益气健脾化瘀方协同5-FU对胃癌的抑制作用及机制

Inhibitory Effect and Mechanism of Yiqi Jianpi Huayu Recipe Combined with 5-FU on Gastric Cancer

目的:观察益气健脾化瘀方协同5-氟尿嘧啶(5-fluorouracil,5-FU)对615小鼠胃癌生长的抑制作用,并通过肿瘤微环境中的肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)极化方向,探讨其可能的作用机制。方法:40只615小鼠随机分为正常组,益气健脾化瘀方(25 g·kg^-1)组,5-FU(25 mg·kg^-1)组,联合(益气健脾化瘀方25 g·kg^-1+5-FU 25 mg·kg^-1)组,每组10只。腋下接种MFC胃癌细胞建立小鼠胃癌移植瘤模型。苏木素-伊红(hematoxylin-eosin staining,HE)染色法观察各组小鼠肿瘤病理的改变;流式细胞仪测定肿瘤组织中M1,M2型TAMs含量;免疫组化观察腋下移植瘤中CD206的表达;实时荧光定量PCR(Real-time PCR)检测瘤组织中TAMs相关基因白细胞介素-1β(interleukin-1β,IL-1β),白细胞介素-12(interleukin-12,IL-12),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),精氨酸酶1(arginase-1,Arg1),几丁质酶3蛋白3(chitinase 3-like 3,Ym1)的mRNA表达水平。Real-time PCR,蛋白免疫印迹法(Western blot)检测瘤组织中上皮间质转化(epithelial mesenchymal transformation,EMT)相关上皮性钙黏附蛋白(E-cadherin),神经性钙黏附蛋白(N-cadherin),锌指转录因子Slug,Snail,波形蛋白(Vimentin)mRNA及蛋白的表达。结果:各组小鼠胃癌细胞排列呈巢状及片状,细胞间质见促纤维结缔组织反应及少量炎症细胞浸润。各药物组可见明显肿瘤凝固性坏死,残留肿瘤细胞量正常组>益气健脾化瘀方组>5-FU组>联合组。免疫组化表明,各给药组均能减少瘤体CD206的表达,以联合组最明显。流式细胞仪检测证实,与正常组比较,各给药组均能减少M2型TAMs的含量(P<0.05,P<0.01),联合组减少M2型TAMs的含量最明显;益气健脾化瘀方组、联合组均能增加M1型TAMs的含量(P<0.05,P<0.01),以益气健脾化瘀方组最为明显。与正常组比较,益气健脾化瘀方组、联合组均能下调Arg1,Ym1 mRNA,上调IL-1β,TNF-α,IL-12 mRNA表达(P<0.05)。与5-FU组比较,联合组能明显升高IL-1β,IL-12,TNF-αmRNA,下调Arg1,Ym1 mRNA表达(P<0.05)。与正常组比较,益气健脾化瘀方组能上调E-cadherin mRNA及蛋白表达(P<0.05),下调N-cadherin,Vimentin mRNA表达(P<0.05);联合组能上调E-cadherin,下调N-cadherin,Slug,Snail,Vimentin mRNA表达(P<0.05),上调E-cadherin,下调N-cadherin,Slug蛋白表达(P<0.01)。与5-FU组比较,联合组能上调肿瘤组织中E-cadherin,下调N-cadherin,Slug,Vimentin mRNA及蛋白表达(P<0.05)。结论:益气健脾化瘀方对胃癌生长有一定的抑制作用,与5-FU联用有较好的协同抑制胃癌生长、改善胃癌上皮间质转化,机制可能与促进胃癌中TAMs从M2表型向M1表型转化有关。

Objective:To observe the synergistic effect and mechanism of Yiqi Jianpi Huayu(YQJPHY recipe combined with 5-fluorouracil(5-FU)on inhibiting gastric cancer growth,and investigate its possible mechanism through polarization direction of tumor-associated macrophages(TAMs)in tumor micro-environment.Method:A total of forty 615 mice were randomly divided into four groups including control group,YQJPHY(25 g·kg^-1)group,5-FU(25 mg·kg^-1)group and combined group(YQJPHY plus 5-FU),with 10 mice in each group.The gastric cancer transplantation model was induced by axillary inoculation of MFC gastric cancer cells.Changes in tumor pathology were observed with hematoxylin-eosin staining(HE)method.The contents of M1 and M2 TAMs in tumor tissues were determined with flow cytometry,while the expression of CD206 in tumor was observed with immunohistochemistry.The mRNA expressions of TAM-related genes including interleukin-1β(IL-1β),interleukin-12(IL-12),tumor necrosis factor-α(TNF-α),arginase-1(Arg1),and chitinase 3-like 3(Ym1)were detected by using quantitative real-time polymerase chain reaction(Real-time PCR).Furthermore,the mRNA and protein expressions of epithelial-mesenchymal transition(EMT)related epithelial calcium adhesion protein(E-cadherin),nervous calcium adhesion protein(N-cadherin),Zinc finger transcription factor Slug,Snail and Waveform protein(Vimentin)were detected with Real-time PCR and Western blot,respectively.Result:The gastric cancer cells in tumors were distributed in nest and/or flaky shape,with fibroblastic connective tissue reaction and inflammatory cells infiltrated in interstitium.Coagulative necrosis of tumor was observed in various treatment groups.The amount of residual cancer cells in mouse was as follows from largest to smallest:control group>YQJPHY group>5-FU group>combined group.Immunohistochemical studies showed that all treatment groups can reduce the expression of CD206 in the tumor,and the effect was most obvious in the combined group.The contents of M2-TAM in all treatment groups were lower than those in control group(P<0.05,P<0.01),and the most significant difference in the content of M2-TAMs was shown between the control group and the combined group.The contents of M1 TAMs were increased in YQJPHY and combined groups(P<0.05,P<0.01),with the largest incremental range in YQJPHY group.The expressions of Arg1 and Ym1 in YQJPHY and combined groups were lower,while IL-1β,TNF-αand IL-12 levels were higher than those in control group(P<0.05).The mRNA expressions of IL-1β,IL-12 and TNF-αin the combined group were significantly higher,while Arg1 and Ym1 mRNA expression levels were lower than those in the 5-FU group(P<0.05).As compared with the control group,the mRNA and protein expression levels of E-cadherin were up-regulated(P<0.05),while the mRNA expression levels of N-cadherin and Vimentin were down-regulation in the YQJPHY group(P<0.05),E-cadherin mRNA and protein expression levels were up-regulated,while N-cadherin,Slug,Snail,Vimentin mRNA expression as well as N-cadherin and Slug protein expression levels were down-regulated in the combined group(P<0.05).As compared with the 5-FU group,the mRNA and protein expression levels of E-cadherin were up-regulated(P<0.05),while the mRNA and protein expression levels of N-cadherin,Slug and Vimentin were down-regulated in the combined group(P<0.05).Conclusion:YQJPHY has an inhibitory effect on the growth of gastric cancer,and after being combined with 5-FU,it has a good synergistic effect on inhibiting the growth of gastric cancer and decreasing EMT.The mechanism may be related to promoting the conversion from M2-TAMs to M1-TAMs in gastric tumor micro-environment.