理冲汤对卵巢癌荷瘤小鼠的免疫调节及抗肿瘤作用  

Immunomodulatory and anti-tumor effects of Lichong Decoction on ovarian cancer-bearing mice

作　　者：周锦宏 吴小兰 张嘉恒 李玥衡 杜玉 刘奇英 刘慧萍[1,4] ZHOU Jinhong;WU Xiaolan;ZHANG Jiaheng;LI Yueheng;DU Yu;LIU Qiying;LIU Huiping(School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China;The Second Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410005,China;Changsha Hospital for Maternal&Child Health Care,Changsha,Hunan 410007,China;School of Medicine,Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南长沙410208 [2]湖南中医药大学第二附属医院,湖南长沙410005 [3]长沙市妇幼保健院,湖南长沙410007 [4]湖南中医药大学医学院,湖南长沙410208

出　　处：《湖南中医药大学学报》2024年第5期754-763,共10页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金项目(2022JJ30035);湖南省中医药科研计划项目(D2022039);湖南省卫生健康委员会科研项目重点指导项目(202305017379);长沙市自然科学基金项目(kq2202038);湖南省高校中医肿瘤重点实验室;湖南省研究生创新基金项目(CX20220784);湖南中医药大学研究生创新项目(2023CX28,2023CX107);国家级大学生创新创业计划训练项目(S202310541063);湖南省大学生创新创业计划训练项目(S202310541063)。

摘　　要：目的探讨理冲汤对卵巢癌荷瘤小鼠的免疫调节及抗肿瘤作用。方法按随机数字表法将48只C57BL/6雌性小鼠随机分为空白组8只和造模组40只。采用腋下注射ID8细胞方法制备卵巢癌荷瘤小鼠模型。造模成功的40只小鼠随机分为模型组、紫杉醇组[10 mg/(kg·3 d)]、理冲汤低剂量组[17.89 g/(kg·d)]、理冲汤中剂量组[35.78 g/(kg·d)]和理冲汤高剂量组[71.56 g/(kg·d)],每组8只。空白组及模型组灌胃蒸馏水[10 mL/(kg·d)]。每组干预21 d。计算抑瘤率及脏器指数,HE染色观察肿瘤组织病理形态变化;流式细胞术检测脾脏中CD4^(+)CD25^(+)Foxp3^(+)调节性T细胞(regulatory T cell,Treg)细胞亚群比例;流式微球阵列技术检测血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-2(interleukin-2,IL-2)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)水平;免疫组织化学法检测肿瘤组织中Wnt家族成员-1(Wnt family member-1,Wnt-1)、β-连环蛋白(β-catenin)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、白细胞分化抗原4(cluster of differentiation 4,CD4)、白细胞分化抗原8(cluster of differentiation 8,CD8)蛋白表达水平;RT-PCR法检测肿瘤组织Wnt-1、β-catenin、GSK-3β、叉头蛋白P3(forkheadbox protein 3,Foxp3)mRNA表达水平。结果与空白组比较,模型组脾脏指数及血清中TNF-α、IL-2水平显著降低(P<0.01),血清中IL-6、IL-10水平及脾脏中CD4^(+)CD25^(+)Foxp3^(+)Treg细胞亚群比例显著升高(P<0.01)。与模型组比较,理冲汤各剂量组脾脏指数升高(P<0.05,P<0.01),各治疗组肿瘤指数显著降低(P<0.01)。模型组小鼠肿瘤组织病理形态学病变显著,各治疗组干预后均有一定程度改善。与模型组比较,各治疗组脾脏中CD4^(+)CD25^(+)Foxp3^(+)Treg细胞亚群比例及血清IL-10水平显著降低(P<0.01);紫杉醇组和理冲汤中剂量组TNF-αObjective To explore the immunomodulatory and anti-tumor effects of Lichong Decoction(LCD)on ovarian cancer-bearing mice.Methods Forty-eight C57BL/6 female mice were randomized into blank group(n=8)and modeling group(n=40)by random number table method,and the ovarian cancer-bearing mouse model was prepared by subaxillary injection of ID8 cells.Forty mice with successful modeling were randomly subdivided into model group,paclitaxel group[10 mg/(kg·3d)],low-,medium-,and high-dose[17.89 g/(kg·d),35.78 g/(kg·d),71.56 g/(kg·d)]LCD groups,with eight mice in each group.The blank group and model group were given distilled water[10 mL/(kg·d)],and each group was intervened for 21 d.The tumor inhibition rate and organ indexes were calculated;HE staining was used to observe histopathological and morphological changes in tumor tissue;the proportion of CD4^(+)CD25^(+)Foxp3^(+)regulatory T cell(Treg)subpopulations in spleen was checked by flow cytometry,Cytometric Bead Array(CBA)was used to determine the serum levels of tumor necrosis factor-α(TNF-α),interleukin-2(IL-2),interleukin-6(IL-6),and interleukin-10(IL-10);immunohistochemistry was performed to examine the protein expression levels of Wnt family member 1(Wnt1),β-catenin,glycogen synthase kinase-3β(GSK-3β),matrix metalloproteinase-9(MMP-9),cluster of differentiation 4(CD4),and cluster of differentiation 8(CD8);the mRNA expression levels of Wnt-1,β-catenin,GSK-3β,and forkheadbox protein 3(Foxp3)were determined by RT-PCR.Results Compared with the blank group,both the spleen index and the serum levels of TNF-αand IL-2 in the model group were significantly lower(P<0.01),while the proportion of CD4^(+)CD25^(+)Foxp3^(+)Treg cell subpopulations in the spleen and the serum levels of IL-6 and IL-10 were significantly higher(P<0.01).Compared with the model group,the spleen index in each dose of LCD group increased(P<0.05,P<0.01),the tumor index in each treatment group significantly reduced(P<0.01),and the histopathological and morphological lesions in tumor tissue of

关 键 词：理冲汤 卵巢癌 WNT/Β-CATENIN通路 肿瘤微环境 免疫逃逸 调节性T细胞 

分 类 号：R271[医药卫生—中医妇科学]