姜黄素和姜黄素联氨基抗肝癌作用比较及机制研究
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篇名: 姜黄素和姜黄素联氨基抗肝癌作用比较及机制研究
TITLE: Comparison of Anti-hepatocarcinoma Effect of Curcumin and Hydrazincurcumin and Mechanism Study
摘要: 目的:比较姜黄素(CUR)和其衍生物联氨基姜黄素(HZC)的体内外抗肝癌作用及机制。方法:采用MTT法检测CUR或HZC(2.5、5、10、20、40、80μmol/L)对人肝癌HepG2细胞增殖的影响;采用流式细胞术检测CUR或HZC(10、20、40μmol/L)对HepG2细胞周期分布和凋亡情况的影响;采用Westernblotting法检测CUR或HZC(10、20、40μmol/L)对HepG2细胞凋亡相关蛋白表达的影响。将雄性SD大鼠随机分为正常对照组(n=10)、CUR对照组(n=10)、HZC对照组(n=10)、肝癌模型组(n=30)、CUR防护组(n=30)、HZC防护组(n=30)。CUR对照组和HZC对照组大鼠分别腹腔注射CUR或HZC(剂量均为80mg/kg);模型组、CUR防护组和HZC防护组大鼠均腹腔注射对二乙基亚硝胺(50mg/kg)建立肝癌模型,同时2种药物防护组大鼠分别腹腔注射CUR或HZC(剂量均为80mg/kg),每周2次,连续给药12周。给药期间,记录大鼠体质量变化和死亡情况;24周后,计算大鼠肝脏指数并观察其外观,统计肝癌结节数;采用苏木精-伊红染色法观察大鼠肝组织病理学变化并计算肝癌细胞核分裂指数;采用免疫组化法检测大鼠肝组织增殖细胞核抗原(PCNA)指数。结果:CUR和HZC均能显著升高HepG2细胞的增殖抑制率、G0/G1期细胞周期百分比和凋亡率(P<0.05);均能显著降低细胞中磷酸化蛋白酪氨酸激酶2(p-JAK2)、磷酸化信号转导及转录激活蛋白3(p-STAT3)、B淋巴细胞瘤2(Bcl-2)、Bcl-xl蛋白的表达水平,显著升高Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt-c)、胱天蛋白酶9(Caspase-9)、Caspase-3、聚腺苷二磷酸核糖聚合酶(PAPR)蛋白的表达水平(P<0.05);且HZC的上述作用均显著强于CUR(P<0.05)。动物实验结果显示,3个对照组大鼠无死亡,未发生肝脏癌变,肝脏外观和组织切片未见病理变化;体质量及增量、肝脏指数、癌细胞核分裂指数、肝组织PCNA指数组间比较差异均均无统计学意义(P>0.05)。与肝癌模型组比较,CUR防护组和HZC防护组大鼠的存活率均显著升高,肝癌发生率和癌结节数均显著降低(P<0.05);体质量及增量均显著升高,肝脏指数、癌细胞核分裂指数、肝组织PCNA指数均显著降低(P<0.05);肝脏外观和组织切片均有一定程度病变,可见癌灶伴局灶性坏死或伴大斑片状坏死;但2种药物防护组上述指标的改善程度差异均无统计学意义(P>0.05)。结论:HZC能通过抑制JAK2/STAT3信号通路和调节线粒体内源性通路的活化,发挥抑制HepG2细胞增殖、诱导其凋亡的作用,且比CUR具有更强的体外抗肝癌活性;但与CUR比较,HZC对肝癌模型大鼠各项抗肝癌指标的改善未见显著差异。
ABSTRACT: OBJECTIVE:To compare the an ti-hepatocarcinoma effects of curcumin (CUR)and its derivative hydrazincurcumin (HZC),and to explore the mechanism. METHODS :MTT assay was used to detect the effects of CUR or HZC (2.5,5,10,20, 40,80 μmol/L)on the proliferation of HepG 2 cells. Flow cytometry was used to detect the effects of CUR or HZC (10,20,40 μmol/L)on cell cycle distribution and apoptosis of HepG 2 cells. Western blotting assay was used to detect the effects of CUR or HZC(10,20,40 μmol/L)on the expression of apoptosis-related protein in HepG 2 cells. The male SD rats were randomly divided into normal control group (n=10),CUR control group (n=10),HZC control group (n=10),model group (n=30),CUR protection group (n=30)and HZC protection group (n=30). CUR control group and HZC control group were given CUR 85917439。E-mail:zhaoji-an-88@163.com or HZC (80 mg/kg) intraperitoneally. Model group ,CUR protection group and HZC protection group were given diethylnitrosamine (50 mg/kg)intraperitoneally to establish hepatocarcinoma model ;at the same time ,2 protection groups were given CUR or HZC (80 mg/kg)intraperitoneally,twice a day,for consecutive 12 weeks. During medication ,the change of body weight and death of rats were recorded. Twenty four weeks later,liver index of rats was calculated and appearance was observed ;the number of cancer nodules was counted ;HE staining was used to observe the pathological changes of liver tissue and calculate the nuclear division index of hepatocarcinoma ;the proliferating cell nuclear antigen (PCNA)index was detected by immunohistochemistry. RESULTS :CUR and HZC could increase the inhibitory rate of HepG 2 cells(P<0.05),and increased the percentage at G 0/G1 phase and apoptotic rate of HepG 2 cells(P< 0.05). CUR and HZC could significantly decrease the protein expression of p-JAK 2,p-STAT3,Bcl-2 and Bcl-xl ,while increased the protein expression of Bax ,Cyt-c,Caspase-9,Caspase-3 and PAPR (P<0.05). Above effects of HZC were significantly better than those of CUR (P<0.05). The results of animal experiment showed that there was no death ,no liver canceration and no pathological changes in liver appearance and tissue section of the three control groups ;there was no statistical significance in body weight and its increased weight ,liver index ,nuclear division index of carcinoma or PCNA index (P>0.05). Compared with model group, survival rate of rats were increased significantly in CUR protection group and HZC protection group , while hepatocarcinoma incidence and the number of cancer nodules were decreased significantly (P<0.05);body weight and its increased weight were increased significantly ,while liver index ,nuclear division index of carcinoma and PCNA index were decreased significantly (P<0.05). There were some pathological changes in liver appearance and tissue section ;cancerous lesions with focal necrosis or cancerous lesions with patchy necrosis were observed. There was no statistical significance in the improvement of above indexes in 2 protection groups (P>0.05). CONCLUSIONS :HZC could inhibit the proliferation and induce apoptosis of HepG 2 cells by inhibiting JAK 2/STAT3 signaling pathway and regulating the activation of mitochondrial endogenous pathway,which shows stronger anti-hepatocarcinoma effect in vitro than CUR. On the other hand ,there was no significant difference in the improvement of liver caner indexes in hepatic cancer model rats between HZC and CUR.
期刊: 2020年第31卷第22期
作者: 赵冀安,崔丽敏,董梁,聂文佳,刘文聪,李增宁
AUTHORS: ZHAO Ji ’an,CUI Limin,DONG Liang,NIE Wenjia ,LIU Wencong ,LI Zengning
关键字: 肝癌;姜黄素;联氨基姜黄素;蛋白酪氨酸激酶 2/信号转导及转录激活蛋白 3信号通路;HepG2细胞;大鼠;核分裂指数;
KEYWORDS: Hepatocarcinoma;Curcumin;Hydrazinocurcumin;JAK2/STAT3 signaling pathway ;HepG2 cells;Rats;Nuclear
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