金复康口服液含药血清调控肺癌淋巴管新生机制探讨

目的中药制剂金复康口服液治疗肺癌的临床疗效已得到肯定,但机制尚不清楚。本研究探讨金复康口服液含药血清抑制肺癌淋巴管新生方向,探究其抑制肿瘤转移的分子学机制。方法将20只SD大鼠随机分为金复康口服液高剂量组〔15g/(kg·d)〕、中剂量组〔10g/(kg·d)〕、低剂量组〔5g/(kg·d)〕和Control组(生理盐水),每组5只。IL-4+为使用白细胞介素4(interleukin 4,IL-4)刺激的RAW264.7细胞,IL-4-为无任何刺激干预的RAW264.7细胞。流式细胞术分析其细胞表面CD206表达来检测RAW264.7细胞极化成M2型的情况。根据含药血清将共培养模型分为高剂量组、中剂量组、低剂量组、Control组和Blank组(不加含药血清),Elisa法检测共培养模型下室培养基中血管内皮生长因子-C/D(vascular endothelial growth factor C/D,VEGF-C/D)蛋白含量;蛋白质印迹法检测下室Lewis细胞中VEGF-C/D的含量;实时q-PCR检测下室Lewis细胞中VEGF-C/D基因表达量。采用Graphpad prism for Mac(Version 8.1.1)、image J、FlowJo(Version 10.4)对数据进行统计分析。结果 M2型RAW264.7表型鉴定,CD206表达率IL4+为(58.45±1.45)%和IL-4-为(1.98±0.18)%,t=102.25,P<0.001。Elisa检测VEGF-C含量,高剂量组(2 866.07±70.97)pg/mL相对于Control组(3 252.97±94.51)pg/mL明显下降(t=5.47 P<0.01),VEGF-D高剂量组(58.43±17.3)pg/mL相对于Control组(117.7±7.3)pg/mL明显下降,t=5.47,<0.01;蛋白质印迹法检测Lewis细胞中VEGF-C含量,高剂量组(0.11±0.015)%相对于Control组(0.34±0.028)%明显下降(t=12.54,P<0.001),VEGF-D高剂量组(0.38±0.037)%相对于Control组(0.071±0.022)%明显下降,t=12.43,P<0.001;q-PCR测定Lewis细胞高剂量组中VEGF-C基因表达量(0.61±0.095)%相对于Control组(3.89±0.44)%明显下降(t=12.59,P<0.001),VEGF-D高剂量组(0.23±0.025)%相对于Control组(1.78±0.09)%明显下降,t=28.55,P<0.001。成管实验中高剂量组共培养模型下室中的培养基干预的HLEC细胞在Matrigel Matrix胶中形成管腔结构的数量明显减少。结论金复康口服液含药血清能够通过调控Lewis细胞合成VEGF-C/D,减少肺癌淋巴管新生从而达到抑制肿瘤转移。

Mechanism of JFK Oral Solution Containing Serum regulates lymphangiogenesis in lung cancer

OBJECTIVE The research on anti-cancer of traditional Chinese medicine(TCM)develops rapidly,and the clinical efficacy of Jinfukang Oral Liquid,a TCM preparation,in lung cancer has been confirmed,but the mechanism is unclear.In this paper,the molecular mechanism of Jinfukang Oral Liquid-containing serum in inhibiting tumor metastasis will be explored from the inhibition of lymphangiogenesis in lung cancer.METHODS Twenty SD rats were randomly divided into high-dose Jinfukang Oral Liquid group15 g/(kg·d)],medium-dose group10 g/(kg·d)],low-dose group5 g/(kg·d)]and control group(normal saline),with 5 rats in each group.RAW264.7 cells stimulated with interleukin 4(IL-4)were classified into IL-4+group,and RAW264.7 cells without stimulation were classified into IL-4-group.CD206 expression on the surface of stimulated RAW264.7 cells was analyzed by flow cytometry to detect M2 polarization.The stimulated RAW264.7 cells and Lewis cells were inoculated into a 24-well Transwell chamber for co-culture,which was added with Jinfukang Oral Liquid-containing serum till a concentration of 10%.Corresponding to that,co-culture model was divided into high-dose group,middle-dose group,low-dose group,control group and blank group(without drug-containing serum).Vascular endothelial growth factor C/D(VEGF-C/D)protein content in co-culture medium of lower Transwell chamber was determined by using ELISA.VEGF-C/D content in total protein of Lewis cells in co-culture medium was detected by Western blot.VEGF-C/D gene expression in co-culture medium was detected using real-time fluorescence quantitative PCR.RESULTS The phenotype identification of M2-type RAW264.7 cells showed that the CD206+rate was(58.45±1.45)%in the IL-4+group and(1.98±0.18)%in the IL-4-group(t=102.25,P<0.001).In the co-culture model,ELISA revealed that VEGF-C protein expression(2 866.07±70.97)pg/mL]in the culture medium of lower Transwell chamber in the high-dose group was significantly lower than that in the control group(3 252.97±94.51)pg/mL](t=5.67,P<0.01).VEGF-D protein expression in the high-dose group(58.43±17.3)pg/mL]decreased significantly(t=5.47,P<0.01).Western blot demonstrated that VEGF-C protein expression in Lewis cells of the high-dose group(0.11±0.015)%]reduced significantly compared with the control group(0.34±0.028)%](t=12.54,P<0.001).Compared with the control group (0.071±0.022)%],VEGF-D protein expression in the high-dose group (0.38±0.037)%]decreased significantly(t=12.43,P<0.001).q-PCR presented that VEGF-C gene expression in Lewis cells of the high-dose group(0.61±0.095)%]was significantly lower than that in the control group(3.89±0.44)%](t=12.59,P<0.001).Compared with the control group(1.78±0.09)%],VEGF-D gene expression in the high-dose group(0.23±0.025)%]decreased significantly(t=28.55,P<0.001).Additionally,in the high-dose group,the number of tube-like structures of HLEC cells intervened with the co-culture medium of lower Transwell chamber in Matrigel matrix also reduced significantly.CONCLUSION Jinfukang Oral Liquid-containing serum can inhibit the metastasis of lung cancer by regulating VEGF-C/D synthesis in Lewis cells and reducing lymphangiogenesis.