仙连解毒方维持肿瘤相关血管内皮细胞稳态抑制肿瘤血管新生的作用机制

Mechanism of Xianlian Jiedu Prescription in Maintaining Cancer Relative Vascular Endothelial Cell Homeostasis and Inhibiting Tumor Neovascularization

目的:观察仙连解毒方对肿瘤相关内皮细胞(CRE)增殖、凋亡、迁移的影响,并探讨仙连解毒方调控血管生成素2(Ang2)维持肿瘤相关内皮细胞稳态抑制肿瘤新生血管生成的作用机制。方法:人结直肠癌细胞HCT-116条件培养基诱导人脐静脉内皮细胞系(HUVEC-c)为肿瘤相关内皮细胞。设置空白组、条件培养基组、仙连解毒方组(1、2、3 g·L^(-1)),分别作用于肿瘤相关内皮细胞48 h。采用噻唑蓝(MTT)比色法检测CRE细胞增殖能力;倒置显微镜观察CRE细胞形态变化;流式细胞术检测各组细胞凋亡率;细胞伤口愈合实验和Transwell迁移实验检测CRE细胞2D/3D迁移能力;蛋白免疫印迹法(Western blot)检测仙连解毒方对CRE细胞波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)、基质金属蛋白酶-9(MMP-9)、Ang2等蛋白的表达。结果:MTT比色法结果显示,与空白组比较,条件培养基组细胞活力明显增强(P<0.05);与条件培养基组比较,仙连解毒方组细胞增殖率显著降低(P<0.01);细胞形态发生明显改变。流式细胞术结果显示,与条件培养基组比较,仙连解毒方组细胞总凋亡率均显著上升(P<0.01)。细胞伤口愈合实验和Transwell迁移实验结果显示,与空白组比较,条件培养基组2D、3D迁移能力增强(P<0.05,P<0.01);与条件培养基组比较,仙连解毒方组2D迁移能力显著减弱(P<0.01),仙连解毒方组(2、3 g·L^(-1))3D迁移能力显著减弱(P<0.01)。Western blot结果显示,与空白组比较,条件培养基组N-cadherin、Vimentin、MMP-9、Ang2蛋白表达水平明显上升(P<0.05,P<0.01);与条件培养基组比较,仙连解毒方组Ang2蛋白表达水平明显下降(P<0.05,P<0.01),仙连解毒方组(2、3 g·L^(-1))N-cadherin、Vimentin、MMP-9蛋白表达水平显著下降(P<0.01)。结论:仙连解毒方可能是通过降低Ang2在肿瘤相关血管内皮细胞中的表达,抑制肿瘤血管新生,维持血管内皮细胞稳态,从而抑制CRE细胞的增殖、迁移、分化,并诱导其凋亡。

Objective:To observe the effect of Xianlian Jiedu prescription (XLJDP) on the proliferation,apoptosis,and migration of cancer-relative endothelial (CRE) cells,and to decipher the mechanism of XLJDP in regulating angiopoietin2(Ang2)to maintain CRE cell homeostasis and inhibit tumor neovascularization.Method:Human umbilical vein endothelial cell line(HUVEC-c)was induced into CREcells in the human colorectal cancer HCT-116 cell-conditioned medium.The CRE cells were assigned into the blank group,conditioned medium group,and XLJDP groups(1,2,3 g·L^(-1))and treated for 48 h.The proliferation of CRE cells was detected by methyl thiazolyl tetrazolium(MTT)colorimetry.The morphological changes of CRE cells were observed via an inverted microscope.The apoptosis rate was detected by flow cytometry.Wound healing test and Transwell migration assay were employed to detect the 2D/3D migration ability of CRE cells.The protein levels of vimentin,N-cadherin,matrix metalloproteinase-9(MMP-9),and Ang2 in CRE cells were measured by Western blot.Result:The MTT results showed that the cell viability was higher in the conditioned medium group than in the blank group(P<0.05).Compared with the conditioned medium group,XLJDP decreased the cell proliferation rate(P<0.01)and changed the cell morphology.The total apoptosis rates of all the XLJDP groups were higher than that of the conditioned medium group(P<0.01).The2D and 3D migration abilities of the conditioned medium group were higher than those of the blank group(P<0.05,P<0.01).Compared with the conditioned medium group,XLJDP at all the concentrations weakened the2D migration ability(P<0.01)and medium-and high-concentration XLJDP weakened the 3D migration ability(P<0.01).The protein levels of N-cadherin,Vimentin,MMP-9,and Ang2 were up-regulated in the conditioned medium group compared with those in the blank group(P<0.05,P<0.01).Compared with the conditioned medium group,XLJDP at all the concentrations down-regulated the protein level of Ang2(P<0.05,P<0.01),and medium-and high-concentration XLJDP down-regulated those of N-cadherin,vimentin,and MMP-9protein(P<0.01).Conclusion:XLJDP may inhibit the proliferation,migration,differentiation,and apoptosis of CRE cells by down-regulating the expression of Ang2,inhibiting tumor neovascularization,and maintaining the cell homeostasis.