止痛调血方有效组分宫内缓释系统通过 PI3K/Akt/mTOR通路介导自噬抑制血管生成治疗子宫腺肌病的机制研究

The incidence of Adenomyosis (AM) is increasing year by year and getting younger, and it has become a research focus to find effective drugs with few side effects. The intrauterine sustained release system study of effective components of traditional Chinese medicine will provide more accurate research ideas for clarifying the intervention targets of traditional Chinese medicine. PI3K/Akt/mTOR signaling pathway, as a classical autophagy pathway, can inhibit angiogenesis. At the early stage, the research group adopted the empirical prescription named analgesic and blood-regulating prescription to treat AM, the clinical effect was satisfactory. Preliminary experiments confirmed that it could promote autophagy and inhibit angiogenesis. Based on these, this research intends to adopt noninvasive AM rat model established by modified allograft pituitary transplantation、 Human AM focal cells cultured in vitro, to observe the changes of autophagosome and vascular density in AM by transmission electron microscopy, to detect the expression levels of proteins related to autophagy, angiogenesis and the upstream and downstream of PI3K/Akt/mTOR signaling pathway by Western blot, qPCR, immunohistochemistry, immunofluorescence, laser confocal electron microscopy and other methods , to investigate the effect of analgesic and blood regulating prescription effective component on the PI3K/Akt/mTOR pathway, and to identify VEGF as the key target. The results will provide a strong basis for the clinical application of analgesic and blood regulating prescription effective component intrauterine sustained release system in AM.

子宫腺肌病(Adenomyosis,AM)发病率逐年增高并呈年轻化，寻找有效、副作用小的药物成为研究重点，开展中药有效组分宫内缓释系统研究将为明晰中医药干预靶点提供更为精准的研究思路。PI3K/Akt/mTOR通路作为经典的自噬通路可抑制血管生成，课题组前期采用经验方止痛调血方（ZTTXF）治疗AM，临床疗效满意，预实验证实此方可促进自噬并抑制血管生成，基于此，本课题组拟采用无创法建立AM大鼠模型并体外培养人AM病灶细胞，透射电镜观察AM中自噬小体、血管密度变化情况；Western blot、qPCR、免疫组化法、免疫荧光法、激光共聚焦电镜等检测自噬、血管生成及PI3K/Akt/mTOR信号通路上下游相关蛋白表达水平，探讨ZTTXF有效组分宫内缓释系统对PI3K/Akt/mTOR通路的影响，研究结果将为ZTTXF有效组分宫内缓释系统在AM的临床应用提供有力证据。

子宫腺肌病(Adenomyosis,AM)发病率逐年增高并呈年轻化，寻找有效、副作用小的药物成为研究重点，开展中药有效组分宫内缓释系统研究将为明晰中医药干预靶点提供更为精准的研究思路。PI3K/Akt/mTOR通路作为经典的自噬通路可抑制血管生成，课题组前期采用经验方止痛调血方（ZTTXF）治疗AM，临床疗效满意，预实验证实此方可促进自噬并抑制血管生成，基于此，本课题通过建立子宫腺肌病大鼠模型及体外培养人子宫腺肌病异位内膜细胞，进行以下研究：1）采用自噬抑制剂3-MA及自噬促进剂雷帕霉素，在自噬促进或抑制状态下，光镜下观察子宫腺肌病病灶细胞血管密度变化；检测子宫腺肌病异位子宫内膜中血管生成相关蛋白表达，明确了自噬与宫腺肌病血管生成关系密切；2）加入PI3K抑制剂LY294002及mTOR的抑制剂雷帕霉素，在PI3K/Akt/mTOR信号转导通路抑制及促进状态下，光镜观察自噬小体形成及血管密度的变化，检测自噬及血管生成相关蛋白及mRNA表达，明确了PI3K/Akt/mTOR信号通路在子宫腺肌病发病中具有负向调节自噬及促进血管生成的作用；3）以止痛调血方、孕三烯酮作为中西药对照组，透视电镜观察自噬小体形成，光镜下观察血管密度的变化，检测 PI3K/Akt/mTOR 通路上下游蛋白及自噬、血管生成相关蛋白表达，明确了止痛调血方有效组分宫内缓释系统通过调控PI3K/Akt/mTOR 信号通路通过调控细胞自噬抑制子宫腺肌病血管生成，这可能是其治疗本病的重要靶点之一。