基于“炎症诱导血管新生假说”从NF-κB、AQP1信号通路研究肠瑞灌肠剂治疗放射性直肠炎的分子机制

The patients of abdominal and pelvic tumors treated by radiation therapy are often campanied with radiation proctitis, and about 30% patients diagnosed with radiation proctitis have to give up radiation therapy. Radiation proctitis seriously affects the prognosis of these patients. No effective drug or treatment is currently available. The western medicine treatments with anti-inflammatory and hemostatic for radiation proctitis are less effective. The traditional Chinese medicine treatments has been demonstrated to be less effective in some clinical observations of small samples. Therefore there is an urgent need for some novel drugs which have specially good effects on radiation proctitis. We have studied the treatment effect of Changrui enema in radiation proctitis rat mode. Exciting, we found Changrui enema reduced inflammation by inhibiting the expression of IL-1 β mRNA and NF- κB, and improved local microcirculation by up-regulating the level of 6-Kote-PGF1 α and down-regulating the level of TXB2. We have gained the new drug patent of Changrui enema, and have transfered the patent to pharmaceutical campany. The phase II trial of Changrui enema has been carried out recently. We propose the hypothesis that inflammation maybe induce angiogenesis in radiation proctitis. The purpose of this study is to evaluate Changrui enema treatment effect on AQP1 mediated induction angiogenesis in radiation proctitis by using AQP1 gene knockout mice, and to demonstrate the hypothesis molecular mechanism related with NF-κB and AQP1 signaling pathways by immunohistochemical technique, PCR and Western Blot. This study will discover novel treatment of traditional Chinese medicine, find new therapeutic targets, and develop pathogenic mechanisms of radiation proctitis.

放射性直肠炎是盆腹腔肿瘤放疗常见的并发症，约30%患者因此中断放疗而影响预后，目前缺乏特效治疗药物及方法，西医多采用消炎、止血及对症治疗，疗效不佳，中医药有一定优势，多见于小样本临床观察，疗效不满意，因此迫切需要研发特效治疗药物。前期研究发现肠瑞灌肠剂通过下调IL-1βmRNA、NF-κB表达而减轻炎症反应，并通过上调6-Kote-PGF1α水平、下调TXB2水平而改善局部微循环，从而发挥治疗作用，该药获得新药发明专利，并成果转让、进入II期临床试验。本项目在此基础上，提出炎症诱导血管新生假说，观察肠瑞灌肠剂是否对血管新生具有抑制作用？应用AQP1基因敲除小鼠，研究AQP1是否参与放射性直肠炎的血管新生？应用免疫组化、PCR、Western Blot等技术阐明肠瑞灌肠剂调控NF-kB、AQP1相关信号通路发挥治疗放射性直肠炎的分子机制，为发现新药物、新靶点、创新发病机制提供实验依据。

放射性直肠炎（RP）是结直肠癌、卵巢癌、前列腺癌、膀胱癌等腹盆腔恶性肿瘤最常见的放疗并发症，急性放射性直肠炎的发生率为50%～75%、慢性放射性直肠炎的发生率为5%。临床症状主要表现为腹痛、腹泻、粘液脓血便等，严重影响患者的生活质量及预后，甚至导致放疗失败。然而，有效的治疗放射性直肠炎的方法还比较少。肠瑞灌肠剂是一种含7味中药的复方，由地榆30g、仙鹤草15g、三七6g、白芨30g，阿胶12g、大黄10g、儿茶6g组成。本研究前期发现，肠瑞灌肠剂是一种有效的放射性直肠炎的中医辅助疗法。然而，肠瑞灌肠剂的作用机制尚未阐明。.本研究通过观察肠瑞灌肠剂对放射性直肠炎小鼠IL-1β、NF-κB、VEGF、AQP1、AQP3等信号分子的调控，阐明肠瑞灌肠剂是否抑制“炎症诱导血管新生”而发挥治疗放射性直肠炎的分子机制。此外，进一步观察肠瑞灌肠剂含药血清对辐射后人正常结直肠粘膜细胞AQP1、AQP3、AQP4的调节，探讨水通道是否参与肠瑞灌肠剂治疗作用机制。.本研究首次发现中药复方肠瑞灌肠剂对小鼠放射性直肠炎有一定的治疗作用。结果提示，放射性直肠炎小鼠直肠组织中，IL-1β、NF-κB、VEGF、AQP1和p-ERK1/2的表达明显增加，肠瑞灌肠剂可下调这些信号的表达。相反的，AQP3和p-JNK在放射性直肠炎小鼠直肠组织中的表达降低，肠瑞灌肠可上调AQP3、p-JNK的表达。肠瑞灌肠剂含药血清能抑制辐射后人正常结直肠粘膜细胞的凋亡、促进其增殖，其机制与下调AQP1、AQP4表达，增加AQP3表达相关。.综上所述，肠瑞灌肠剂能够有效地治疗放射性直肠炎，其作用机制可能与抑制“炎症诱导血管新生”相关。肠瑞灌肠剂可抑制IL-1β、NF-κB炎性因子的水平，进而抑制血管生成因子VEGF的表达。进一步研究发现，AQP1促进血管生成，而AQP3抑制炎症。肠瑞灌肠剂可通过下调p-ERK1/2抑制AQP1的表达，上调p-JNK增加AQP3的表达，从而发挥治疗作用。此外，肠瑞灌肠剂AQP4的表达也具有抑制作用。因此，不同水通道蛋白在放射性直肠炎治疗中的作用有待于进一步研究。