基于mTOR/HIF-1α/VEGF信号通路探讨抗纤抑癌方对肝癌前病变大鼠模型的调控作用
DOI: 10.12449/JCH241019
Regulatory effect of Kangxian Yiai Prescription in a rat model of precancerous lesions of liver cancer: A study based on the mTOR/HIF-1α/VEGF signaling pathway
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摘要:
目的 研究抗纤抑癌方对肝癌前病变大鼠模型mTOR/HIF-1α/VEGF信号通路的影响。 方法 40只雄性Wistar大鼠,分为正常组、模型组、抗纤抑癌组和鳖甲软肝组,每组各10只。正常组大鼠腹腔注射生理盐水,剂量为0.4 mL/100 g,其他3组大鼠以50 mg/kg剂量腹腔注射二乙基亚硝胺,制备肝癌前病变大鼠模型。通过免疫组化和Western Blot法检测GST-Pi的表达,实时荧光定量PCR和Western Blot法检测mTOR、HIF-1α、VEGF、M2型丙酮酸激酶(PKM2)、葡萄糖载体蛋白1(GLUT-1) mRNA及蛋白的表达。计量资料多组间比较采用单因素方差分析或Kruskal-Wallis H秩和检验,进一步两两比较采用LSD-t检验。 结果 与正常组比较,模型组大鼠肝组织GST-Pi蛋白表达显著升高(P<0.01);与模型组比较,抗纤抑癌组GST-Pi蛋白表达水平显著降低(P<0.05)。与正常组比较,模型组大鼠肝组织GLUT1及PKM2 mRNA的表达均显著升高(P值均<0.01);与模型组比较,鳖甲软肝组及抗纤抑癌组GLUT1 mRNA的表达均显著降低(P值均<0.05)。与正常组比较,模型组大鼠肝组织GLUT1及PKM2的蛋白表达均显著升高(P值均<0.01)。与正常组比较,模型组大鼠肝组织mTOR、HIF-1α及VEGF的mRNA表达均显著升高(P值均<0.01);与模型组比较,鳖甲软肝组mTOR及VEGF的mRNA的表达均显著降低(P值均<0.05),抗纤抑癌组mTOR及VEGF mRNA的表达亦显著降低(P值均<0.01)。与正常组比较,模型组大鼠肝组织mTOR、HIF-1α、VEGF的蛋白表达均显著升高(P值均<0.01);与模型组相比,鳖甲软肝组只有mTOR的蛋白表达显著降低(P<0.01),抗纤抑癌组mTOR、HIF-1α、VEGF的蛋白表达均显著降低(P值均<0.05);与鳖甲软肝组相比,抗纤抑癌组mTOR的蛋白表达较高(P<0.01)。 结论 抗纤抑癌方可通过调控mTOR/HIF-1α/VEGF信号抑制肝癌前病变。 -
关键词:
- 肝肿瘤 /
- 信号传导 /
- 抗纤抑癌方 /
- 大鼠, Wistar
Abstract:Objective To investigate the effect of Kangxian Yiai Prescription (KXYA) on the mTOR/HIF-1α/VEGF signaling pathway in a rat model of precancerous lesions of liver cancer. Methods A total of 40 male Wistar rats were divided into normal group, model group, KXYA group, and Biejia Rangan Tablets (BJRG) group, with 10 rats in each group. The rats in the normal group were given intraperitoneal injection of normal saline at a dose of 0.4 mL/100 g, and those in the other three groups were given intraperitoneal injection of diethylnitrosamine at a dose of 50 mg/kg to establish a rat model of the precancerous lesions of liver cancer. Immunohistochemistry and Western Blot were used to measure the expression level of GST-Pi, and quantitative real-time PCR and Western Blot were used to measure the mRNA and protein expression levels of mTOR, HIF-1α, VEGF, PKM2, and GLUT1. A one-way analysis of variance or the Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Compared with the normal group, the model group had a significant increase in the protein expression level of GST-Pi in liver tissue (P<0.01), and compared with the model group, the KXYA group had a significant reduction in the protein expression level of GST-Pi (P<0.05). Compared with the normal group, the model group had significant increases in the mRNA expression levels of GLUT1 and PKM2 in liver tissue (P<0.01), and compared with the model group, the BJRG group and the KXYA group had a significant reduction in the mRNA expression level of GLUT1 (P<0.05). Compared with the normal group, the model group had significant increases in the protein expression levels of GLUT1 and PKM2 in liver tissue (P<0.01). Compared with the normal group, the model group had significant increases in the mRNA expression levels of mTOR, HIF-1α, and VEGF in liver tissue (P<0.01); compared with the model group, the BJRG group had significant reductions in the mRNA expression levels of mTOR and VEGF (P<0.05), and the KXYA group also had significant reductions in the mRNA expression levels of mTOR and VEGF (P<0.01). Compared with the normal group, the model group had significant increases in the protein expression levels of mTOR, HIF-1α, and VEGF in liver tissue (P<0.01); compared with the model group, the BJRG group had a significant reduction in the protein expression level of mTOR (P<0.01), and the KXYA group had significant reductions in the protein expression levels of mTOR, HIF-1α, and VEGF (P<0.05); compared with the BJRG group, the KXYA group had a significantly higher protein expression level of mTOR (P<0.01). Conclusion KXYA can inhibit the precancerous lesions of liver cancer by regulating the mTOR/HIF-1α/VEGF signaling pathway. -
Key words:
- Liver Neoplasms /
- Signal Transduction /
- Kangxian-Yiai Formula /
- Rats, Wistar
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肝癌作为全球范围内发病率和死亡率较高的恶性肿瘤,其发病机制涉及病毒感染、酒精滥用、肥胖以及不洁饮食等[1-2]。肝癌前病变与肝癌的发生密切关联[3]。肝癌前病变缺氧微环境的形成与能量代谢异常密切相关。糖酵解在缺氧条件下发挥着关键作用,与肿瘤的发生和发展密切相关[4-5]。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/缺氧诱导因子1α(hypoxia inducible factor1α,HIF-1α)/血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路作为细胞适应缺氧环境的重要调控网络,近年引起广泛关注。
mTOR参与细胞生长、增殖和代谢的调控[6]。在低氧环境中,mTOR与HIF-1α协同作用,共同参与调控细胞对缺氧的适应性反应[7-8]。HIF-1α通过调控多个基因的表达,包括VEGF,参与调节血管生成、细胞存活和炎症反应等生物学过程[9-10]。VEGF作为一个重要的促血管生成因子,在包括肝癌在内的多种肿瘤的血管生成中发挥关键作用[11-12]。
抗纤抑癌方是叶永安教授治疗肝癌及其癌前病变的经验方,临床疗效显著[13-14]。然而,其在分子水平上对肝癌前病变的调控机制仍然不清楚。因此,本研究探讨抗纤抑癌方对mTOR/HIF-1α/VEGF信号通路的调控作用,深入研究其对肝癌前病变的影响,以期为肝癌前病变的预防和治疗提供新的理论与实验基础。
1. 材料与方法
1.1 实验动物
40只健康雄性Wistar大鼠(SPF级),体质量(175±20)g,由北京维通利华公司购得[实验动物生产许可证:SCXK(京)2016-0006]。在东直门医院动物房(SPF级)进行常规饲养(恒温、恒湿、自由饮食饮水),实验动物使用许可证:SYXK(京)2015-0001。
1.2 实验药品
抗纤抑癌方颗粒剂成分包括柴胡、山药、白芥子、黄芪等,由南宁培力药业供应,通过质控鉴定确保为同一批次。复方鳖甲软肝片(批准文号:Z19991011,中国内蒙古福瑞中蒙药科技公司生产);二乙基亚硝胺(N0756,美国Sigma公司)。
1.3 主要试剂与仪器
Anti-HIF1α (ab1,英国abcam公司),Anti-PKM2 (3198S,美国CST公司),Anti-mTOR (2972S,美国CST公司),Anti-VEGF (ab53465,英国abcam公司),Anti-GLUT1 (1293S,美国CST公司),Anti-GSTPi (ab53943,英国abcam公司),GAPDH(ab8245,英国abcam公司),Trizol(R401-1,南京诺唯赞生物科技有限公司),M-MLV反转录试剂盒(A2791,美国Promega公司),Real-time PCR扩增试剂盒(Q121-02,南京诺唯赞限公司),DAB显色试剂盒(DA1010,北京索莱宝公司)。Western Blot电泳系统(美国Bio-rad公司),CFX96 Q-PCR仪(美国Bio-rad公司),NanoDrop分光光度计(美国Malcom公司),PCR引物由美国life technology公司代工合成。
1.4 实验方法
1.4.1 分组与模型制备
采用随机数字表法,分为正常组、模型组、抗纤抑癌方组和鳖甲软肝组,每组10只。制备基于肝硬化基础上的肝癌前病变动物模型[15]。正常组大鼠腹腔注射生理盐水,剂量为0.4 mL/100 g,其他3组大鼠以50 mg/kg剂量腹腔注射二乙基亚硝胺,每周1次,连续14周后成功制备模型。
1.4.2 给药
造模后第9周,抗纤抑癌方组和鳖甲软肝组大鼠开始药物灌胃,剂量分别相当于抗纤抑癌方、复方鳖甲软肝片临床剂量的7倍。每次用药体积均按1 mL/100 g的剂量给药,每天1次,连续给药,共6周。正常组和模型组大鼠灌胃对应量的蒸馏水,每天1次,连续给药,共6周。
1.4.3 标本采集
在实验的第14周末,停止给药24 h后,以0.33 mL/100 g的剂量给予10%水合氯醛腹腔注射麻醉,从腹主动脉采集大鼠血液。在距离最大叶肝脏约1 cm处,取得约1 cm×1 cm×0.3 cm的组织样本,随后浸泡于4%多聚甲醛溶液中固定。同时,迅速将部分肝组织存放于液氮中,以备进行实时荧光定量PCR和Western Blot分析。
1.4.4 免疫组化法检测大鼠肝组织中胎盘型谷胱甘肽转移酶(GST-Pi)表达
将切片置于二甲苯中浸泡脱蜡,浸入乙醇溶液中水化;置于抗原修复液中煮沸修复;滴加3% H2O2溶液以及一抗(稀释度1∶150),放入湿盒4 ℃过夜;加二抗以及DAB显色,显微镜下观察,苏木素复染,细胞核变蓝终止;按常规进行脱水、透明、封片。
1.4.5 实时荧光定量PCR法检测大鼠肝组织中GLUT1、PKM2、mTOR、HIF-1α和VEGF的mRNA表达
使用Trizol提取组织中总RNA,用二步法进行mRNA表达的检测;按试剂盒说明书进行反转录,合成cDNA,以cDNA为模板进行实时荧光定量PCR反应。反应条件:预变性95 ℃ 4 min、95 ℃ 10 s、60 ℃ 10 s、72 ℃ 20 s,39个循环。以GAPDH作为内参照,采用2-ΔΔCT法计算mRNA相对表达量。引物序列见表1。
表 1 实时荧光定量PCR引物序列Table 1. Real time fluorescence quantitative PCR primer sequence引物名称 引物序列(5'-3') 扩增产物长度(bp) Rat-mTOR F:TGTCAGCCTGTCAGAATCCA 74 R:CCATGTTGACCAGCATTTCA Rat-HIF-1α F:TGGAAGCACTAGACAAAGCTCA 78 R:TTGACCATATCGCTGTCCAC Rat-VEGF F:GAGTTAAACGAACGTACTTGCAGA 90 R:TCTAGTTCCCGAAACCCTGA Rat-PKM2 F:GGAGAAGTGCGATGAGAACAT 141 R:TCTGTCACCAGGTAGTCAGCAC Rat-GLUT1 F:GTATCCTGTTGCCCTTCTGC 95 R:TCGAAGCTTTTTCAGCACAC GAPDH F:TCATTGACCTCAACTACATGG 131 R:TCGCTCCTGGAAGATGGTG 1.4.6 Western Blot法检测大鼠肝组织GLUT1、PKM2、mTOR、HIF-1α和VEGF的蛋白表达
用蛋白裂解液于冰上裂解组织,按BCA蛋白浓度测定试剂盒测定蛋白浓度;将蛋白样品分装到离心管中,加上样缓冲液,煮沸5 min;制备12% SDS-PAGE分离胶和5%浓缩胶,上样,电泳4~5 h,转膜,用5%脱脂奶粉室温封闭,加入一抗4 ℃封闭过夜,孵育二抗1 h;ECL发光显影,用Image J软件对各条带的灰度值进行分析。
1.5 统计学方法
采用SPSS 25.0统计软件进行数据分析。计量资料多组间比较采用单因素方差分析或Kruskal-Wallis H秩和检验,进一步两两比较采用LSD-t检验。P<0.05为差异有统计学意义。
2. 结果
2.1 大鼠肝组织GST-Pi免疫组化及蛋白表达
2.1.1 GST-Pi免疫组化
GST-Pi阳性灶为胞浆中棕黄色不规则形团块。正常组未见明显阳性表达,模型组则见较多阳性灶,肝小叶内及汇管区周围均可见,染色深;抗纤抑癌及鳖甲软肝组的阳性灶较模型组减少,染色较浅(图1)。
2.1.2 GST-Pi蛋白表达
与正常组比较,大鼠肝组织GST-Pi蛋白在模型组的表达显著升高(P<0.01);与模型组比较,抗纤抑癌组GST-Pi蛋白的表达水平显著降低(P<0.05)(图2)。结果表明抗纤抑癌方的应用显著降低了大鼠GST-Pi的表达。
2.2 抗纤抑癌方对大鼠肝组织GLUT1和PKM2的影响
2.2.1 GLUT1和PKM2 mRNA表达
与正常组比较,模型组大鼠肝组织GLUT1及PKM2 mRNA的表达均显著升高(P值均<0.01);与模型组比较,鳖甲软肝组及抗纤抑癌组GLUT1 mRNA的表达均显著降低(P值均<0.05)(图3)。
2.2.2 GLUT1和PKM2蛋白表达
与正常组比较,模型组大鼠肝组织GLUT1及PKM2的蛋白表达均显著升高(P值均<0.01);与模型组比较,鳖甲软肝组和抗纤抑癌组GLUT1及PKM2的蛋白表达无统计学差异(P值均>0.05);鳖甲软肝组与抗纤抑癌组GLUT1和PKM2的蛋白表达无显著差异(P值均>0.05)(图4)。
2.3 抗纤抑癌方对大鼠肝组织mTOR、HIF-1α、VEGF的影响
2.3.1 mTOR、HIF-1α、VEGF mRNA的表达
与正常组比较,模型组大鼠肝组织mTOR、HIF-1α及VEGF的mRNA表达均显著升高(P值均<0.01);与模型组比较,鳖甲软肝组mTOR及VEGF的mRNA的表达均显著降低(P值均<0.05),抗纤抑癌组mTOR及VEGF mRNA的表达亦显著降低(P值均<0.01)。鳖甲软肝组与抗纤抑癌组mTOR、HIF-1α、VEGF的mRNA的表达无显著差异(P值均>0.05)(图5)。
2.3.2 mTOR、HIF-1α、VEGF蛋白的表达
与正常组比较,模型组大鼠肝组织mTOR、HIF-1α、VEGF的蛋白表达均显著升高(P值均<0.01);与模型组相比,鳖甲软肝组只有mTOR的蛋白表达显著降低(P<0.01),抗纤抑癌组mTOR、 HIF-1α、VEGF的蛋白表达均显著降低(P值均<0.05);与鳖甲软肝组相比,抗纤抑癌组mTOR的蛋白表达较高(P<0.01),HIF-1α、VEGF的蛋白表达无明显差异(图6)。
3. 讨论
中医药在肝癌前病变的防治中发挥积极作用。临床研究[16-18]表明,中药单体及其组分发挥抗炎和抗氧化、调节免疫、抑制肿瘤血管生成、抑制细胞增殖等作用。中药复方通过抑制上皮间质转化、抑制血管生成,抑制细胞增殖、调节自噬、诱导细胞凋亡、阻滞细胞周期和调节免疫功能等作用有效预防肝细胞癌变[16]。课题组前期研究[13-14,19]表明抗纤抑癌方可抑制肝细胞异常增生。
研究[20-22]表明,靶向mTOR/HIF-1α/VEGF是治疗横纹肌肉瘤、卵巢透明细胞腺癌和乳腺癌的有效策略。在肝癌方面,索拉非尼通过抑制mTOR相关信号通路,进而抑制HIF-1α的转录和蛋白表达,下调VEGF的表达[15]。本研究评估了抗纤抑癌方对mTOR/HIF-1α/VEGF途径的影响,实时荧光定量PCR及Western Blot均证实抗纤抑癌方可抑制mTOR、HIF-1α和VEGF的表达。
mTOR/HIF-1α/VEGF通路在肝癌前病变的血管生成中发挥关键作用。肝癌前病变大鼠肝组织中mTOR的高表达与HIF-1α和VEGF上调提示该信号通路的活化。这一结果强调了mTOR/HIF-1α/VEGF通路在肝癌前病变血管生成中的潜在作用,为相关治疗策略的制订提供了新的见解。
此外,本研究观察到抗纤抑癌方可明显降低PKM2和GLUT-1及其上游mTOR/HIF-1α的蛋白表达水平,提示在缺氧环境的刺激下,mTOR/HIF-1α信号通路异常活化,上调糖酵解相关的基因,促进PKM2、GLUT-1的表达。本研究表明糖酵解是大鼠肝癌前病变缺氧微环境的代谢特征,参与肝癌前病变的进展,因此抑制糖酵解,改善局部微环境,可阻断具有恶变潜能的癌前病变组织。
本研究的局限性:首先,抗纤抑癌方的主要生物活性成分有待进一步研究确定。其次,仅在体内实验对抗纤抑癌方治疗肝癌前病变的作用机制进行了探讨,尚未进行细胞实验对其机制进行进一步评估验证。
综上所述,通过探讨抗纤抑癌方在肝癌前病变中的作用,揭示了其对mTOR/HIF-1α/VEGF信号通路的抑制效应,进一步确认了其在阻止肝癌前病变进展方面的潜在作用。为深入研究提供了有力支持,同时也为开发更为精准的肝癌前病变干预策略奠定了基础。有望通过深化对抗纤抑癌方机制的解析,推动更具前瞻性的治疗策略的发展,为肝癌前病变的有效干预提供新的方向和可能性。
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表 1 实时荧光定量PCR引物序列
Table 1. Real time fluorescence quantitative PCR primer sequence
引物名称 引物序列(5'-3') 扩增产物长度(bp) Rat-mTOR F:TGTCAGCCTGTCAGAATCCA 74 R:CCATGTTGACCAGCATTTCA Rat-HIF-1α F:TGGAAGCACTAGACAAAGCTCA 78 R:TTGACCATATCGCTGTCCAC Rat-VEGF F:GAGTTAAACGAACGTACTTGCAGA 90 R:TCTAGTTCCCGAAACCCTGA Rat-PKM2 F:GGAGAAGTGCGATGAGAACAT 141 R:TCTGTCACCAGGTAGTCAGCAC Rat-GLUT1 F:GTATCCTGTTGCCCTTCTGC 95 R:TCGAAGCTTTTTCAGCACAC GAPDH F:TCATTGACCTCAACTACATGG 131 R:TCGCTCCTGGAAGATGGTG -
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