加味桂枝茯苓丸通过PTEN/PI3K/Akt通路调控线粒体凋亡途径预防小鼠结直肠腺瘤的作用机制

Mechanism of Modified Guizhi Fulingwan in Regulating Mitochondrial Apoptosis Pathway Through PTEN/PI3K/Akt Pathway to Prevent Colorectal Adenoma in Mice

目的:探讨加味桂枝茯苓丸通过磷酸酯酶与张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路调控线粒体凋亡途径预防小鼠结直肠腺瘤(CRA)的作用及机制。方法:将60只SPF级C57BL/6雄性小鼠随机分为正常组、模型组、加味桂枝茯苓丸低、中、高剂量组(13、26、52 g·kg^(-1)·d^(-1)),阳性药阿司匹林组(0.015g·kg^(-1)·d^(-1)),通过氧化偶氮甲烷(AOM)/葡聚糖硫酸钠(DSS)化学诱导CRA小鼠模型,造模同时灌胃给予加味桂枝茯苓丸或阿司匹林。给药期间,每周检测各组小鼠体质量;9周后,观察腺瘤形成数目;酶联免疫吸附测定法(ELISA)测定血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)水平;苏木素-伊红(HE)染色观察腺瘤组织病理变化;免疫组织化学法(IHC)检测细胞周期蛋白D1(Cyclin D1)和细胞增殖核抗原(Ki67)的表达;原位末端转移酶标记法(TUNEL)检测腺瘤组织细胞凋亡情况;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)观察PTEN、PI3K、Akt、磷酸化PI3K(p-PI3K)、磷酸化Ak(p-Akt)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、胱天蛋白酶-9(Caspase-9)、胱天蛋白酶-3(Caspase-3)mRNA和蛋白表达水平。结果:与正常组比较,模型组小鼠体质量在第1周至第2周无明显变化,第3周至第9周明显降低(P<0.05,P<0.01);结直肠长度显著缩短,结直肠重量显著增加,黏膜表面可见大小不等的瘤样突起(P<0.01);血清TNF-α、IL-6、IL-1β水平升高(P<0.01);小鼠肠上皮腺体结构紊乱,细胞核明显拉长且可见病理性核分裂,固有层可见大量淋巴细胞小灶性浸润;Cyclin D1、Ki67阳性表达率显著升高(P<0.01);腺瘤细胞凋亡率显著降低(P<0.01);腺瘤组织PI3K、Akt、Bcl-2 mRNA和蛋白表达显著增强(P<0.01),p-PI3K、p-Akt蛋白表达显著增强(P<0.01),PTEN、Bax、Cyt C、Caspase-9、Caspase-3 mRNA和蛋白表达明显降低(P<0.05,P<0.01)。与模型组比较,各给药组小鼠体质量上升(P<0.01);肠道重量减轻,结直肠长度增加,腺瘤数量明显减少(P<0.05,P<0.01);血清TNF-α、IL-6、IL-1β含量显著降低(P<0.01);肠上皮腺体结构紊乱及黏膜固有层中性粒细胞浸润程度有所改善;Cyclin D1、Ki67阳性表达率显著下降(P<0.01);腺瘤组织细胞凋亡率明显增加(P<0.05,P<0.01);腺瘤组织PI3K、Akt、Bcl-2 mRNA和蛋白表达下降(P<0.05,P<0.01),p-PI3K、p-Akt蛋白表达明显降低(P<0.05,P<0.01),PTEN、Bax、Cyt C、Caspase-9、Caspase-3 mRNA和蛋白表达明显升高(P<0.05,P<0.01),以加味桂枝茯苓丸高剂量组干预效果最为显著。结论:加味桂枝茯苓丸可能通过调控PTEN/PI3K/Akt信号通路,进而诱导线粒体凋亡途径发挥预防小鼠CRA的作用。

Objective:To investigate the effect and mechanism of modified Guizhi Fulingwan in preventing colorectal adenoma(CRA)in mice by regulating mitochondrial apoptosis pathway through the regulation of the phosphatase and tensin homolog(PTEN)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Method:Sixty SPF-grade male C57BL/6 mice were randomly divided into six groups:Normal group,model group,low,medium,and high dose groups of modified Guizhi Fulingwan(13,26,52 g·kg^(-1)·d^(-1)),and positive control aspirin group(0.015 g·kg^(-1)·d^(-1)).A mouse model of CRA was chemically induced using azoxymethane(AOM)and dextran sulfate sodium(DSS).During the modeling process,mice received modified Guizhi Fulingwan or aspirin.Body weight of mice was measured weekly during the treatment.After 9 weeks,the number of adenomas formed was observed.Serum levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β)were determined by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining was used to observe the histopathologic changes in adenoma tissues.The expression of Cyclin D1 and proliferative nuclear antigen(Ki67)was detected by immunohistochemistry(IHC).Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)was used to assess the apoptosis in adenoma tissues.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to observe the mRNA and protein expression levels of PTEN,PI3K,Akt,phosphorylated PI3K(p-PI3K),phosphorylated Akt(p-Akt),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cytochrome C(Cyt C),Caspase-9,and caspase-3.Result:Compared with the normal group,the model group showed no significant change in body weight from week 1 to week 2,but a significant decrease from week 3 to week 9(P<0.05,P<0.01).The colorectal length was significantly shortened,and the colorectal weight increased with visible varying sized tumor-like protrusions on the mucosal surface(P<0.01).Serum levels of TNF-α,IL-6,and IL-1βwere elevated(P<0.01).Histopathology showed disordered epithelial gland structure,elongated nuclei with pathological mitosis,and numerous lymphocytic infiltrations in the lamina propria.The positive expression rates of Cyclin D1 and Ki67 were significantly increased(P<0.01),while the apoptosis rate of adenoma cells was significantly decreased(P<0.01).Expression levels of PI3K,Akt,Bcl-2 mRNA and proteins,as well as p-PI3K and p-Akt proteins,were significantly increased(P<0.01),whereas PTEN,Bax,Cyt C,Caspase-9,and Caspase-3 mRNA and protein levels were significantly decreased(P<0.05,P<0.01).Compared with the model group,all drug treatment groups showed an increase in body weight(P<0.01),decreased intestinal weight,increased colorectal length,reduced number of adenomas significantly(P<0.05,P<0.01),and significantly lowered serum levels of TNF-α,IL-6,and IL-1β(P<0.01).Histopathology indicated improved glandular structure and reduced neutrophil infiltration in the mucosal lamina propria.The positive expression rates of Cyclin D1 and Ki67 significantly decreased(P<0.01),while the apoptosis rate of adenoma cells significantly increased(P<0.05,P<0.01).Expression levels of PI3K,Akt,Bcl-2 mRNA and proteins,and p-PI3K and p-Akt proteins were significantly reduced(P<0.05,P<0.01),while PTEN,Bax,Cyt C,Caspase-9,and Caspase-3 mRNA and protein levels significantly increased(P<0.05,P<0.01).The high-dose modified Guizhi Fulingwan group exhibited the most significant intervention effects.Conclusion:Modified Guizhi Fulingwan may prevent CRA in mice by regulating the PTEN/PI3K/Akt signaling pathway and inducing the mitochondrial apoptosis pathway.