基于“正虚伏毒”理论探讨金复康有效组分调控NK细胞功能抑制Lewis肺癌细胞的转移  

Study on the effective components of Jinfukang in regulating the function of NK cells and inhibiting the metastasis of lung cancer Lewis cells based on"Deficiency of Vital Qi and Hidden Toxin"theory

作　　者：于盼 田建辉 陆鑫熠 罗斌 阙祖俊 YU Pan;TIAN Jianhui;LU Xinyi;LUO Bin;QUE Zujun(Cancer Clinical Medical Center,Shanghai Municipal Hospital of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200071,China;Instituteof Oncology,Shanghai Municipal Hospital of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200071,China)

机构地区：[1]上海市中医医院暨上海中医药大学附属市中医医院肿瘤临床医学中心,上海200071 [2]上海市中医医院暨上海中医药大学附属市中医医院肿瘤研究所,上海200071

出　　处：《中国肿瘤生物治疗杂志》2023年第11期957-964,共8页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金项目(No.81973517,No.82174245,No.8214943,No.82174017);上海市卫生健康委领军人才基金(No.2022LJ014);上海市中医医院未来计划科技发展基金项目(No.WL-HBRC-2021001K);上海市进一步加快中医传承创新发展三年行动计划[No.ZY(2021-2023)-0211]。

摘　　要：目的:基于“正虚伏毒”理论,探讨金复康中有效的“扶正”和“蠲毒”组分黄芪甲苷Ⅳ联合重楼皂苷Ⅶ抑制Lewis肺癌细胞转移的免疫学机制。方法:C57BL/6J小鼠尾静脉注射Lewis细胞建立肺癌转移动物模型,将小鼠按随机表法随机分为治疗组和对照组(每组8只),治疗组采用金复康有效组分黄芪甲苷Ⅳ(25 mg/kg)联合重楼皂苷Ⅶ(2.5 mg/kg)进行干预,4周后观察小鼠肺和肝转移情况,H-E染色观察肺和肝转移灶病理组织变化,流式细胞术检测外周血和脾组织中NK细胞的比例,ELISA法检测血清中TNF-α、IFN-γ水平,免疫组化法检测转移灶组织中Ki-67蛋白的表达水平,免疫荧光法检测转移灶组织中NK细胞的浸润和IFN-γ分泌水平。免疫磁珠法分选健康小鼠脾内的NK细胞,然后与黄芪甲苷Ⅳ(5μmol/L)、重楼皂苷Ⅶ(0.5μmol/L)及两药联合干预后的Lewis细胞共培养,流式细胞术检测共培养后NK细胞的脱颗粒水平,LDH法检测NK细胞对Lewis细胞的杀伤活性。结果:与对照组相比,经黄芪甲苷Ⅳ联合重楼皂苷Ⅶ干预后的小鼠肺转移和肝转移灶的数目明显减少(均P<0.05),且转移负荷也显著降低(均P<0.05);转移灶中Ki-67蛋白的表达水平显著降低(P<0.05),转移灶组织中NK细胞的浸润水平显著增加,且IFN-γ从NK细胞的胞内释放至胞外。此外,模型小鼠脾组织中的NK细胞比例显著降低(P<0.05)而外周血中的NK细胞水平却显著升高(P<0.05),小鼠血清中IFN-γ的水平显著减少而TNF-α的水平却显著升高(均P<0.05)。体外研究发现,黄芪甲苷Ⅳ和重楼皂苷Ⅶ单药或联合干预后,NK细胞的脱颗粒水平显著增加(P<0.05),但并未提高NK细胞对Lewis细胞的杀伤活性(P>0.05)。结论:金复康中的有效组分黄芪甲苷Ⅳ联合重楼皂苷Ⅶ可通过促进NK细胞浸润到肿瘤组织内和增强IFN-γ分泌以抑制Lewis肺癌细胞的肺转移和肝转移,研究结果为“正虚伏毒”理论提供了科学依Objective:To explore the immunological mechanism of the effective components in Jinfukang,astragalosideⅣand paris saponinsⅦ,in inhibiting lung cancer Lewis cells metastasis based on the theory of"Deficiency of Vital Qi and Hidden Toxin".Methods:The animal model of lung cancer metastasis was established by injecting Lewis cells into the tail vein of C57BL/6J mice.The model mice were randomly divided into a treatment group and a control group according to the random table method,8 mice per group.The treatment group was intervened with Jinfukang effective components,astragalosideⅣ(25 mg/kg)and with paris saponinsⅦ(2.5 mg/kg).Four weeks later,the lung and liver metastases were observed.The histopathological changes of lung and liver metastatic foci were observed by H-E staining.The proportions of NK cells in peripheral blood and spleen tissues were detected by flow cytometry.The levels of TNF-αand IFN-γin serum were detected by ELISA.The protein expression of Ki-67 in the metastatic foci was detected by immunohistochemical method,and the infiltration of NK cells and the secretion of IFN-γin the metastatic foci were detected by immunofluorescence.The NK cells in the spleen of healthy mice were separated by immunomagnetic beads and co-cultured with astragalosideⅣ(5μmol/L),paris saponinsⅦ(0.5μmol/L),and Lewis cells that intervened by both drugs.The degranulation level of NK cells was detected by flow cytometry,and the killing activity of NK cells against Lewis cells was detected by LDH method.Results:Compared with the control group,the number and load of lung and liver metastases were significantly decreased(both P<0.05),The transfer load is also significantly reduced(both P<0.05),and the expression of Ki-67 protein in the metastatic foci was significantly reduced(P<0.05)in the mice treated with astragalosideⅣand paris saponinsⅦ;the infiltration level of NK cells in metastatic tumor tissue was significantly increased,and IFN-γwas released from intracellular to extracellular of NK cells.In addition,

关 键 词：肺癌 Lewis细胞 金复康 正虚伏毒 转移 NK细胞 重楼皂苷Ⅶ 黄芪甲苷Ⅳ 

分 类 号：R734.2[医药卫生—肿瘤] R228[医药卫生—临床医学] R730.52