Nrf2抑制剂ML-385腹腔注射对小鼠乳腺癌骨转移的抑制作用及其与骨桥蛋白的关系

Inhibitory effect of intraperitoneal injection of Nrf2 inhibitor ML-385 on bone metastasis of mouse breast cancer and its relationship with osteopontin

目的 观察核因子红细胞相关因子2(Nrf2)抑制剂ML-385对乳腺癌小鼠骨转移的抑制作用,并探讨其机制是否与骨桥蛋白(OPN)有关。方法 将40只BABL/c小鼠随机分为空白组、假手术组、模型组、治疗组,每组10只,模型组、治疗组均采用胫骨种植小鼠亲骨性乳腺癌高转移细胞株4T1.2的方法建立乳腺癌骨转移模型,假手术组仅打孔,空白对照组正常饲养;治疗组建模后第1天腹腔注射ML385 20 mg/kg,空白组、假手术组、模型组腹腔注射等量生理盐水,1次/天,连续注射28 d。将各组小鼠处死,记录模型组和治疗组的成瘤情况,并称取胫骨瘤重;采用Micro-CT检查观察各组小鼠胫骨组织骨质破坏情况,比较其骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)以及骨小梁间隙(Tb.Sp);HE染色后观察胫骨组织病理变化,免疫组化法检测胫骨组织Nrf2、OPN阳性率。结果 模型组及治疗组小鼠胫骨成瘤率均为100%,胫骨瘤重分别为(0.921 0±0.358 2)、(0.575 1±0.228 3)g,两组比较P<0.05。空白组及假手术组骨质无明显改变,模型组存在明显骨质破坏,治疗组骨质破坏程度较模型组减轻。各组小鼠胫骨组织Tb.N比较差异无统计学意义(P均>0.05)。与假手术组和空白组比较,模型组胫骨组织BV/TV、Tb.Th、Tb.Sp及Nrf2、OPN阳性率均升高(P均<0.05)。与模型组比较,治疗组胫骨组织BV/TV、Tb.Th、Tb.Sp及Nrf2、OPN阳性率均降低(P均<0.05)。HE染色结果显示,空白组与假手术组小鼠胫骨组织未见明显异常;模型组小鼠胫骨骨髓腔内可见大量核大、深染、核仁明显的细胞,细胞质空,附近可见坏死区,伴出血,骨皮质见编织骨大量形成;治疗组小鼠胫骨组织的上述病理改变较模型组减轻。结论 Nrf2抑制剂ML-385可通过下调OPN表达而抑制乳腺癌小鼠的骨转移。

Objective To observe the inhibitory effect of nuclear factor erythroid2-related factor 2(Nrf2) inhibitor ML-385 on bone metastasis in mice with breast cancer and to investigate whether the mechanism is related to osteopontin(OPN).Methods Forty BABL/c mice were randomly divided into four groups:control group,sham operation group,model group,and treatment group,with 10 mice in each group.In the model group and the treatment group,we established the breast cancer bone metastasis models by using the hypermetastatic cell line 4 T1.2 of osteophilic breast cancer in tibial implant mice.The mice in the sham operation group were only perforated,and the mice in the control group were fed normally.ML385(20 mg/kg) was injected intraperitoneally on the 1 st day after modeling in the treatment group,and the equal volume of normal saline was injected intraperitoneally in the mice of the control group,sham operation group and model group,once/day,for 28 d.The mice in each group were sacrificed,the tumor formation in the model group and the treatment group was recorded,and the weight of the tibial osteoma was weighed.Micro-CT was used to observe the bone destruction of the tibial tissues of the mice in each group.Bone volume/tissue volume(BV/TV),trabecular bone thickness(Tb.Th),trabecular bone number(Tb.N) and trabecular bone separation(Tb.Sp) were compared.HE staining was used to observe the pathological changes of tibial tissues.The positive rates of Nrf2 and OPN were detected by immunohistochemistry.Results The tibial neoplasia rate in both model and treatment groups was 100%.The tibioma weight was(0.921 0±0.358 2) and(0.575 1±0.228 3) g,respectively,with statistically significant difference between the two groups(P0.05).Compared with the sham operation group and the blank group,the BV/TV,Tb.Th,Tb.Sp,and the positive rates of Nrf2 and OPN were higher in the model group(all P<0.05).Compared with the model group,BV/TV,Tb.Th,Tb.Sp,and the the positive rates of Nrf2 and OPN decreased in the treatment group(all P<0.05).The results of HE staining showed that there was no obvious abnormality in the tibia tissues of the mice in the blank group and the sham operation group;a large number of cells with large nuclei,deep staining and obvious nucleoli were visible in the bone marrow cavity,the cytoplasm was empty,and necrotic areas were seen nearby accompanied by hemorrhage,and the cortical bone had massive formation of woven bone in the model group;the above pathological changes in the tibia tissues of the mice in the treatment group were lighter than those in the model group.Conclusion Nrf2 inhibitor ML-385 inhibits bone metastasis in mice with breast cancer by down-regulating OPN expression.