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

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-α水平升高(P<0.05);理冲汤高剂量组TNF-α、IL-2水平升高(P<0.01),IL-6水平降低(P<0.05)。与模型组比较,理冲汤各剂量组肿瘤组织中GSK-3β、CD4蛋白的表达升高(P<0.05,P<0.01),Wnt-1、MMP-9蛋白表达降低(P<0.05,P<0.01);理冲汤中、高剂量组CD8蛋白表达显著升高(P<0.01),β-catenin蛋白表达降低(P<0.05,P<0.01)。与模型组比较,各治疗组GSK-3βmRNA表达水平升高(P<0.05,P<0.01),Wnt-1、β-catenin、Foxp3 mRNA表达水平显著降低(P<0.01)。结论理冲汤具有抑制卵巢癌荷瘤小鼠肿瘤生长的作用,该作用可能与降低CD4^(+)CD25^(+)Foxp3^(+)Treg细胞亚群比例,提高机体免疫,调节Wnt/β-catenin信号通路有关。

Objective 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 the model group were significant,and there were variable degrees of improvement in all treatment groups after intervention.Compared with the model group,the proportion of CD4^(+)CD25^(+)Foxp3^(+)Treg subpopulations in the spleen and the serum level of IL-10 were significantly reduced in each treatment group(P<0.01),the TNF-αlevel in the paclitaxel and medium-dose LCD groups increased(P<0.05),and the serum levels of TNF-αand IL-2 in high-dose LCD group increased(P<0.01),while the IL-6 level decreased(P<0.05).Compared with the model group,the protein expressions of GSK-3βand CD4 in tumor tissue of mice in each dose of LCD group increased(P<0.05,P<0.01),the protein expressions of Wnt-1 and MMP-9 decreased(P<0.05,P<0.01),and the CD8 protein expression in the medium-and high-dose LCD groups significantly increased(P<0.05,P<0.01),whileβ-catenin protein expression reduced(P<0.05,P<0.01).Compared with the model group,the GSK-3βmRNA expression level in each treatment group increased(P<0.05,P<0.01),while the mRNA expression levels of Wnt-1,β-catenin,and Foxp3 decreased(P<0.01).Conclusion Lichong Decoction can inhibit tumor growth in ovarian cancer-bearing mice,which may be related to reducing the proportion of CD4^(+)CD25^(+)Foxp3^(+)Treg cell subpopulations,improving immunity,and regulating Wnt/β-catenin signaling pathway.