薯蓣丸调控M1/M2型肿瘤相关巨噬细胞极化抗大肠癌进展的作用机制

Shuyuwan Regulates M1/M2 Polarization of Tumor-associated Macrophages to Inhibit Progression of Colorectal Cancer

目的:探讨薯蓣丸调控肿瘤相关巨噬细胞(TAMs)极化抑制大肠癌(CRC)进展的分子机制。方法:构建结肠癌原位移植瘤裸鼠模型,雄性BALB/c-nu裸鼠(n=28,4周龄),随机分成4组,每组7只,模型组(生理盐水)、薯蓣丸低、中、高剂量组(1.725、2.310、2.895 g·kg^(-1)·d^(-1))。每只裸鼠灌胃剂量为15 mL·kg^(-1),于接种瘤块后第9天开始灌胃处理,每日灌胃1次,每周6 d,第7天不灌胃,连续干预2周后处死裸鼠,剥离肿瘤标本;取部分结肠组织和肿瘤组织制作切片,苏木素-伊红(HE)染色进行病理观察;免疫组化(IHC)检测肿瘤组织中诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测肿瘤组织中白细胞介素-12(IL-12)、表皮细胞生长因子(EGF)、转化生长因子-β(1 TGF-β1)mRNA表达;蛋白免疫印迹法(Western blot)检测肿瘤组织中iNOS、IL-12、EGF、TGF-β1蛋白表达。结果:与模型组比较,薯蓣丸能抑制裸鼠结肠癌肿瘤细胞的生长,病理可见不同程度的肿瘤细胞坏死。其中,薯蓣丸高剂量组对肿瘤细胞生长的抑制作用最强,肿瘤细胞形态基本消失;与模型组比较,薯蓣丸组明显上调了M1型TAMs相关细胞因子iNOS、IL-12表达(P<0.05),下调了M2型TAMs相关细胞因子Arg-1、EGF、TGF-β1的表达(P<0.05),提示薯蓣丸处理后TAMs向M2型极化减弱,向M1型极化增强。结论:薯蓣丸能够抑制结肠癌原位移植瘤的生长,其作用机制可能与阻断TAMs极化为M2型、促进TAMs向M1型极化有关。

Objective:To explore the molecular mechanism of Shuyuwan regulating polarization of tumor-associated macrophages(TAMs)to inhibit the progression of colorectal cancer(CRC).Method:The nude mouse model of orthotopic transplantation of colon cancer was established.Male BALB/c-nu nude mice(n=28,4 weeks old)were randomly assigned into 4 groups(n=7):Model group(normal saline)and low-,medium-,and high-dose(1.725,2.310,2.895 g·kg^(-1)·d^(-1),respectively)Shuyuwan groups.On day 9 after the tumor block was inoculated,the mice were administrated by gavage with corresponding agents at a dose of 15 mL·kg^(-1)once a day,6 days a week,and no agent on the 7th day.After two consecutive weeks of intervention,the nude mice were sacrificed and the tumor samples were collected.A part of the colon tissue and the tumor tissue was used to prepare sections,and hematoxylin-eosin(HE)staining was performed for pathological observation.The expression of inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)in the tumor tissue was detected by immunohistochemistry(IHC).The mRNA levels of interleukin-12(IL-12),epidermal growth factor(EGF),and transforming growth factor-β1(TGF-β1)in the tumor tissue were determined by Realtime polymerase chain reaction(PCR).Western blot was employed to determine the protein levels of iNOS,IL-12,EGF,and TGF-β1 in the tumor tissue.Result:Compared with the model group,Shuyuwan inhibited the growth of colon cancer cells in nude mice and caused the tumor cell necrosis in different degrees.The high-dose Shuyuwan group had the strongest inhibitory effect on the growth of tumor cells,which basically lost the normal morphology.Furthermore,Shuyuwan up-regulated the expression of iNOS and IL-12 in M1-type macrophages(P<0.05)and down-regulated the expression of Arg-1,EGF,and TGF-β1 in M2-type macrophages(P<0.05),which indicated the weakened polarization of macrophages toward M2 type and the enhanced polarization toward M1 type after treatment with Shuyuwan.Conclusion:Shuyuwan can inhibit the growth of orthotopically transplanted colon tumor by blocking the polarization of TAMs to M2 type and promoting the polarization of TAMs to M1 type.