medium and low concentration(8%,5%,2%) groups and solvent control group(2% Tween-80). Except the blank group, the other groups were induced by azoxymethane(AOM)/dextran sodium sulfate(DSS) to construct the CAC model, and were intervened by aerosol inhalation of Huajiao(Zanthoxylum bungeanum) essential oil at different concentrations. The general conditions of mice were observed and recorded, and the changes of body mass and disease activity index(DAI) were monitored. Hematoxylin eosin(HE) staining was used to observe colonic histopathology and screen the best drug intervention concentration. The relative expression of interleukin-6(IL-6) mRNA in colon tissue was detected by real-time fluorescence quantitative PCR(RT-qPCR). The activities of acetylcholinesterase(AChE) and acetylcholine transferase(ChAT) in colon were detected by colorimetry. Western blot was used to detect α Nicotinic acetylcholine receptor(α-7 nachr) protein expression level. Results(1)HE staining showed that compared with those of the blank group, the pathological damage of colon tissue in the model group was significant and the structure of epithelial cells was disordered. The atypical hyperplasia and a large number of inflammatory cell infiltration appeared in the model group. Compared with those of the model group, the colonic mucosal damage, structural disorder, atypical hyperplasia and inflammatory infiltration in the Huajiao(Zanthoxylum bungeanum) high and low concentration groups were lighter. The colon mucosa of mice in Huajiao(Zanthoxylum bungeanum) medium concentration group was slightly damaged. There was no well differentiated adenocarcinoma like change and no obvious hyperplasia. It was determined that the optimal intervention concentration of Huajiao(Zanthoxylum bungeanum) essential oil was the medium concentration.(2)Compared with those of the blank group, the AChE activity was increased(P<0.01) and the chat activity decreased(P<0.05) in the model group. The expression of α-7 nachr protein was decreased(P<0.01) and the relative expression of IL-6 mRNA increased(P<0.01). Compared with those of the model group, the AChE activity was decreased(P<0.01) and chat activity increased(P<0.05),the expression of α-7 nachr protein increased(P<0.05),and the relative expression of IL-6 mRNA decreased in the Huajiao(Zanthoxylum bungeanum) medium concentration group(P<0.05). Conclusion The aerosol inhalation of Huajiao(Zanthoxylum bungeanum) essential oil can delay the process of inflammatory and cancer transformation in CAC mice, and its mechanism may be related to the activation of cholinergic anti-inflammatory pathway and the down-regulation of IL-6 expression.