消癥湯對大鼠子宮平滑肌瘤細胞增殖和遷移的影響及機制

吳艷麗 范楷 孔守芳 趙秀芳 歐陽江華

中圖分類號 R285;R361+.3 文獻標志碼 A 文章編號 1001-0408（2021）20-2450-09

DOI 10.6039/j.issn.1001-0408.2021.20.04

摘 要 目的：初步研究消癥湯對大鼠子宮平滑肌瘤細胞增殖和遷移的影響及作用機制。方法：將雌性SD大鼠隨機分為正常對照組、模型組、化學藥陽性對照組（米非司酮片2.25 mg/kg）、中藥陽性對照組（桂枝茯苓膠囊200 mg/kg）和消癥湯低、中、高劑量組（1.4、2.8、5.6 g/kg），每組15只。除正常對照組外，其余各組大鼠均肌內注射雌激素和孕激素建立子宮平滑肌瘤模型。自建模第2天起，各給藥組大鼠灌胃相應藥液，正常對照組和模型組大鼠灌胃等體積生理鹽水，每天1次，連續4個月。末次給藥后，摘取子宮并觀察其形態，計算子宮系數;分離培養子宮平滑肌瘤細胞或子宮平滑肌細胞，采用MTT法檢測細胞增殖率，細胞劃痕實驗檢測子宮平滑肌瘤細胞遷移率，流式細胞術檢測細胞凋亡率，熒光定量聚合酶鏈反應技術檢測細胞中高遷移率族蛋白B1（HMGB1）mRNA表達情況，酶聯免疫吸附測定法檢測細胞中磷酸化蛋白激酶B（p-Akt）、核因子κB抑制蛋白α（IκBα）、磷酸化轉化生長因子β激活激酶1（p-TAK1）的表達水平，Western blot法檢測細胞質內HMGB1、磷脂酰肌醇3激酶（PI3K）、p-Akt蛋白和細胞核內核因子κB p65（NF-κB p65）蛋白的表達水平。結果：與正常對照組比較，模型組大鼠子宮肌層明顯增厚，子宮平滑肌細胞明顯增多且大小不一，部分區域的肌纖維排列紊亂，子宮系數和細胞中HMGB1 mRNA及其蛋白的相對表達水平均顯著升高（P<0.01）。與模型組比較，消癥湯各劑量組以及各陽性對照組大鼠的子宮平滑肌層增厚等情況均有不同程度改善，子宮系數以及子宮平滑肌瘤細胞增殖率、相對遷移率和細胞中HMGB1 mRNA及其蛋白的相對表達水平，p-Akt、IκBα的表達水平，細胞質中PI3K、p-Akt蛋白的相對表達水平（除消癥湯低劑量組外）均顯著降低（P<0.05或P<0.01），子宮平滑肌瘤細胞凋亡率和細胞中p-TAK1的表達水平、細胞核內NF-κB p65蛋白的相對表達水平均顯著升高（P<0.05或P<0.01），且消癥湯的作用有劑量依賴的趨勢。結論：消癥湯可能通過下調HMGB1、PI3K、p-Akt表達，上調NF-κB p65表達，進而抑制子宮平滑肌瘤細胞的增殖和遷移，并促使其凋亡。

關鍵詞 消癥湯;子宮平滑肌瘤;高遷移率族蛋白B1;磷脂酰肌醇3激酶/蛋白激酶B;核因子κB;大鼠

Effects and Mechanism of Xiaozheng Decoction on the Proliferation and Migration of Uterine Leiomyoma Cells in Rat

WU Yanli，FAN Kai，KONG Shoufang，ZHAO Xiufang，OUYANG Jianghua（Dept. of Gynaecology， Qingdao Hospital of TCM， Shandong Qingdao 266033， China）

ABSTRACT? ?OBJECTIVE： To study the effects and mechanism of Xiaozheng decoction on the proliferation and migration of uterine leiomyoma cells in rat. METHODS： Female SD rats were randomly divided into normal control group， model group， chemical medicine positive control group （Mifepristone tablets， 2.25 mg/kg）， TCM positive control group （Guizhi fuling capsules， 200 mg/kg）， Xiaozheng decoction low-dose， medium-dose and high-dose groups （1.4， 2.8， 5.6 g/kg）， with 15 rats in each group. Except for normal control group， other groups were given intramuscular injection of estrogen and progesterone to induce uterine leiomyomas model. On the second day after modeling， rats in administration groups were given relevant medicine intragastrically; normal control group and model group were given constant volume of normal saline intragastrically， once a day， for consecutive 4 months. After last administration， the uterus was removed and its morphology was observed; the uterine coefficient was calculated. Uterine leiomyoma cells or uterine smooth muscle cells were isolated and cultured. The proliferation rate and migration rate of cells were detected by MTT method and cell scratch test; flow cytometry was used to detect the cell apoptosis rate; mRNA expression of HMGB1 were detected by RT-qPCR. The expression of phosphorylated protein kinase B （p-Akt）， nuclear factor κB inhibitor α? （IκBα） and phosphorylated transforming growth factor β-activated kinase 1 （p-TAK1） were detected by ELISA; the protein expression of HMGB1， phospholipid 3 kinase （PI3K）， p-Akt in cytoplasm and nuclear factor κB p65（NF-κB p65） in nucleus were detected by Western blot assay. RESULTS： Compared with normal control group， the myometrium of the model group was significantly thickened， the number of uterine smooth muscle cells were significantly increased and the sizes were different， the arrangement of muscle fibers in some areas was disordered， the uterine coefficient， and the relative expression of HMGB1 mRNA and protein were increased significantly （P<0.01）. Compared with model group， the thickening of uterine myometrium and other symptoms were improved to different extents in? Xiaozheng decoction groups and positive control groups; the uterine coefficient， cell proliferation rate， migration rate， mRNA and protein expression of HMGB1， the expression of p-Akt and IκBα， protein expression of PI3K and p-Akt （except for Xiaozheng decoction low-dose group） in cytoplasm were all decreased significantly （P<0.05 or P<0.01）; the cell apoptosis rate， the expression of p-TAK1， protein expression of NF-κB p65 in nucleus were all increased significantly （P<0.05 or P<0.01）. The effects of Xiaozheng decoction showed a dose-dependent trend. CONCLUSIONS： Xiaozheng decoction can inhibit the proliferation and migration of uterine leiomyoma cells by down-regulating the expression of HMGB1， PI3K and p-Akt， up-regulating the expression of NF-κB p65， so as to promote cell apoptosis.