基于SHH/GLI-1信号通路探讨齐墩果酸抑制结直肠癌小鼠移植瘤生长与炎症反应的作用机制

Inhibition of Oleanolic Acid on Growth and Inflammatory Response in Colorectal Cancer Transplanted Tumors in Mice Based on SHH/GLI-1 Signaling Pathway

目的基于SHH/GLI-1信号通路探讨齐墩果酸(OA)对结直肠癌小鼠移植瘤生长与炎症反应的影响。方法选择SPF级6周龄雄性BALB/c裸鼠12只,采用皮下注射HT-29细胞构建结直肠癌移植瘤小鼠模型,随机分为对照组与干预组各6只。干预组按12.5 mg/(kg·d)予腹腔注射OA溶液,对照组按500μL/(kg·d)予腹腔注射生理盐水,给药6 d停止1 d,共干预16 d。干预期间每2 d测量小鼠体质量和肿瘤体积,干预结束后测量肿瘤重量;免疫组化法检测肿瘤组织增殖标志物Ki67、细胞周期相关蛋白[细胞周期素依赖性激酶抑制因子(p21)、周期蛋白依赖性激酶4(CDK4)、细胞周期蛋白D1(Cyclin D1)]、细胞凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2相关X(Bax)、磷酸化-B淋巴细胞瘤-2死亡拮抗蛋白(p-Bad)、死亡因子(Fas)、死亡因子配体(FasL)]、炎症相关蛋白[环氧化酶-2(COX-2)、一氧化氮合酶(iNOS)]以及SHH/GLI-1信号通路相关蛋白[音猬因子(SHH)、补丁受体(PTCH)、光滑蛋白(SMO)、胶质瘤相关癌基因同源物1(GLI-1)]的表达;TUNEL法检测肿瘤细胞的凋亡情况;Bio plex检测2组小鼠血清炎症因子白介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)的表达。结果2组不同时间段体质量比较,差异无统计学意义(P>0.05)。与对照组比较,干预组干预7、9、11、13、15、17、19 d小鼠肿瘤体积均明显减小(P<0.05),干预后肿瘤重量明显降低(P<0.05)。与对照组比较,干预组肿瘤组织Ki67、CDK4、Cyclin D1、Bcl-2、COX-2、iNOS、SHH、PTCH、SMO、GLI-1和血清IL-1β、IL-6、TNF-α表达均明显降低(P<0.05),p21、Bax、p-Bad、Fas、FasL表达均明显升高(P<0.05);凋亡细胞比例明显增加(P<0.05)。结论OA可以抑制结直肠癌小鼠移植瘤的生长和炎症反应,可能与调控SHH/GLI-1信号通路相关。

Objective:To explore effects of oleanolic acid(OA)on tumor growth and inflammatory response in colorectal cancer transplanted tumors in mice based on SHH/GLI-1 signaling pathway.Methods:A total of twelve SPF grade 6-week-old male BALB/c nude mice were subcutaneously injected with HT-29 cells to establish a colorectal cancer transplant tumor mouse model,and randomly divided into control group and intervention group,with 6 mice in each group.The intervention group received intraperitoneal injection of OA solution at a dose of 12.5 mg/(kg·d),while the control group received intraperitoneal injection of physiological saline at a dose of 20 mL/(kg·d).The treatment lasted for 6 days and was stopped for 1 day,for a total of 16 days.During the intervention period,the body mass and tumor volume of mice were measured every 2 days,while tumor weight was measured after the intervention.Immunohistochemistry was used to detect the expression of tumor tissue proliferation markers Ki67,cycle-associated proteins[cyclin dependent kinase inhibitor(p21),cyclin dependent kinase 4(CDK4),Cyclin D1],apoptosis-associated proteins[B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),phosphorylated Bcl-2 antagonist of cell death(p-Bad),death factor(Fas),death factor ligand(FasL)],inflammation-associated proteins[cyclooxygenase-2(COX-2),nitric oxide synthase(iNOS)]and the expression of SHH/GLI-1 signaling pathway associated proteins[Sonic hedgehog(SHH),patch receptor(PTCH),smooth protein(SMO),glioma associated oncogene homolog 1(GLI-1)].TUNEL method was used to detect the apoptosis of tumor cells.Bio plex was used to detect the expression of inflammatory factors interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α)in the serum of the two groups of mice.Results:There was no significant difference in body weight between the two groups at different time periods(P>0.05).Compared with the control group,the tumor volume in mice at 7,9,11,13,15,17,and 19 days of intervention in the intervention group significantly decreased(P<0.05),while the tumor weight after intervention significantly decreased(P<0.05).Compared with the control group,the expression of Ki67,CDK4,Cyclin D1,Bcl-2,COX-2,iNOS,SHH,PTCH,SMO and GLI-1 in tumor tissue in the intervention group significantly decreased,as well as IL-1β,IL-6,and TNF-α in serum(P<0.05),while the expression of p21,Bax,p-Bad,Fas and FasL in tumor tissue and the proportion of apoptotic cells significantly increased(P<0.05).Conclusion:OA can inhibit the growth and inflammatory responses of colorectal cancer transplanted tumors in mice,which may be related to the regulation of the SHH/GLI-1 signaling pathway.