高脂低碳水化合物饮食联合放疗对肺癌移植瘤小鼠肿瘤微环境的影响

Effects of high-fat and low-carbohydrate diet combined with radiotherapy on tumor microenvironment of Lewis lung cancer bearing mice

目的探讨高脂低碳水化合物饮食联合放疗对肺移植瘤小鼠肿瘤微环境的影响。方法选用C57BL/6J小鼠建立Lewis肺癌模型,分为普通饮食组、高脂低碳水化合物饮食组、普通饮食+放疗组和高脂低碳水化合物饮食+放疗组,每组18只。普通饮食组和普通饮食+放疗组小鼠予以普通饮食(脂肪供能比例为12.11%)饲养,高脂低碳水化合物饮食组和高脂低碳水化合物饮食+放疗组小鼠予以高脂低碳水化合物饮食(脂肪供能比例为45.00%)饲养。第12~14天对普通饮食+放疗组和高脂低碳水化合物饮食+放疗组小鼠的肿瘤部位进行放射治疗,照射剂量为24 Gy/3f,观察和监测小鼠体重、肿瘤体积、血糖和血酮值、肝肾功能、生存情况。采用免疫组织化学染色检测CD34和淋巴管内皮透明质酸受体1(LYVE-1),采用天狼星染色检测胶原纤维,采用多重免疫荧光检测CD8和程序性死亡蛋白1(PD-1),采用流式细胞术检测免疫细胞表型。结果接种后第27天,普通饮食组小鼠的体重为(24.78±2.22)g,高于普通饮食+放疗组[(22.15±0.48)g,P=0.030]和高脂低碳水化合物饮食+放疗组[(22.02±0.77)g,P=0.031]。在接种后第15天,高脂低碳水化合物饮食+放疗组的肿瘤体积为(220.88±130.05)mm^(3),小于普通饮食组[(504.37±328.48)mm^(3),P=0.042]和高脂低碳水化合物饮食组[(534.26±230.42)mm^(3),P=0.016],但与普通饮食+放疗组[(274.64±160.97)mm^(3)]差异无统计学意义(P>0.05)。第4周高脂低碳水化合物饮食组小鼠的血糖值为(8.00±0.36)mmol/L,低于普通饮食组[(9.57±0.40)mmol/L,P<0.05]。第2周和第3周高脂低碳水化合物饮食组小鼠的血酮值分别为(1.00±0.20)mmol/L和(0.90±0.17)mmol/L,均高于普通饮食组[分别为(0.63±0.06)mmol/L和(0.70±0.10)mmol/L,均P<0.05]。接种后第30天,普通饮食组与高脂低碳水化合物饮食组小鼠的天冬氨酸氨基转移酶、丙氨酸氨基转移酶、肌酐和尿素等指标差异均无统计学意义(均P>0.05),各组小鼠的心、肝、脾、肺、肾均未见明显毒性改变及肿瘤转移。普通饮食组、高脂低碳水化合物饮食组和普通饮食+放疗组小鼠的中位生存时间分别为38、41和55 d,高脂低碳水化合物饮食+放疗组小鼠的中位生存时间未达到。高脂低碳水化合物饮食+放疗组小鼠肿瘤组织中CD8表达上调,PD-1、CD34、LYVE-1和胶原纤维表达下调。高脂低碳水化合物饮食+放疗组的肿瘤旁淋巴结中CD8^(+)T细胞比例[(25.13±0.97)%]高于普通饮食组[(20.60±2.23)%,P<0.05]和普通饮食+放疗组[(19.26±3.07)%,P<0.05],但与高脂低碳水化合物饮食组[(22.03±1.75)%]差异无统计学意义(P>0.05)。普通饮食+放疗组肿瘤旁淋巴结中CD4^(+)T细胞比例[(31.33±5.16)%]和高脂低碳水化合物饮食+放疗组肿瘤旁淋巴结中CD4^(+)T细胞比例[(30.63±1.70)%]高于普通饮食组[(20.27±2.15)%,P<0.05]和高脂低碳水化合物饮食组(23.70±2.62,P<0.05)。普通饮食+放疗组的肿瘤旁淋巴结中Treg细胞在T细胞中占比最高[(16.58±5.10)%],但与普通饮食组、高脂低碳水化合物饮食组和高脂低碳水化合物饮食+放疗组比较,差异均无统计学意义(均P>0.05)。结论高脂低碳水化合物饮食联合放疗可促进肺癌肿瘤微环境中免疫效应细胞的募集并增强其功能,抑制肿瘤微血管生成,从而抑制肿瘤生长。

Objective To investigate the effect of high-fat and low-carbohydrate diet combined with radiotherapy on the tumor microenvironment of mice with lung xenografts.Methods C57BL/6J mice were selected to establish the Lewis lung cancer model,and they were divided into the normal diet group,the high-fat and low-carbohydrate diet group,the normal diet+radiotherapy group,and the high-fat and low-carbohydrate diet+radiotherapy group,with 18 mice in each group.The mice in the normal diet group and the normal diet+radiotherapy group were fed with the normal diet with 12.11%fat for energy supply,and the mice in the high-fat and low-carbohydrate diet group and the high-fat and low-carbohydrate diet+radiotherapy group were fed with high-fat and low-carbohydratediet with 45.00%fat for energy.On the 12th to 14th days,the tumor sites of the mice in the normal diet+radiotherapy group and the high-fat and low-carbohydrate diet+radiotherapy group were treated with radiotherapy,and the irradiation dose was 24 Gy/3f.The body weight,tumor volume,blood glucose and blood ketone level,liver and kidney function,and survival status of the mice were observed and monitored.Immunohistochemical staining was used to detect the tumor-associated microangiogenesis molecule(CD34)and lymphatic endothelial hyaluronan receptor 1(LYVE-1),Sirius staining was used to detect collagen fibers,and multiplex immunofluorescence was used to detect CD8 and programmed death-1(PD-1).Expression of immune cell phenotypes(CD3,CD4,CD8,and Treg)was detected by flow cytometry.Results On the 27th day after inoculation,the body weigh of the common diet group was(24.78±2.22)g,which was significantly higher than that of the common diet+radiotherapy group[(22.15±0.48)g,P=0.030]and high-fat low-carbohydrate diet+radiotherapy group[(22.02±0.77)g,P=0.031].On the 15th day after inoculation,the tumor volume of the high-fat and low-carbohydrate diet+radiotherapy group was(220.88±130.05)mm^(3),which was significantly smaller than that of the normal diet group[(504.37±328.48)mm^(3),P=0.042]and the high-fat,low-carbohydrate diet group[(534.26±230.42)mm^(3),P=0.016],but there was no statistically significant difference compared with the normal diet+radiotherapy group[(274.64±160.97)mm^(3)].In the 4th week,the blood glucose values of the mice in the high-fat and low-carbohydrate diet group were lower than those in the normal diet group,with the value being(8.00±0.36)mmol/L and(9.57±0.40)mmol/L,respectively,and the difference was statistically significant(P<0.05).The blood ketone values of the mice in the high-fat and low-carbohydrate diet group were higher than those in the normal diet group,with the value being(1.00±0.20)mmol/L and(0.63±0.06)mmol/L,respectively,in the second week.In the third week,the blood ketone values of the two groups of mice were(0.90±0.17)mmol/L and(0.70±0.10)mmol/L,respectively,and the difference was statistically significant(P<0.05).On the 30th day after inoculation,there were no significant differences in aspartate aminotransferase,alanine aminotransferase,creatinine,and urea between the normal diet group and the high-fat,low-carbohydrate diet group(all P>0.05).The hearts,livers,spleens,lungs,and kidneys of the mice in each group had no obvious toxic changes and tumor metastasis.In the high-fat and low-carbohydrate diet+radiotherapy group,the expression of CD8 was up-regulated in the tumor tissues of mice,and the expressions of PD-1,CD34,LYVE-1,and collagen fibers were down-regulated.The proportion of CD8^(+)T cells in the paratumoral lymph nodes of the high-fat and low-carbohydrate diet+radiotherapy group was(25.13±0.97)%,higher than that of the normal diet group[(20.60±2.23)%,P<0.050]and the normal diet+radiotherapy group[(19.26±3.07)%,P<0.05],but there was no statistically significant difference with the high-fat and low-carbohydrate diet group[(22.03±1.75)%,P>0.05].The proportion,of CD4^(+)T cells in the lymph nodes adjacent to the tumor in the normal diet+radiotherapy group(31.33±5.16)%and the high-fat and low-carbohydrate diet+radiotherapy group(30.63±1.70)%were higher than that in the normal diet group[(20.27±2.15)%,P<0.05]and the high-fat and low-carbohydrate diet group(23.70±2.62,P<0.05).Treg cells accounted for the highest(16.58±5.10)%of T cells in the para-tumor lymph nodes of the normal diet+radiotherapy group,but compared with the normal diet group,the high-fat and low-carbohydrate diet group,and the high-fat and low-carbohydrate diet+radiotherapy group,there was no statistically significant difference(all P>0.05).Conclusion High-fat and low-carbohydrate diet plus radiotherapy can enhance the recruitment and function of immune effector cells in the tumor microenvironment,inhibit tumor microangiogenesis,and thus inhibit tumor growth.