益肾祛痛颗粒灌服对小鼠乳腺癌骨转移灶的改善作用及其机制

Inhibitory effect and mechanism of Yishen Qutong granules on bone metastasis of breast cancer in mice

目的观察益肾祛痛颗粒灌服对小鼠乳腺癌骨转移灶的改善作用,并探讨其可能的机制。方法将84只雌性BABL/c小鼠随机分为7组,每组12只。其中模型组、ML-385组及低、中、高剂量组均采用左侧胫骨种植小鼠亲骨性乳腺癌高转移细胞系4T1.2细胞悬液的方法建立乳腺癌骨转移模型,种植后第1天低、中、高剂量组分别给予益肾祛痛颗粒水溶液2.275、4.55、9.1 g/kg灌服并腹腔注射生理盐水,ML-385组给予蒸馏水灌服并腹腔注射溶于生理盐水的细胞核因子E2相关因子2(Nrf2)抑制剂ML-38520 mg/kg,空白组、假手术组、模型组给予等体积蒸馏水灌服并腹腔注射生理盐水,1次/天,连续给药28 d。记录各组小鼠分组处理1~4周的体质量,连续给药28 d后处死,称取肿瘤质量。取各组小鼠左侧胫骨,micro-CT扫描后分析骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数目(Tb.N)及骨小梁间隙(Tb.Sp),HE染色后观察组织病理情况,酒石酸酸性磷酸酶染色后分析破骨细胞数量及破骨细胞与骨组织接触面长度,免疫组化法检测Kelch样环氧氯丙烷相关蛋白1(Keap1)、Nrf2蛋白阳性表达率。结果分组处理1~4周,各组小鼠体质量呈持续上升趋势;分组处理第4周,模型组体质量低于其余各组(P均<0.05)。与模型组比较,各组肿瘤质量均降低,但仅ML-385组和中剂量组P均<0.05。空白组和假手术组左侧胫骨组织病理学观察均正常,模型组左侧胫骨有明显骨转移瘤形成、细胞成分以成骨性骨转移为主,ML-385组、中剂量组左侧胫骨骨转移瘤形成及骨质破坏程度均较模型组减轻,低、高剂量组与模型组相近。与空白组及假手术组比较,模型组左侧胫骨组织BV/TV、Tb.N、Tb.Sp、Tb.Th、破骨细胞数量、破骨细胞接触面长度及Keap1、Nrf2阳性表达率均升高(P均<0.05);与模型组比较,ML-385组左侧胫骨组织BV/TV、Tb.N、Tb.Sp、Tb.Th及Keap1、Nrf2阳性表达率均降低,中剂量组左侧胫骨组织Tb.N、Tb.Sp、Tb.Th均降低,低、高剂量组Tb.Th均降低,低、中、高剂量组小鼠左侧胫骨组织破骨细胞数量、破骨细胞接触面长度、Nrf2阳性表达率均降低而Keap1阳性表达率升高(P均<0.05)。结论益肾祛痛颗粒灌服可抑制小鼠乳腺癌骨转移瘤生长及骨质破坏,以中等剂量效果最佳,其机制可能与调控Keap1/Nrf2信号通路而减轻氧化应激反应有关。

Objective To observe the inhibitory effect of intragastric administration of Yishen Qutong granules(YSQTG)on breast cancer bone metastasis in mice,and to explore its possible mechanism of action.Methods Eightyfour female BABL/c mice were randomly divided into seven groups,with 12 mice in each group.Mice in the model group,ML-385 group,and low,medium,and high dose groups were all implanted with the bone-affine breast cancer high meta⁃static cell line 4T1.2 cell suspension in the left tibia to create the breast cancer bone metastasis models.On the first day af⁃ter implantation,mice in the low,medium,and high dose groups were given YSQTG aqueous solution at 2.275,4.55,and 9.1 g/kg,respectively,by gavage,followed by intraperitoneal injection of normal saline.Mice in the ML-385 group were given distilled water by gavage and intraperitoneal injection of nuclear factor E2-related factor 2(Nrf2)inhibitor ML-385(20 mg/kg)dissolved in normal saline.Mice in the blank group,sham operation group,and model group were given equal volume of distilled water by gavage and intraperitoneal injection of normal saline.The dose was administered once a day,for 28 consecutive days.The body weight of mice in each group was recorded from 1 to 4 weeks after treatment,and after 28 days of continuous administration,the mice were sacrificed and the tumor mass was weighed.The left tibia of mice in each group was harvested,and after micro-CT scanning,the bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),and trabecular space(Tb.Sp)of cancellous and cortical bone were analyzed.Histopath⁃ological conditions were observed after HE staining,and the number of osteoclasts and the length of contact surface between osteoclasts and bone tissues were analyzed after tartrate acid phosphatase staining.Immunohistochemical method was used to detect the positive expression rates of Kelch-like ECH-associated protein 1(Keap1)and Nrf2 proteins.Results During the 1 to 4 weeks of treatment,the body weight of mice in each group showed a continuous upward trend.At the 4th week of treatment,the body weight of the model group was lower than that of the other groups(all P<0.05).Compared with the model group,the tumor mass decreased,but only significant difference was found between the ML-385 group and the medi⁃um dose group(P<0.05).The histopathological examination of the left tibia in the blank group and the sham operation group was normal.In the model group,significant bone metastasis was observed on the left tibia,with the main cellular components of osteogenic bone metastasis.In the ML-385 group and the medium dose group,the formation of bone metas⁃tasis and the degree of bone destruction on the left tibia were less severe in comparison with those of the model group,while the low and high dose groups were similar to the model group.Compared with the blank group and the sham operation group,the BV/TV,Tb.N,Tb.Sp,Tb.Th,osteoclast numbers,osteoclast contact surface length,and the positive ex⁃pression rates of Keap1 and Nrf2 in the left tibia tissues of the model group increased(all P<0.05);compared with the model group,the BV/TV,Tb.N,Tb.Sp,Tb.Th,and the positive expression rates of Keap1 and Nrf2 in the left tibia tis⁃sues of the ML-385 group decreased,the Tb.N,Tb.Sp,and Tb.Th in the left tibia tissue of the medium dose group de⁃creased,the Tb.Th decreased in the low and high dose groups,and the numbers of osteoclasts,the length of osteoclast contact surfaces,and the positive expression rate of Nrf2 in the left tibia tissues decreased,while the positive expression rate of Keap1 increased in the low,medium,and high dose groups(all P<0.05).Conclusions Intragastric administra⁃tion of YSQTG can inhibit the growth of bone metastatic tumors and bone destruction in mice with breast cancer,with the medium dose showing the best effect.The mechanism may be related to the regulation of the Keap1/Nrf2 signaling pathway and the reduction of oxidative stress.