2024 年第 5 期 第 19 卷

理冲生髓饮有效组分调控相关信号通路对卵巢癌自噬的影响研究

Study on the effects of active components of Lichong Shengsui decoction on autophagy in ovarian cancer by regulating related signaling pathways

作者：刘芳媛1 丁丹妮2 郭滢1 沈影1 李佳1 韩凤娟1

英文作者：Liu Fangyuan1 Ding Danni2 Guo Ying1 Shen Ying1 Li Jia1 Han Fengjuan1

单位：1黑龙江中医药大学附属第一医院妇科三科，哈尔滨150040；2黑龙江中医药大学第一临床医学院，哈尔滨150040

英文单位：1Third Department of Gynecology First Affiliated Hospital Heilongjiang University of Chinese Medicine Harbin 150040 China; 2First Clinical Medical College Heilongjiang University of Chinese Medicine Harbin 150040 China

关键词：卵巢癌；理冲生髓饮；细胞自噬；信号通路

英文关键词：Ovariancancer;LichongShengsuidecoction;Cellautophagy;Signalingpathway

• 摘要：

• 目的  观察理冲生髓饮有效组分（LCSSY）调控腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/丝氨酸苏氨酸蛋白激酶-失调51样激酶1(ULK1)信号通路对卵巢癌自噬的影响。方法  体外培养卵巢癌SKOV3细胞，制备LCSSY含药血清，采用随机数字表法将SD大鼠分为空白对照组和LCSSY低、中、高剂量组。细胞计数盒8法检测卵巢癌SKOV3细胞增殖抑制率；蛋白质印迹法检测计算自噬相关蛋白重组人自噬效应蛋白（Beclin-1）、人微管相关蛋白轻链3Ⅱ与小鼠微管相关蛋白轻链3Ⅰ比值（LC3Ⅱ/Ⅰ）、自噬接头蛋白（P62）以及AMPK/mTOR/ULK1信号通路相关蛋白的表达，并采用AMPK激活剂验证其作用机制。结果  给药24、48 h，LCSSY低、中、高剂量组对SKOV3细胞增殖的抑制率均大于空白对照组，且随着药物浓度的增加抑制程度逐渐加强（均P<0.05）；另外，给药48 h LCSSY低剂量组、LCSSY中剂量组抑制率均大于本组给药24 h时（均P<0.05）。与空白对照组比较，LCSSY低、中、高剂量组均能够下调Beclin-1和LC3Ⅱ/Ⅰ的表达，上调P62的表达，且呈剂量依赖性［（0.98±0.02）、（0.59±0.02）、（0.55±0.02）比（1.46±0.12），（4.45±0.21）、（4.00±0.25）、（2.95±0.21）比（5.98±0.23），（0.67±0.02）、（0.94±0.05）、（1.53±0.13）比（0.30±0.02）］（均P<0.05）。与空白对照组比较，LCSSY低、中、高剂量组均可以降低AMPK和ULK1表达，升高mTOR复合物1表达，且呈剂量依赖性（均P<0.05）。AMPK激活剂逆转AMPK/mTOR/ULK1信号通路后，与单独LCSSY高剂量组相比，LCSSY高剂量联合AMPK激活剂组使AMPK、ULK1蛋白表达量升高，而mTOR复合物1蛋白表达量降低（均P<0.05）。结论  LCSSY可能通过调控AMPK/mTOR/ULK1信号通路抑制卵巢癌细胞自噬。

• Objective  To observe the effect of Lichong Shengsui decoction (LCSSY) on autophagy in ovarian cancer by regulating adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/uncoordinated 51 like kinase-1(ULK1) signaling pathway. Methods  Ovarian cancer SKOV3 cells were cultured in vitro and serum containing LCSSY was prepared. SD rats were divided into blank control group and LCSSY low, medium and high dose groups by random number table method. Cell count kit-8 method was used to detect the proliferation inhibition rate of ovarian cancer SKOV3 cells. Western blotting was used to detect the expression of autophagy related proteins recombinant human autophagy effector protein(Beclin-1), ratio of human microtubule associated protein light chain 3 Ⅱ to mouse microtubule associated protein light chain 3 Ⅰ(LC3Ⅱ/Ⅰ), autophagy junction protein(P62), as well as AMPK/mTOR/ULK1 pathway related proteins. AMPK activator was used to verify its mechanism of action. Results  At 24 and 48 h after administration, the inhibition rates of SKOV3 cell proliferation in the low, medium and high dose LCSSY groups were higher than those in the blank control group, and the inhibition degree was gradually strengthened with the increase of drug concentration(all P<0.05). In addition, the inhibition rates of low dose LCSSY group and medium dose LCSSY group at 48 h were higher than those at 24 h (both P<0.05). Compared with the blank control group, the expression of Beclin-1 and LC3Ⅱ/Ⅰ was down-regulated in the low, medium, and high dose LCSSY groups in a dose-dependent manner［(0.98±0.02), (0.59±0.02), (0.55±0.02) vs (1.46±0.12); (4.45±0.21), (4.00±0.25), (2.95±0.21) vs (5.98±0.23); (0.67±0.02), (0.94±0.05), (1.53±0.13) vs (0.30±0.02)］(all P<0.05). Compared with the blank control group, the expression of AMPK and ULK1 was decreased, and the expression of mTOR complex 1 was increased in low, medium and high dose LCSSY groups in a dose-dependent manner(all P<0.05). AMPK activator reversed the AMPK/mTOR/ULK1 signaling pathway, and compared with the high dose LCSSY group, the high dose of LCSSY combined with AMPK activator increased the protein expressions of AMPK and ULK1, while the protein expression of mTOR complex 1 decreased(all P<0.05). Conclusion  LCSSY may inhibit autophagy in ovarian cancer cells by regulating AMPK/mTOR/ULK1 signaling pathway.