白屈菜碱对小鼠肺癌细胞皮下移植瘤生长和血管生成的抑制作用及其机制

Inhibitory effect of leucovorin on growth and angiogenesis of subcutaneous transplanted tumors in mouse lung cancer cells and its mechanism

目的:探讨白屈菜碱对肺癌移植瘤小鼠肿瘤生长和血管生成的影响,并阐明其作用机制。方法:选取32只健康C57BL/6小鼠,制备Lewis肺癌移植瘤小鼠模型。将小鼠随机分为模型组(0.9%氯化钠)、低剂量白屈菜碱组(25 mg·kg^(-1)白屈菜碱)、高剂量白屈菜碱组(50 mg·kg^(-1)白屈菜碱)和阳性对照组(60 mg·kg^(-1)环磷酰胺),每组8只。称量各组小鼠移植瘤质量并计算抑瘤率,计算各组小鼠胸腺指数和脾脏指数,采用酶联免疫吸附测定(ELISA)法检测各组小鼠血清中白细胞介素2(IL-2)、γ干扰素(INF-γ)和肿瘤坏死因子α(TNF-α)水平,免疫组织化学法检测各组小鼠肿瘤组织中血管内皮生长因子(VEGF)蛋白表达情况并计算微血管密度(MVD)及VEGF蛋白表达评分,Westernblotting法检测各组小鼠肿瘤组织中核因子κB(NF-κB)和缺氧诱导因子1α(HIF-1α)蛋白表达水平。结果:与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠移植瘤质量均降低(P<0.05);与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠移植瘤质量均降低(P<0.05),抑瘤率均升高(P<0.05);与高剂量白屈菜碱组比较,阳性对照组小鼠移植瘤质量均降低(P<0.05),抑瘤率升高(P<0.05)。与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠脾脏指数和胸腺指数均升高(P<0.05);与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠脾脏指数及胸腺指数均升高(P<0.05);与高剂量白屈菜碱组比较,阳性对照组小鼠脾脏指数和胸腺指数均升高(P<0.05)。ELISA法,与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠血清中IL-2、INF-γ和TNF-α水平均升高(P<0.05);与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠血清中IL-2、INF-γ和TNF-α水平均升高(P<0.05);与高剂量白屈菜碱组比较,阳性对照组小鼠血清中IL-2、INF-γ和TNF-α水平均升高(P<0.05)。与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠肿瘤组织MVD降低(P<0.05);与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠肿瘤组织MVD降低(P<0.05);与高剂量白屈菜碱组比较,阳性对照组小鼠肿瘤组织中MVD降低(P<0.05)。与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠肿瘤组织中VEGF蛋白表达量减少;与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠肿瘤组织中VEGF蛋白表达量减少;与高剂量白屈菜碱组比较,阳性对照组小鼠肿瘤组织中VEGF蛋白表达量减少;模型组、低剂量白屈菜碱组、高剂量白屈菜碱组和阳性对照组小鼠肿瘤组织VEGF蛋白表达评分比较差异有统计学意义(P<0.05)。Western blotting法,与模型组比较,低和高剂量白屈菜碱组及阳性对照组小鼠肿瘤组织中NF-κB和HIF-1α蛋白表达水平降低(P<0.05);与低剂量白屈菜碱组比较,高剂量白屈菜碱组和阳性对照组小鼠肿瘤组织中NF-κB和HIF-1α蛋白表达水平降低(P<0.05);与高剂量白屈菜碱组比较,阳性对照组小鼠肿瘤组织中NF-κB和HIF-1α蛋白表达水平降低(P<0.05)。结论:白屈菜碱能够抑制Lewis肺癌移植瘤小鼠肿瘤组织生长、保护免疫器官和抑制肿瘤血管生成,可能通过靶向NF-κB/HIF-1α信号通路和下调NF-κB及HIF-1α蛋白表达水平发挥治疗作用。

Objective:To discuss the effects of leucovorin on tumor growth and angiogenesis in the mice with lung cancer transplantation tumor,and to elucidate its mechanism.Methods:Thirty-two healthy C57BL/6 mice were selected to prepare the Lewis lung cancer transplantation tumor models.The mice were randomly divided into model group(0.9% NaCl),low dose of leucovrin group(25 mg·kg~(-1)leucovrin),high dose of leucovrin group(50 mg·kg~(-1) leucovrin) and positive control group(60 mg·kg~(-1)cyclophosphamide),and there were 8 mice in each group.The levels of interleukin-2(IL-2),interferon-γ(INF-γ) and tumor necrosis factor-α(TNF-α) in serum of the mice in various groups were detected by enzyme linked immunosorbent assay(ELISA) method;the expression of vascular endothelial growth factor(VEGF) in tumor tissue of the mice in various groups were detected by immunohistochemistry,and the microvascular density(MVD) and VEGF protein expression scores were calculated;the expression levels of nuclear factor-κB(NF-κB) and hypoxia inducible factor-1α(HIF-1α) in tumor tissue of the mice in various groups were detected by Western blotting method.Results:Compared with model group,the transplantation tumor masses of the mice in low and high doses of leucovorin groups and positive control group were decreased(P<0.05);compared with low dose of leucovorin group,the transplantation tumor masses of the mice in high dose of leucovorin group and positive control group were decreased(P<0.05),and the tumor inhibitory rates were increased(P<0.05);compared with high dose of leucovorin group,the transplantation tumor mass of the mice in positive control group was decreased(P<0.05),and the tumor inhibitory rate was increased(P<0.05).Compared with model group,the spleen indexes and thymus indexes of the mice in low and high doses of leucovorin groups and positive control group were increased(P<0.05);compared with low dose of leucovorin group,the spleen indexes and thymus indexes of the mice in high dose of leucovorin group and positive control group were increased(P<0.05);compared with high dose of leucovorin group,the spleen index and thymus index of the mice in positive control group were increased(P<0.05).The ELISA method results showed that compared with model group,the levels of IL-2,INF-γ and TNF-α in serum of the mice in low and high doses of leucovorin groups and positive control group were increased(P<0.05);compared with low dose of leucovorin group,the levels of IL-2,INF-γ and TNF-α in serum of the mice in high dose of leucovorin group and positive control group were increased(P<0.05);compared with high dose of leucovorin group,the levels of IL-2,INF-γ and TNF-α in serum of the mice in positive control group were increased(P<0.05).Compared with model group,the MVD in tumor tissue of the mice in low and high dose of leucovorin groups and positive control group was decreased(P<0.05);compared with low dose of leucovorin group,the MVD in tumor tissue of the mice in high dose of leucovorin group and positive control group was decreased(P<0.05);compared with high dose of leucovorin group,the MVD in tumor tissue of the mice in positive control group was decreased(P<0.05).Compared with model group,the expression amounts of VEGF protein in tumor tissue of the mice in low and high dose of leucovorin groups and positive control group were decreased;compared with low dose of leucovorin group,the expression amounts of VEGF protein in tumor tissue of the mice in high dose of leucovorin group and positive control group were decreased;compared with high dose of leucovorin group,the expression amount of VEGF protein in tumor tissue of the mice in positive control group was decreased.The differences in VEGF protein expression scores between model group,low dose of leucovorin group,high dose of leucovorin group and positive control group were statistically significant(P<0.05).Compared with model group,the expression levels of NF-κB and HIF-1α in tumor tissue of the mice in low and high doses of leucovorin groups and positive control group were decreased(P<0.05);compared with low dose of leucovorin group,the expression levels of NF-κB and HIF-1α in tumor tissue of the mice in high dose of leucovorin group and positive control group were decreased(P<0.05);compared with high dose of leucovorin group,the expression levels of NF-κB and HIF-1 α in tumor tissue of the mice in positive control group were decreased(P<0.05).Conclusion:Leucolorin can inhibit the tumor growth,protect the immune organs and suppress the tumor angiogenesis in the Lewis lung cancer transplantation tumor mice,and may exert the therapeutic effect by targeting NF-κB/HIF-1α signaling pathway and down-regulating the expression levels of NF-κB and HIF-1α proteins.