中药药理与临床

目的:探讨通消阳和颗粒联合多柔比星对4T1乳腺癌荷瘤小鼠肿瘤的抑制作用及机制，并验证其缓解多柔比星治疗4T1乳腺癌荷瘤小鼠心脏毒性的机制，为临床乳腺癌的中西医结合治疗提供理论依据及实验基础。方法:建立4T1乳腺癌荷瘤小鼠模型。将造模成功的小鼠分为模型对照组、通消阳和颗粒6.82 g/kg组、多柔比星2.5 mg/kg组、多柔比星+通消阳和颗粒6.82、13.64 g/kg组，每组8只，另选8只作为正常对照组，连续给药28 d。记录一般状况、体质量、肿瘤体积、瘤质量、心脏质量。HE染色观察肿瘤、肺及心脏组织的病理改变；ELISA法检测炎性因子的含量；生化法检测心肌酶的活性；Western blot法检测肿瘤组织中NF-κB、IKKα、E-Cadherin、Vimentin及MMP-9蛋白的表达情况，并检测心肌组织中IKKα、INOS的蛋白表达。结果:与正常对照组比较，模型对照组血清中TNF-α、IL-6、IFN-γ含量明显增加(P<0.05),肿瘤组织多形态，显示出细胞异型性和局部纤维组织增生，肺组织肿瘤浸润和组织破坏，显示致密炎性；与模型对照组比较，通消阳和颗粒6.82 g/kg组、各联合组肿瘤质量显著降低，抑瘤率显著升高(P<0.01),血清中TNF-α、IL-6、IFN-γ的含量、LDH、AST、CK的活力明显降低，肿瘤组织Vimentin、MMP-9、NF-κB及IKKα的蛋白表达明显下调(P<0.05),E-Cadherin的蛋白表达显著上调(P<0.01),肿瘤组织结构异常，伴肿瘤细胞坏死，并伴胞核碎裂，固缩深染，肺组织肿瘤细胞浸润减少，肺转移减少；联合用药组上述指标降低更明显(P<0.05);多柔比星2.5 mg/kg组小鼠的心质量指数明显增加、心肌组织结构明显异常(P<0.01),血清TNF-α、IL-6、IFN-γ含量、LDH、AST、CK的活力明显升高，心肌组织中IKKα及INOS的蛋白表达显著上调(P<0.01),提示心肌损伤；与多柔比星2.5 mg/kg组相比，联合组心质量指数降低，肿瘤质量降低，抑瘤率升高，血清TNF-α、IL-6、IFN-γ、LDH、AST、CK含量或活力降低，肿瘤组织IKKα、NF-κB、Vimentin、MMP-9蛋白表达下调，E-Cadherin蛋白表达上调(P<0.05或P<0.01),心肌组织IKKα及INOS的蛋白表达下调(P<0.01)。结论:通消阳和颗粒能通过调节NF-κB、INOS、IKKα、Vimentin、MMP-9及E-Cadherin的表达与多柔比星发挥协同作用，抑制4T1小鼠乳腺癌增殖、肺转移及多柔比星诱导的心脏毒性。

Objective:To investigate the inhibitory effect and mechanism of Tongxiao Yanghe(通消阳和) Granules combined with doxorubicin on tumors in 4T1 breast cancer-bearing mice and validate the mechanism of Tongxiao Yanghe Granules in alleviating doxorubicin-induced cardiotoxicity in mice, thus providing theoretical and experimental bases for breast cancer treatment with integrated traditional Chinese and Western medicine. Methods:A 4T1 breast cancer-bearing mouse model was established. Successfully modeled mice were assigned into model control, Tongxiao Yanghe Granules(6.82 g/kg),doxorubicin(2.5 mg/kg),doxorubicin + Tongxiao Yanghe Granules(6.82 g/kg and 13.64 g/kg) groups(n=8). Additionally, 8 normal mice were selected as the normal control group, and the treatment lasted for 28 days. The general status, body weight, tumor volume, tumor weight, and heart weight were recorded. Hematoxylin-eosin staining was employed to observe pathological changes in the tumor, lung, and heart tissue. ELISA was employed to measure inflammatory factor levels. Biochemical assays were conducted to measure myocardial enzyme activities. Western blot was employed to assess the protein levels of nuclear factor(NF)-κB,inhibitor of kappa B kinase alpha(IKKα),E-Cadherin, Vimentin, and matrix metalloproteinase(MMP-9) in the tumor tissue and IKKα and inducible nitric oxide synthase(INOS) in the myocardial tissue. Results:Compared with the normal control group, the serum levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6,and interferon(IFN)-γ elevated in the model group(P<0.05),and tumor tissues are polymorphic, showed cellular atypia and local fibrous tissue hyperplasia, tumor cell infiltration and tissue destruction in lung tissue,showed dense inflammation. Compared with the model control group,the Tongxiao Yanghe Granules(6. 82 g/kg) group and all the combined treatment groups presented reduced tumor mass,increased tumor inhibition rate(P<0. 01),declined serum levels of TNF-α, IL-6,and IFN-γ,weakened activities of lactate dehydrogenase(LDH),aspartate aminotransferase(AST),and creatine kinase(CK),down-regulated protein levels of Vimentin,MMP-9,NF-κB,and IKKα(P<0. 05),up-regulated protein level of E-Cadherin(P<0. 01),abnormal tumor structures,tumor cell necrosis,karyorrhexis,karyopyknosis,and reduced tumor cell infiltration and metastasis in the lung tissue. The combined treatment groups demonstrated more significant changes in the above indexes(P< 0. 05),the doxorubicin(2. 5 mg/kg) group demonstrated increased cardiac mass index,and abnormal cardiac muscle structure(P<0. 01), elevated serum levels or activities of TNF-α,IL-6,IFN-γ,LDH,AST,and CK,up-regulated protein levels of IKKαand INOS in myocardial tissue(P< 0. 01),which showed myocardial injuryand. Compared with the doxorubicin(2. 5 mg/kg)group,the combined treatment groups demonstrated decreased cardiac mass index and tumor mass,increased tumor inhibition rate,declined serum levels or activities of TNF-α,IL-6,IFN-γ,LDH,AST,and CK,down-regulated protein levels of IKKα,NF-κB,Vimentin and MMP-9, up-regulated protein level of E-Cadherin in tumor tissue(P<0. 05 or P<0. 01),and down-regulated protein levels of IKKαand INOS in myocardial tissue(P<0. 01).Conclusion:Tongxiao Yanghe Granules combined with doxorubicin can effectively inhibit the proliferation and lung metastasis of 4T1 breast cancer and doxorubicin-induced cardiotoxicity by regulating the expression of NF-κB, INOS, IKKα, Vimentin, MMP-9, and E-Cadherin.