基于EGFR表达与细胞定位研究姜黄素联合银杏内酯B治疗多囊肾病的作用机制

Autosomal dominant polycystic kidney disease (ADPKD), a common disease with high incidence ratio, often causes great harm to people. However, no effective drug is currently available. The overexpression and abnormal celluar location of EGFR are the key factors in the pathogenesis of polycystic kidney disease, cancer, diabetes and other diseases. The novel roles of curcumin and ginkgolide B were both found in inhibition of cyst formation in vitro by regulating the downstream of EGFR such as MAPK signaling pathway. We found that the shapes of cysts were different after curcumin or ginkgolide B treatment. The cysts treated with curcumin often became small thick-walled cysts, the cysts exposed to ginkgolide B often turned into small thin-walled cysts, and the cysts after coinhibition of curcumin and ginkgolide B often became round shaped colonies, which suggest the combination of two drugs is more effective than singel drug. Foreign study and our study have confirmed the cyst inhibition effects of curcumin and ginkgolide B on polycystic kidney disease mice. The cyst inhibition mechanism hypothesis of curcumin and ginkgolide B were raised based on these findings. Curcumin may up-regulate calcium level and Annexin expression, then regulate the expression, dimer structure and cellular localization of EGFR. Ginkgolide B may inhibit EGFR expression, but unaffect its cellular localization.The purpose of this study is to evaluate the renal cyst coinhibition of curcumin and ginkgolide B by using PKD1 knockout mice and to demonstrate their inhibition mechanism hypothesis related with EGFR expression and cellular location by immunohistochemistry. The study will provide experimental evidence and ideas for us to discover novel drugs, find new therapeutic targets, develop pathogenic mechanisms of ADPKD, and contribute the prevention and treatment of diseases related with EGFR.

多囊肾病ADPKD发病率高，危害大，目前尚无特效治疗药物。EGFR过度表达与异常细胞定位是多囊肾病、肿瘤、糖尿病等疾病的发病关键因素。前期研究发现姜黄素、银杏内酯B调控EGFR下游MAPK信号通路抑制体外囊泡生长，姜黄素、银杏内酯B、姜黄素联合银杏内酯B作用后囊泡形态分别多为厚壁小囊泡、薄壁小囊泡、圆形细胞集落，两药联合的囊泡抑制作用优于单一用药。国外及前期研究随后证实两药均抑制多囊肾病小鼠体内肾囊泡生长。本课题在此基础上，提出了两药囊泡抑制作用的机制假说，即姜黄素上调钙离子、后者激活Annexin而调控EGFR的表达、二聚体构成及细胞定位，银杏内酯B抑制EGFR的表达而不影响其细胞定位，将通过应用多囊肾病PKD1基因敲除小鼠评价两药联合的囊泡抑制作用，应用免疫组化等技术研究作用机制假说，为研发新的治疗多囊肾病药物、发现新的治疗靶点、创新发病理论、防治EGFR相关疾病等提供实验依据及思路。

本项目应用MDCK囊泡模型，研究姜黄素联合银杏内酯对MDCK囊泡的抑制作用，发现银杏内酯B（0.125μM、0.5μM、2μM）、姜黄素（0.4μM、2μM、10μM）联合均可显著减少MDCK囊泡的生成，MDCK囊泡抑制率高达90.46%（单用姜黄素、银杏内酯B分别仅为63.82%、66.73%）。应用多囊肾病PKD1基因敲除小鼠模型，研究姜黄素联合银杏内酯对小鼠肾囊泡的抑制作用，银杏内酯B（20mg/kg、40mg/kg、80mg/kg）、姜黄素（80mg/kg、160mg/kg、320mg/kg）联合均可显著减少小鼠肾囊泡的生长，小鼠肾囊泡抑制率高达87.93%（而单用姜黄素、银杏内酯B分别仅为41.22%、46.43%），小鼠生存期延长率61.40%（而单用姜黄素、银杏内酯B分别仅为20.18%、7.02%）。应用Western blot、免疫组化等技术检测囊泡相关信号蛋白，发现小鼠多囊肾组织中磷酸化的EGFR、Cerb-B2表达均增加，姜黄素、银杏内脂B均可抑制磷酸化EGFR、Cerb-B2表达，且姜黄素联合银杏内酯B优于单一用药。结合前期及本项目研究，姜黄素、银杏内脂B可通过抑制EGFR介导Ras-ERK/JNK MAPK信号通路发挥囊泡抑制作用。此外，从小鼠肾囊泡形态观察，姜黄素组囊泡壁较厚，银杏内酯B组囊泡壁较薄，姜黄素联合银杏内酯B组囊泡壁较厚，其机制仍需进一步研究。. 基于以上结果，获新药发明专利1项：姜黄素与银杏内酯B配伍组合物及其应用（专利号ZL201310568288.8），成果鉴定为国际先进，获山西省科技进步三等奖（证书号2015-J-3-072）。2014年美国实验生物学年会（EB2014）收录成果摘要。现已发表相关文章2篇，并参与编著人民卫生部出版社书籍1部：《中药药效研究方法学》。