目的 基于腺苷5'-单磷酸活化蛋白激酶（ AMPK）/转化生长因子-β1（ TGF-β1）/Smad同源物（ Smads）信号通路，探讨少腹逐瘀汤（ SFZY）含药血清对永生化人子宫内膜异位症细胞（ 12Z）纤维化形成的影响及作用机制。方法 制备SFZY大鼠含药血清，体外培养12Z细胞，CCK-8法筛选SFZY含药血清（ 5%、10%、15%、20%）的作用体积分数及时间；实时荧光定量PCR（ qRT-PCR）法检测细胞的Ⅰ型胶原蛋白（ COL1A1）、α-平滑肌肌动蛋白（ α-SMA）和结缔组织生长因子（ CTGF）mRNA水平，Western blotting法检测细胞α-SMA的蛋白表达水平，确定TGF-β1（ 5、10、15 ng·mL^-1）诱导细胞呈纤维化模型的最佳质量浓度； SFZY含药血清（ 5%、10%、15%）干预造模后的12Z细胞，采用Western blotting实验检测各组细胞的α-SMA蛋白表达水平，确定含药血清抑制纤维化的最佳作用体积分数。将12Z细胞分为对照组、模型组、SFZY（添加10%的含药血清）组、CC（添加AMPK抑制剂Compound C 10 μmol·L^-1）组、SFZY＋ CC组，除对照组外，其余各组采用TGF-β1诱导细胞纤维化，造模的同时给药，CC给药前2 h添加；干预结束后采用qRT-PCR法检测AMPK、TGF-β1、COL1A1、α-SMA和CTGF的mRNA表达水平，采用免疫荧光细胞化学法检测p-AMPK/AMPK、p-Smad2/3/Smad2/3、COL1A1和α-SMA的蛋白表达水平。结果 SFZY含药血清5%、10%、15%作用48 h对细胞存活率无明显影响；与对照组比较，5 ng·mL^-1的TGF-β1干预后的CTGF、COL1A1、α-SMA的mRNA表达升高最明显，α-SMA的蛋白表达升高最明显（ P ＜ 0.01、0.001），确定为造模质量浓度；与模型组相比，5%、10%、15%含药血清干预后的α-SMA的蛋白表达均明显降低（ P ＜ 0.001），10%组降低最明显，确定为后续给药体积分数。与对照组相比，模型组的AMPK mRNA及p-AMPK/AMPK的蛋白表达显著降低（ P ＜ 0.01、0.001），TGF-β1和CTGF mRNA表达显著升高（ P ＜ 0.01、0.001），p-Smad2/3/Smad2/3的蛋白表达明显上调（ P ＜ 0.01），COL1A1和α-SMA的mRNA和蛋白表达水平均显著升高（ P ＜ 0.05、0.01、0.001）；与模型组相比，SFZY组的AMPK mRNA及p-AMPK/AMPK的蛋白表达水平明显升高（ P ＜ 0.001），TGF-β1和CTGF的mRNA表达均显著降低（ P ＜ 0.001），p-Smad2/3/Smad2/3的蛋白表达明显降低（ P ＜ 0.01），COL1A1和α-SMA的mRNA及蛋白表达水平显著降低（ P ＜ 0.01、0.001）； CC可显著抵消SFZY的上述作用。结论 SFZY含药血清可显著抑制TGF-β1刺激后12Z细胞纤维化因子的表达，该作用可能与激活AMPK磷酸化从而抑制TGF-β1/Smads通路的活化有关。

Objective Based on the adenosine monophosphate-activated protein kinase (AMPK)/transforming growth factor-β1 (TGF-β1)/Smad homologs (Smads) signaling pathway, the effect and mechanism of Shaofu Zhuyu Decoction (SFZY)-containing serum on fibrosis formation in immortalized human endometriosis cells (12Z) were investigated. Methods SFZY-containing serum from rars was prepared, and 12Z cells were cultured in vitro. The action volume fraction and time of SFZY drug-containing serum (5%, 10%, 15%, and 20%) were screened by CCK-8 method. The mRNA levels of type I collagen (COL1A1), α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) in cells were detected by real-time fluorescence quantitative PCR (qRT-PCR), and the protein expression level of α-SMA was detected by Western blotting to determine the optimal mass concentration of TGF-β1 (5, 10, and 15 ng·mL^-1) for inducing fibrosis in cells. SFZY drug-containing serum (5%, 10%, 15%) was used to intervene in the 12Z cells after modeling, and the protein expression level of α-SMA in each group of cells was detected by Western blotting to determine the optimal volume fraction of drug-containing serum for inhibiting fibrosis. 12Z cells were divided into the control group, model group, SFZY (adding 10% drug-containing serum) group, CC (adding AMPK inhibitor Compound C 10 μmol·L^-1) group, and SFZY + CC group. Except for the control group, the other groups were induced to fibrosis by TGF-β1, and the drugs were administered simultaneously during modeling, with CC administered 2 h before. After intervention, the mRNA expression levels of AMPK, TGF-β1, COL1A1, α-SMA, and CTGF were detected by qRT-PCR, and the protein expression levels of p-AMPK/AMPK, pSmad2/3/Smad2/3, COL1A1, and α-SMA were detected by immunofluorescence cell chemistry. Results SFZY-containing serum at 5%, 10%, and 15% for 48 h had no significant effect on cell survival rate; compared with control group, the mRNA expression of CTGF, COL1A1, and α-SMA was significantly increased after intervention with 5 ng·mL^-1 TGF-β1, and the protein expression of α- SMA was significantly increased (P < 0.01, 0.001), which was determined as the modeling concentration; compared with the model group, the protein expression of α-SMA was significantly decreased after intervention with 5%, 10%, and 15% drug-containing serum (P < 0.001), with the 10% group was the most significant, which was determined as the subsequent administration volume fraction. Compared with the control group, the mRNA expression of AMPK and the protein expression of p-AMPK/AMPK in the model group were significantly decreased (P < 0.01, 0.001), the mRNA expression of TGF-β1 and CTGF was significantly increased (P < 0.01, 0.001), the protein expression of p-Smad2/3/Smad2/3 was significantly upregulated (P < 0.01), and the mRNA and protein expression levels of COL1A1 and α-SMA were significantly increased (P < 0.05, 0.01, 0.001). Compared with the model group, the mRNA expression of AMPK and the protein expression level of p-AMPK/AMPK in the SFZY group were significantly increased (P < 0.001), while the mRNA expression of TGF-β1 and CTGF were significantly decreased (P < 0.001), and the protein expression of pSmad2/3/Smad2/3 was significantly reduced (P < 0.01). The mRNA and protein expression levels of COL1A1 and α-SMA were also significantly decreased (P < 0.01, 0.001). CC could significantly counteract the above effects of SFZY. Conclusion SFZYDcontaining serum significantly inhibits the expression of fibrotic factors in TGF-β1-stimulated 12Z cells, potentially by activating AMPK phosphorylation to suppress the TGF-β1/Smads pathway.

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国家自然科学基金资助项目（82104920）；山东省自然科学基金资助项目（ ZR2021QH129）； 山东省中医药重点实验室资助项目（鲁卫涵[2022]4 号）