【作者】 余畅；

【导师】 闵苏；

【作者基本信息】 重庆医科大学 ， 麻醉学， 2023， 博士

【摘要】 背景营养支持是加速术后康复(Enhanced Recovery After Surgery,ERAS)理念的基本要求。以术前口服碳水化合物溶液(Preoperative Oral Carbohydrate,POC)为例,其可为择期手术患者提供能量,且不增加麻醉诱导期间反流误吸风险。碳水化合物,也称糖类,是生命活动的主要供能物质。然而,进食糖类引起的血糖波动可能增加糖尿病、癌症和高龄等糖代谢紊乱高风险患者手术相关并发症的发生率,但长时间的空腹可能导致患者低血糖,重则休克、甚至出现不可逆的神经系统损伤。因此,重视并优化糖类在围手术期的运用,在满足供能的同时减少应激反应,是ERAS实施的重要策略。目前糖类在临床实践中的应用并不规范,种类、浓度等还不统一。葡萄糖、果糖和麦芽糊精的单独使用,以及采用前述3种成分按比例配制的混合制剂,均有报道,饮用不同糖溶液分别对血糖有何影响,尚不清楚。另一方面,糖类也为肿瘤细胞供能。正常生理状态下,细胞产生ATP的主要方式为有氧氧化。肿瘤细胞依赖ATP产生速度快但效率低的糖酵解途径以满足自身快速增殖,该现象被称为Warburg效应,为肿瘤发生发展的重要驱动因素。不同糖的代谢途径存在差异,其分别对肿瘤的生长有何影响,还缺乏研究。研究表明,高糖饮食可能损害胰岛素敏感性,且具有促炎潜质,故进一步诱导肿瘤细胞的恶性进展。“限糖”可能是有效的抑癌方式,但供能不足可加剧患者的营养不良状态,进一步削弱其对治疗的耐受力。因此,如何在满足“供能“的同时“抑癌”,成了一大难题。维生素C(Vitamin C,VC)安全无毒,为人体必需营养素之一。因摄入不足,伴机体消耗增加,肿瘤患者多存在VC减少甚至缺乏的现象,经外源性补充VC有利于缓解躯体不适。近年来,高浓度VC被报道具有“选择性”抑制肿瘤的作用,大量临床试验结果也表明,其可作为抑癌辅助治疗因子,为肿瘤的治疗增效,且无明显不良反应。VC抑制肿瘤的能力与Warburg效应间有何联系,其对糖类促瘤生长作用的影响有何特点,还不清楚。糖类作为最主要的能量载体,以满足患者能量需求为前提,联合VC能否产生辅助“抑癌式供能”的作用,成为本研究关注的重点。目的明确临床实践中常见糖类对血糖水平的影响,研究优化的肿瘤患者的用糖方案,探索VC在营养补充中的潜在价值。本研究将为糖类在围手术期的合理选用及营养支持提供新思路。研究方法第一部分不同糖对C57BL/6N小鼠血糖及多种肿瘤生长的影响1.1不同糖对C57BL/6N小鼠血糖波动的影响选用6-8周龄雌性C57BL/6N小鼠,研究不同糖溶液灌胃对血糖水平的影响。含糖溶液浓度均设定为10%,混合糖配置比例参考商用口服碳水化合物饮品。经鼠尾静脉采血,记录各组小鼠0 h、0.25 h、0.5 h、1 h、2 h及24 h的血糖水平。1.2不同糖对多种肿瘤生长的影响本小节实验纳入结肠癌HCT116,肺癌A549,胃癌MGC-803,肝癌Hep-3B,乳腺癌MDA-MB-231及卵巢癌SKOV3,共6种常用的不同器官来源的人源性肿瘤细胞系,分别经离体实验和BALB/c裸鼠荷瘤模型进行研究。在离体层面,配制含不同糖的培养基,含糖浓度设定为0.2%,通过Cell-Counting-Kit 8(CCK-8)法和划痕实验,研究不同糖对各肿瘤细胞系增殖和迁移能力的影响。选用结肠癌细胞系HCT116,经克隆形成实验和Transwell实验,研究集落形成及侵袭能力的改变;检测ATP和乳酸含量,经Western Blot检测葡萄糖转运蛋白-1(Glucose transporter 1,GLUT1)和M2型丙酮酸激酶(Pyruvate kinase-M2 type,PKM2)、AMPK-mTOR信号通路相关蛋白的表达,以研究不同糖对Warburg效应的影响。选用4-6周龄雌性BALB/c裸鼠,建立前述6种人源性肿瘤细胞系的皮下荷瘤模型,研究饮用不同糖溶液对肿瘤生长的影响。含糖溶液浓度设定为10%。取同期饲养的健康BALB/c裸鼠为C组,作空白对照。连续干预21天,记录体重、肿瘤体积变化。干预期结束后,测空腹血糖(Fasting Blood Glucose,FBG),牺牲裸鼠取血清、肿瘤、肝、肠备用。取结肠癌HCT116建立的肿瘤组织,H/E染色和Ki67染色比较各组形态学特征和细胞增殖活性;Western Blot比较各组肿瘤组织Warburg效应相关蛋白表达情况;酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)检测各组裸鼠血清空腹胰岛素水平(Fasting insulin level,FINS),并计算胰岛素抵抗指数(Homeostatic Model Assessment for Insulin Resistance,HOMA-IR)及胰岛素敏感指数(Homeostasis Model Assessment for Insulin Sensitivity Index,HOMA-ISI),探索性的比较胰岛素敏感性的改变;ELISA检测裸鼠血清及肿瘤组织中促炎细胞因子肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)、白细胞介素-6(Interleukin 6,IL-6)和抑炎细胞因子白细胞介素-10(Interleukin 10,IL-10)水平,比较炎症水平的变化。第二部分VC对肿瘤生长及Warburg效应的调控作用本部分离体实验纳入:肿瘤细胞系(结肠癌HCT116、肺癌A549和胃癌AGS),以及对应的正常细胞系(肠道上皮FHC,肺上皮BEAS-2B和胃黏膜GES-1)。2.1 肿瘤细胞与正常细胞间糖代谢特征的比较Western Blot比较前述细胞系糖酵解相关蛋白GLUT1和PKM2表达量。进一步采用含0.2%葡萄糖的细胞培养基干预,动态检测各细胞系葡萄糖消耗量,葡萄糖含量,乳酸及ATP含量,并绘制变化曲线,明确肿瘤细胞的代谢特点。2.2 VC对肿瘤生长及对Warburg效应的调控在离体层面,MTS法、EdU细胞增殖检测试剂盒研究不同浓度VC对细胞活力、增殖能力的影响,克隆形成实验研究VC对各肿瘤细胞系集落形成能力的调控;检测各细胞系ATP和乳酸含量变化,Western Blot检测VC干预对各细胞系Warburg效应相关蛋白含量的影响。选用4-6周龄BALB/c雌性裸鼠,建立结肠癌HCT116裸鼠荷瘤模型,研究不同途径给予VC(灌胃i.g.vs腹腔注射i.p.)对肿瘤生长的影响。VC干预均为4g/kg,200μL/10g,1天2次,连续干预21天。记录体重、肿瘤体积变化。干预期结束,测FBG,牺牲裸鼠取血清、肿瘤、肝、肠备用。肿瘤组织H/E染色和Ki67染色比较各组形态学特征和细胞增殖活性;Western Blot比较各组肿瘤组织Warburg效应相关蛋白表达;ELISA探索胰岛素敏感性和炎症水平的变化。第三部分 VC对糖类促瘤作用的影响及机制本部分离体实验所纳入细胞系与第二部分一致。模拟“营养补充”状态,研究VC在高糖环境对肿瘤细胞生长的影响。在离体层面,经MTS法检测细胞活力,EdU试剂盒分析增殖能力,克隆形成实验比较各肿瘤细胞系集落形成能力;检测各组葡萄糖消耗量、葡萄糖含量,乳酸及ATP含量变化;Western Blot检测各肿瘤细胞系Warburg效应相关蛋白的表达情况;取4-6周龄雌性BALB/c裸鼠,研究联合i.p VC对饮用10%葡萄糖或10%混合糖溶液荷瘤裸鼠的影响。连续干预21天,记录裸鼠体重、肿瘤体积。干预期结束后,测FBG,牺牲裸鼠取空腹血清、肿瘤、肝、肠备用。肿瘤组织H/E染色和Ki67染色比较各组形态学特征和细胞增殖活性;Western Blot比较各组肿瘤组织Warburg效应相关蛋白表达情况,并研究裸鼠胰岛素敏感性和炎症水平的改变。结果第一部分不同糖对C57BL/6N小鼠血糖及多种肿瘤生长的影响1.1不同糖对C57BL/6N小鼠血糖水平的影响蒸馏水灌胃对小鼠的血糖水平几乎无影响。含糖溶液灌胃造成一过性血糖波动,变化趋势为:血糖浓度均在灌胃后上升,于0.25 h达到峰值,约2 h回归正常血糖水平。其中,以葡萄糖和麦芽糊精所致的血糖水平波动最为显著;果糖灌胃在各时点与蒸馏水无显著差异;果糖与混合糖在各时点均无统计学差异。1.2不同糖对多种肿瘤生长的影响:不同糖均显著促进了各肿瘤细胞增殖和迁移,而不同肿瘤细胞系对不同糖的亲和性存在差异,表现为增殖活力和迁移能力不同程度上调,其中以葡萄糖作用最显著。在结肠癌细胞系HCT116,不同糖干预显著上调了集落形成能力及迁移能力;ATP及乳酸含量在各含糖组均显著增加,以葡萄糖最显著,果糖组含量增幅最小;Western Blot结果显示,除果糖外,葡萄糖、麦芽糊精和混合糖均显著上调了GLUT1表达量;不同糖均上调PKM2蛋白表达量,p-AMPK/AMPK比值降低,p-mTOR/mTOR比值上调,葡萄糖与果糖组间差异有统计学意义,即不同糖的促瘤生长作用与Warburg效应激活有关,在结肠癌HCT116细胞,葡萄糖对Warburg效应的激活显著强于果糖。在BALB/c裸鼠荷瘤模型,饮用不同糖溶液均促进肿瘤的生长,表现为肿瘤生长更快,体积更大。结肠癌HCT116肿瘤切片染色显示,各含糖溶液组肿瘤细胞排列紧密,病理性核分裂象显著增多,Ki67阳性率增加,其中以葡萄糖和麦芽糊精最为显著;肿瘤组织Western Blot结果显示,除果糖外,葡萄糖、麦芽糊精和混合糖均显著上调了GLUT1表达量;不同糖均上调PKM2蛋白表达量,pAMPK/AMPK比值降低,p-mTOR/mTOR比值上调,葡萄糖与果糖组间差异有统计学意义。荷瘤状态降低了裸鼠胰岛素敏感性,而连续饮用21天含糖溶液致荷瘤裸鼠胰岛素敏感性进一步降低,其中以葡萄糖组受损最显著。荷瘤状态上调了裸鼠炎症水平,饮用含糖溶液致炎症水平进一步增加,以葡萄糖溶液促炎作用最强,麦芽糊精次之,果糖较弱。提示对于肿瘤患者,应尽可能减少葡萄糖的直接摄入。第二部分VC对肿瘤生长及Warburg效应的调控作用2.1 肿瘤细胞与正常细胞间的糖代谢特征肿瘤细胞系GLUT1和PKM2表达量显著增加,葡萄糖消耗量、乳酸及ATP含量显著多于正常细胞系;在含糖培养基干预后,各细胞系葡萄糖含量、乳酸及ATP生成量均升高,于1 h时达峰值,后逐步回降至基线水平,前述指标变化幅度在各肿瘤细胞系显著大于正常细胞系,表明其糖代谢活性更强,对外源性葡萄糖刺激更敏感。2.2 VC对肿瘤生长及Warburg效应的影响VC显著抑制了各肿瘤细胞系细胞活力、增殖能力和集落形成能力,ATP和乳酸含量显著降低,Warburg效应相关蛋白在肿瘤细胞的表达受到下调,且呈浓度依赖性。在一定浓度范围内,VC对正常细胞无显著影响。在结肠癌细胞系HCT116 BALB/c裸鼠荷瘤模型,i.g.或i.p.VC显著延缓肿瘤生长,以i.p.抑瘤作用更强。肿瘤切片H/E染色显示,VC干预组肿瘤细胞核深染细胞数减少,排列稀疏无序,出现空泡和部分坏死,坏死区域见炎性细胞浸润,且Ki67阳性率显著降低。肿瘤组织Western Blot结果显示,i.g.或i.p.VC下调Warburg效应相关蛋白的表达,以i.p.VC作用更显著。此外,i.g.和i.p.VC对健康裸鼠的胰岛素敏感性和炎症水平无显著影响,但显著抑制了荷瘤状态所致的胰岛素敏感性受损和炎症水平升高。第三部分VC对糖类促瘤作用的影响及机制高糖干预显著上调了肿瘤细胞的细胞活力、增殖能力及集落形成能力,各肿瘤细胞葡萄糖消耗量、葡萄糖含量、乳酸及ATP含量显著增加,且Warburg效应相关蛋白表达得到进一步激活,而该作用被VC有效拮抗,前述干预对正常细胞无显著影响。在结肠癌HCT116细胞系建立的BALB/c荷瘤裸鼠模型,与第一部分结果一致,饮用含糖溶液促进了肿瘤生长,肿瘤组织Ki67阳性率增加,上调了Warburg效应相关蛋白表达,葡萄糖促瘤作用强于混合糖,联合i.p.VC有效抑制前述改变,VC干预各组肿瘤细胞排列稀疏无序,出现空泡和坏死,坏死区域可见大量炎性细胞浸润,Ki67阳性率显著降低,肿瘤增殖活性受抑制。饮用含糖溶液加重了裸鼠胰岛素敏感性的受损,上调裸鼠炎症反应,联合i.p.VC有效抑制了前述改变。结论(1)饮用含糖溶液可造成血糖波动,波动幅度排序为:葡萄糖≥麦芽糊精>混合糖(葡萄糖+果糖+麦芽糊精)≥果糖。提示增加果糖在含糖溶液中的占比,或减少葡萄糖和麦芽糊精的使用,更有利于减少血糖异常风险,为患者在围手术期的血糖管理增益。(2)不同糖均促进肿瘤的增殖、迁移,糖类促瘤生长机制与Warburg效应激活有关,其中以葡萄糖作用最显著,表明肿瘤患者应尽量减少葡萄糖的直接使用。不同肿瘤细胞对同种糖的亲和性存在差异,提示未来对不同肿瘤患者个体在糖类的选用应更个性化、精细化。(3)与正常细胞相比,肿瘤细胞对微环境中葡萄糖的刺激更敏感,对葡萄糖的消耗及利用能力更强。在一定浓度范围内,VC对正常组织细胞无显著影响,但可发挥Warburg效应抑制剂的作用,限制肿瘤细胞对葡萄糖的摄取和利用,延缓肿瘤的生长。(4)VC对维持胰岛素敏感性及炎症水平的稳态具有保护作用。更多还原

【Abstract】 BackgroundCarbohydrate,or known as sugar,is crucial energy source.Preoperative oral carbohydrate(POC)solution is able to reduce the selective surgery patients’ discomfort,attenuate perioperative hemodynamic fluctuation and restrain postoperative insulin resistance,is thought to be an important part of Enhance Recovery After Surgery(ERAS).However,the blood glucose variation induced by sugar intake is not easy for blood glucose management on patients with cancer,diabetes and the old.However,the risk of hypoglycemia is not neglectable,which may lead to dizzy,week and even shock.Thus,it is worth to pay more attention on the utility of sugar on those who are in high risk of glucose abnormality.Due to the material restriction and insufficient cognition of medical staffs and patients,the utility of sugar in clinical work is uniformed.Glucose,fructose and maltodextrin were common seen carbohydrate,and the mixed sugar the most applied form.The concentration of sugary drinks is not uniformed,too.The effect of common-seen sugary drinks on blood glucose level is not known.On the other hand,sugar feeds not only normal cells but also cancer cells.The main route to generate ATP is by aerobic oxidation in physiological condition,but cancer cells tend to rely on glycolysis,a route which generate ATP inefficiently.The reprogrammed metabolism is called Warburg effect.The metabolic process is differed among sugar,but the effect of common-seen sugar on cancer progress is lack of investigation.To restrict the sugar usage may be an effective way on cancer management,but lack of energy may attenuate patients’ tolerance on treatment.Thus,how to meet the energy needs of body while reducing the impact on cancer progression is a topic worth exploring.Vitamin C(VC),a natural,safe and essential nutrient,is reported to selectively kill cancer cells.In addition,due to insufficient intake and increased consumption,VC deficiency tends to exist in most cancer patients.However,VC supplement was reported to enhance the body condition.At present,little research has been made on the relationship between VC and Warburg effect,so did its impact on sugar-associated oncogenic effect.As sugar the main and most crucial energy source,pay attention to the optimized usage of sugar on patients for the aim of reducing stress,to meet body’s energy demand with limiting cancer progression,is a matter of concern.ObjectThe objective of this study is to illustrate the impact of common-used sugar on blood glucose level and explore an optimal sugar application protocol for cancer patients.This research will facilitate a theoretical foundation for optimizing blood glucose control and provide novel approaches for nutritional supplementation and scientific energy supply for cancer patients.MethodsSECTION 1 The impact of different sugar on glycemic spikes of C57BL/6N mice and cancer progression.1.1 Impact of sugary drinks on glycemic spike of C57BL/6N miceSPF graded 6-8-week-old female C57BL/6 N mice were used.Mice were treated with specific sugary solution(150μL/10 g per mouse)by oral gavage.The concentration of sugar solution was set as 10%.Blood glucose was recorded at 0 h,0.25 h,0.5 h,1 h,2 h and 24 h after administration.1.2 Impact of different sugar on cancer cell growth.Six kinds of human cancer cell lines were included for experiments in vitro and in vivo: colon cancer cell line HCT116,lung cancer cell line A549,hepatocellular cancer cell line Hep3 B,gastric cancer cell line MGC-803,breast cancer cell line MDA-MB-231 and ovarian cancer cell line SKOV3.In vitro exploration,sugar concentration was set as 0.2%.Cell counting kit-8(CCK-8)and wound-healing assay were used to assess proliferation and migration ability.Colonic formation and Transwell assay were applied on colon cancer cell HCT116 to study the change of clonogenicity and invasion capacity.ATP and lactate content were assessed.Western Blot was used to assess the expression of glycolytic key enzymes(GLTU1 and PKM2)and AMPK-mTOR related protein.SPF grade 4-6-week-old female BALB/c nude mice were used for xenograft model that inoculated by 6 kinds of cancer cells mentioned above.The concentration of sugary drink was set as 10%.The intervention lasted for 21 days.The weight and tumor volume were assessed.On the 21 st day,fasting blood glucose(FBG)was recorded.Then,mice were euthanized,the serum,tumor tissue,liver and small intestine were harvest.The tumor tissue of HCT116 was used for H/E staining and IHC(Ki67),and Warburg related protein expression in tumor were assessed by Western blot.Homeostasis model assessment for insulin resistance(HOMA-IR)and insulin sensitive index(HOMA-ISI)between groups was analyzed using FBG and fasting insulin level(FINS).Enzyme linked immunosorbent assay(ELISA)was used to for detect the level of inflammatory factors(TNF-α,IL-6 and IL-10).SECTION 2 The impact of VC on cancer growth and its modulation on Warburg effectThree kinds of human cancer cells lines(colorectal carcinoma cell HCT116,non-small cell lung carcinoma cell A549 and gastric adenocarcinoma cell AGS)and corresponding 3 kinds of normal cell lines(fetal colon epithelial cell FHC,bronchial epithelial cell BEAS-2B and gastric epithelial cell GES-1)were used for experiments in vitro.2.1 Comparison of glucose metabolic characteristics between cancer cell and normal cellsGLUT1 and PKM2 expression were analyzed by Western Blot.Cells were incubated in fresh culture medium containing 0.2% glucose.The glucose consumption,intra-cellular glucose content,lactate and ATP content of cells were detected at 0 h,1 h,3 h,6 h,12 h and 24 h.2.2 The impact of Vitamin C on cancer growth and its modulation on Warburg effectFor exploration in vitro,MTS assay was used to analyze the cell viability of cells treated with 0 m M、0.625 m M、1.25 m M、2.5 m M、5m M、10 m M or 20 m M VC.EdU assay was used to assess the impact of VC on proliferation ability between cancer cells and normal cells.Also,colonic formation capacity of cancer cells was studied after VC intervention.ATP and lactate content were assessed after cells been treated with VC,and the expression of Warburg effect related protein were analyzed using Western blot.SPF grade 4-6-week-old female BALB/c nude mice were used for colon cancer cell line HCT116 xenograft model.The effect of VC administration via different route(gavage,i.g.or intraperitoneal injection,i.p.)was studied.The mice weight and tumor volume were assessed.On the 21 st day,FBG was recorded.After been euthanized,the serum,tumor tissue,liver and small intestine were harvest and stored.Tumor slices were used for H/E staining and IHC(Ki67).Warburg effect related protein expression in tumor obtained from mice was assessed using Western Blot.Insulin sensitivity and inflammation level were studied using ELISA.SECTION 3 The impact of VC on sugar-induced oncogenic effectCells used in this section were the same to section 2.MTS assay was for cell viability.EdU cell proliferation assay was for proliferation capacity.Also,colonic formation capacity of cancer cells was studied after specific intervention.Glucose consumption,intra-cellular glucose content,ATP and lactate generation between groups were assessed,and the protein expression of Warburg effect in cancer cells were analyzed using Western Blot.SPF grade 4-6-week-old female BALB/c nude mice were used for colon cancer cell line HCT116 xenograft model,to study the cointervention of VC on mice that intaking 10% glucose solution or mixed solution.Weight and tumor volume were collected during the 21-day period.On the 21 st day,FBG was recorded.After been euthanized,the serum,tumor tissue,liver and small intestine were harvest and stored.Tumor slices were used for H/E staining and IHC(Ki67).Protein expression of Warburg effect in tumor was assessed using Western Blot.ELISA was used to study the change of insulin sensitivity and inflammation level.ResultsSECTION 1 The impact of different sugar on glycemic spike and cancer growth.1.1 The impact of different sugar on C57BL/6N mice glycemic spikeDistilled water induced negligible blood glucose fluctuation.The trend among sugary solution was similar: blood glucose levels increased promptly and reached the apex around 0.25 h after gavage,and backed to baseline about 2 h-post gavage,the fluctuation in glucose and maltodextrin group was most extraordinary.No difference was found between distilled water and fructose.Also,there was no difference between fructose and mixed sugar according to the results.1.2 The impact of different sugar on cancer cell growth.In vitro,different sugars significantly promoted the proliferation and migration of tumor cells,while the affinity of different tumor cell lines to sugars was discrepant,but glucose was the strongest oncogenic factor.In colon cancer cell line HCT116,different sugar interventions significantly upregulated colony formation and migration ability,and glucose was stronger than maltodextrin,fructose and mixed sugars,with fructose the least.In colon cancer cell line HCT116,clonogenicity and invasion ability were significantly increased by sugar treatment,with glucose the strongest oncogenic factor and fructose the least.The ATP and lactate content were significantly increased after been treated with different sugar,with glucose the most powerful one,while fructose the least.Except of fructose,the expression of GLUT1 was increased in sugary groups.PKM2 was significantly upregulated in all the sugary groups.As for AMPK-mTOR related protein,p-AMPK/AMPK in sugary groups was decreased and pmTOR/mTOR increased,and the difference between glucose and fructose was significant.Indicating that the oncogenic effect of different sugar is related to the intensification of Warburg effect,with glucose the strongest factor.In BALB/c nude mice xenograft model,intaking different sugar solutions promoted tumor growth,presenting as faster tumor growth and larger volume.Colon cancer HCT116 tumor slide staining showed that tumor cells in sugary solution group were closely arranged,pathological nuclear fission images increased significantly,and the positive rate of Ki67 increased,indicating an increase in tumor proliferative activity,among which glucose and maltodextrin were the most significant,the oncogenic effect of fructose and mixed sugars was weaker.Based on the bulks of HCT116,glucose,maltodextrin and mixed sugars significantly upregulated the expression of GLUT1 in addition to fructose.The expression of PKM2 protein was upregulated in all the sugary treated groups and pAMPK/AMPK ratio was reduced,with increased p-mTOR/mTOR ratio.The difference between glucose and fructose was statistically significant,but no significant difference was found between glucose maltodextrins.Results in vivo experiments also suggest that different sugar solutions promote tumor growth,which is related to the upregulation of the Warburg effect,with glucose and maltodextrin being the most powerful oncogenic factors.The tumor-bearing status reduced the insulin sensitivity of nude mice and further reduced via drinking sugar-containing solution,which was most significant in glucose group.The tumor-bearing state upregulated the level of inflammation in nude mice,and was further intensified via drinking sugary drinks,with glucose showed the most pro-inflammation effect,followed by maltodextrin,and fructose the weakest.SECTION 2 The impact of VC on cancer growth and its modulation on Warburg effect2.1 Comparison of glucose metabolic characteristics between cancer cell and normal cellsCompared with normal cells,the expression of GLUT1 and PKM2 in tumor cell lines was significantly increased,and the contents of glucose consumption,lactate and ATP were significantly higher than those of normal cell lines.After the intervention of medium containing 0.2%glucose,cells showed the trend: the glucose content,lactate and ATP content first increased,reached the apex at 1 h,and then gradually dropped back to the baseline level,and the content changes of the indexes were significantly greater in cancer cell lines.The above results showed that glucose metabolism gap between cancer cells and normal cells.2.2 The impact of Vitamin C on cancer growth and its modulation on Warburg effectIn vitro,VC significantly inhibited cancer cell viability,proliferation and colony formation ability in a concentration-dependent manner.Reduced the ATP and lactate contents of 3 kinds of cancer cell line,and significantly downregulated the expression of Warburg effect-related protein in cancer cells Within a certain concentration range,VC showed little adverse effect on normal cell activity.In vivo,i.g.or i.p.VC significantly delay tumor growth,and i.p.showed significantly stronger inhibition effect on cancer growth.H/E staining of tumor sections showed that the number of hyperchromatic cells in VC intervention group was reduced,the arrangement of cancer cells was sparse and disorderly,vacuoles and partial necrosis appeared,immune cell infiltration was seen in the necrotic area,and the positive rate of Ki67 was significantly reduced,indicating that the proliferation activity of cancer was inhibited.Western Blot results showed that i.g.or i.p.VC deregulated the Warburg effect of tumor,with i.p.VC the more effective.In addition,i.g.and i.p.VC had no significant effect on insulin sensitivity and inflammation in healthy nude mice,but inhibited impairment of insulin sensitivity and upregulation of inflammation level caused by tumor-bearing status.SECTION 3 The impact of VC on sugar-induced oncogenic effectIn vitro,the high-glucose intervention significantly upregulated the cancer cell viability,proliferation ability and colony formation ability,upregulated the glucose consumption,glucose content,lactic acid and ATP content of cancer cells,and the expression of Warburg effect-related proteins was further intensified,but the change was effectively inhibited by VC,and the intervention had negligible effect on normal cells.In BALB/c nude mice xenograft,intaking sugary drinks promoted tumor growth,the positive rate of Ki67 in tumor tissue was increased significantly,and oncogenic effect of glucose was stronger than that of mixed sugar.Combined use of VC effectively inhibited tumor growth,which presenting as slower tumor growth and smaller tumor volume.Sparse and disorderly arrangement of cancer cells in VC treated tumor,combining with vacuoles and necrosis,and numerous inflammatory cell infiltration in the necrotic area.The positive rate of Ki67 of tumor in VC treat tumor indicating that tumor proliferative activity was inhibited.Meanwhile,intaking sugary solution significantly intensified Warburg effect,while combination of i.p VC significantly inhibited the changes.What’s more,consistent with the results of the section 1,drinking sugarsweetened solution aggravated the impairment of insulin sensitivity and upregulated the inflammatory level of nude mice,while the combined with i.p.VC effectively inhibited the above changes.Suggesting that VC is beneficial in maintaining homeostasis of insulin sensitivity and inflammatory level.Conclusion(1)Sugary drinks intaking induce transient blood glucose fluctuation,sugar included can be ordered based on the variation of blood glucose fluctuation,as: glucose ≥ maltodextrin > mixed sugar(glucose +fructose + maltodextrin)≥ fructose.Thus,increased the proportion of fructose in solution,or reduce the content of glucose and maltodextrin may be benefit on blood glucose management.(2)Glucose,fructose,maltodextrin and mixed sugar promoted cancer progression,which were related to the upregulated Warburg effect.Glucose is the most powerful pro-oncogenic factor,which indicating that cancer patients should forbid the directly usage of glucose in diet.What’s more,the affinity to a certain kind of sugar was different among cancer cell lines included in this study,which suggest that the application of sugar on different cancer patients may be refined and personalized in the future.(3)Compared with normal cells,cancer cells have stronger glucose metabolism activity,which is manifested as more sensitive to glucose stimulation,and stronger ability of glucose consumption and utility,that is,the Warburg effect.In a certain concentration range,VC has no significant effect on normal cells,but may role as Warburg effect inhibitor,delay tumor growth,and effectively inhibit the sugar-induced oncogenic effect.(4)VC supplement has a protective effect on maintaining homeostasis of insulin sensitivity and inflammation level.更多还原