中华中医药学刊

目的 观察加味芍药甘草汤(加芍甘汤)治疗人来源性子宫腺肌病裸鼠的效果并探讨其对铁死亡相关基因表达的影响。方法 45只雌性SPF级BALB/C-nu鼠作为研究对象，随机选取其中10只作为对照组(A组),其余35只用于造模。对照组腹腔移植CINⅢ患者子宫肌层组织，模型鼠腹腔移植子宫腺肌病(adenomyosis, AM)患者病灶组织。成模后随机抽取5只AM模型鼠用于鉴定，其余30只随机分入AM模型组(B组，10只)、加芍甘汤组(C组，10只)、加芍甘汤联合抑制剂组(D组，10只)。A和B组予生理盐水灌胃，C和D组均根据成人等效剂量的两倍予加芍甘汤灌胃治疗，D组在治疗同时予Ferrostatin-1腹腔注射。疗程满3周即取材，比较4组一般情况、体质量变化、瘤体大小；比较不同组别DMT1、FPN、FTL、GPX4基因表达情况以及谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)、铁的含量。结果 成模后，随机抽取5只AM模型鼠取移植瘤HE染色见AM典型病理变化，提示造模成功。3周疗程结束后，各组瘤体体积均有不同程度缩小。A组和C组的瘤体体积均明显小于B组，差异具有统计学意义(P<0.01)。D组瘤体大于C组(P<0.01),D与B组比较差异无统计学意义(P>0.05)。RT-qPCR结果显示，B组二价金属转运体(divalent metal transporter, DMT1)、铁转运蛋白(ferroportin, FPN or SLC40A1)、铁蛋白轻链(ferritin light chain, FTL)、谷胱甘肽过氧化物酶(glutathione peroxidase, GPX4) mRNA相对表达量显著高于A组和C组(P<0.01)。GPX4基因mRNA表达C组低于D组(P<0.01)。蛋白免疫印迹法(Western blot, WB)结果显示，DMT1、FPN、GPX4 3个基因的蛋白相对表达量B组均高于A组和C组(P<0.05),但FTL表达量在3组间差别无统计学意义(P>0.05)。D组的FPN的蛋白相对表达量高于C组(P<0.05)。在4组中，GSH的含量在B组最高，显著高于其他3组(P<0.01),其他组别两两比较差异均无统计学意义(P>0.05)。MDA的含量在C组最高，显著高于其他3组，D组和B组的含量高于A组(P<0.01),其他组别比较差异无统计学意义。铁含量B组、C组和D组显著高于A组(P<0.01),其他组别两两比较差异均无统计学意义(P>0.05)。结论 人来源性AM裸鼠模型可重复性高。加芍甘汤可缩小AM模型鼠的瘤体大小。加芍甘汤可能通过调控细胞铁死亡机制发挥作用。

Objective To observe the effect of Jiawei Shaoyao Decoction(加味芍药甘草汤,JWSY) in the treatment of human-derived adenomyosis(AM) in nude mice and explore its effect on the expressions of ferroptosis-related genes. Methods Forty-five female SPF BALB/C-nude mice were the study subjects, 10 of which were randomly selected as the control group(group A),and the other 35 were used for modeling. Myometrium of cervical intraepithelial neoplasia(CIN)Ⅲ patients was transplanted into the abdomen of the control group, and the focal tissue of AM patients was transplanted into the abdomen of the model mice. After modeling, five AM model mice were randomly selected for identification, and the other 30 mice were randomly divided into AM model group(group B,10 mice),JWSY group(group C,10 mice)and JWSY combined with inhibitor group(group D,10 mice). Group A and B were given saline intragastric administration, group C and D were given JWSY by intragastric administration(double the adult human equivalent dose),and group D was given ferrostatin-1 intraperitoneal injection at the same time. The subjects were sampled after 3 weeks of treatment, and the general conditions, body weight changes and tumor size of the four groups were compared. The expressions of divalent metal transporter(DMT1),ferroportin(FPN),ferritin light chain(FTL) and glutathione peroxidase(GPX4) genes and the contents of glutathione(GSH,malondialdehyde(MDA) and Fe in different groups were compared. Results After modeling, 5 AM model mice were randomly selected for HE staining and typical AM pathological changes were observed, indicating successful modeling. After 3 weeks of treatment, the tumor volume of each group was reduced to varying degrees. The tumor volume in group A and C was significantly smaller than that in group B,with statistical significance(P<0.01). The tumor volume of group D was larger than that of group C(P<0.01),but there was no significant difference between group D and group B(P>0.05). RT-qPCR results showed that the mRNA relative expression levels of DMT1,FPN,FTL and GPX4 genes in group B were significantly higher than those in groups A and C(P<0.01). The mRNA expression of GPX4 gene in group C was lower than that in group D(P<0.01). WB results showed that the relative protein expressions of DMT1,FPN and GPX4 genes in group B were higher than those in group A and C(P<0.05),but there was no difference in FTL protein expression among the three groups(P>0.05). The relative expression level of FPN protein in group D was higher than that in group C(P<0.05). Among the four groups, GSH content in group B was the highest, which was significantly higher than that in the other three groups(P<0.01),but there was no statistical significance in the pair comparison of other groups(P>0.05). The MDA content in group C was the highest, significantly higher than that in the other three groups. The MDA contents in group D and B were higher than that in group A(P<0.01),but there was no statistical significance in other groups. The Fe content in group B,C and D was significantly higher than that in group A(P<0.01),but there was no statistical significance in other groups(P>0.05). Conclusion Human-derived AM nude mice model has high reproducibility. JWSY can reduce the volume of tumor in AM model mice. JWSY may play a role by regulating ferroptosis-related mechanism of cells.