肺瘤平膏联合环磷酰胺化疗对肺癌的抑瘤作用和酸性微环境的影响

Effect of Fei-Liu-Ping ointment combined with cyclophosphamide on lung cancer cell proliferation and acidic microenvironment

目的:观察肺瘤平膏和化疗药环磷酰胺对肺癌小鼠的作用,并从酸性微环境和细胞凋亡角度探索其内在作用机制。方法:建立Lewis肺癌小鼠模型,分别给予环磷酰胺、肺瘤平膏、环磷酰胺+肺瘤平膏相应处理,以生理盐水组作为对照,观察各组小鼠的肿瘤体积、肿瘤增殖率、食物消耗量。给药15 d后处死小鼠,取肿瘤组织,用乳酸试剂盒检测肿瘤组织的乳酸相对浓度,免疫组织化学法检测葡萄糖转运蛋白4(glucose transporter 4,GLUT4)、己糖激酶1(hexokinase 1,HK1)、葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、碳酸酐酶-Ⅸ(carbonic anhydrase-Ⅸ,CA-Ⅸ)的蛋白表达,流式细胞术检测肿瘤细胞凋亡率和调节性T细胞(regulatory T cells,Treg)比例,Wes-tern blot法检测缺氧诱导因子1-α(hypoxia-inducible factor-1α,HIF-1α)、Bcl-2、Bax、Caspase-3、白细胞介素2(interleukin-2,IL-2)的蛋白表达。结果:肺瘤平膏+环磷酰胺组小鼠的肿瘤生长受到抑制最为明显,肿瘤增殖率最低,与生理盐水组相比差异有统计学意义(P<0.05),食物的消耗量也最多,但差异并不具有统计学意义(P>0.05)。进一步的分子生物学检测发现,肺瘤平膏+环磷酰胺组肿瘤组织的乳酸水平在各组中最低,并伴有GLUT4、HK1、GRP78、CA-Ⅸ等蛋白表达和Treg细胞比例的下降,与生理盐水组相比差异具有统计学意义(P<0.05)。此外,与生理盐水组相比,肺瘤平膏+环磷酰胺组肿瘤细胞凋亡显著增加,而HIF-1α、Bcl-2、Bax、Caspase-3、IL-2等蛋白的表达与生理盐水组相比存在明显差异。结论:化疗药环磷酰胺与肺瘤平膏在抑制肺癌生长、提高荷瘤小鼠一般状况上具有协同作用,这些作用部分是因为二者联用改善组织缺氧,抑制HIF-1α表达,进而调控下游GLUT4、HK1、GRP78、CA-Ⅸ等蛋白的表达,降低肿瘤组织局部的乳酸水平,改善肿瘤组织酸性微环境,从而诱导肿瘤细胞凋亡,抑制T细胞向Treg细胞分化。

Objective:To observe the effects of Fei-Liu-Ping ointment and chemotherapy on mice with lung cancer,and to explore the inherent mechanism of action from the point of acidic microenvironment and apoptosis.Methods:First of all,the Lewis lung cancer transplanted mouse model was established.Therefore,they were treated by Fei-Liu-Ping ointment,cyclophosphamide,Fei-Liu-Ping ointment+cyclophosphamide and the saline as control.All the groups’tumor size,tumor growth rate and food consumption were recorded.The mice were sacrificed and the tumors were took out after 15 days’interventions.Then lactate relative concentrations were detected with lactate kits and the protein expressions of glucose transporter 4(GLUT4),hexokinase 1(HK1),glucose-regulated protein 78(GRP78),carbo-nic anhydrase-Ⅸ(CA-Ⅸ)were detected through immunohistochemical staining.Flow cytometry was adopted to detect the percentage of apoptotic tumor cells and regulatory T cells(Treg),and the expression of hypoxia-inducible factor-1α(HIF-1α),Bcl-2,Bax,Caspase-3,interleukin-2(IL-2)were tested through western blot.Results:The strongest inhibition effect and the lowest tumor growth rate was found in Fei-Liu-Ping ointment+cyclophosphamide group.There were significant differences between Fei-Liu-Ping ointment+cyclophosphamide group and saline group(P<0.05).And the highest food consumption was found in Fei-Liu-Ping ointment+cyclophosphamide group while there were no significant differences between Fei-Liu-Ping ointment+cyclophosphamide group and saline group(P>0.05).Further molecular biological detections found that the lowest lactate level and regulatory T cells ratio were found in Fei-Liu-Ping ointment+cyclophosphamide group and these expressions of GLUT4,HK1,GRP78,CA-Ⅸwere suppressed.There were significant differences between Fei-Liu-Ping ointment+cyclophosphamide group and saline group(P<0.05).In addition,the Fei-Liu-Ping ointment+cyclophosphamide group’s cell apoptosis increased significantly compared with saline group and there were significant differences on expressions of HIF-1α,Bcl-2,Bax,Caspase-3,IL-2 for this group compared with saline group.Conclusion:Chemotherapy and Fei-Liu-Ping ointment had the synergistic effect on inhibiting tumor growth and improving the general conditions of tumor-bearing mice.The effect was partly owed to the improvement on tissue hypoxia,the inhibition of HIF-1αexpression and the regulations on its downstream proteins,such as GLUT4,HK1,GRP78,and CA-Ⅸ.And then all these alterations led to the modulation tumor acidic microenvironment,the induced tumor cells apoptosis and suppression of T cells to regulatory T cells differentiation.