Nicotinamide N-methyltransferase decreases 5-fluorouracil sensitivity in human esophageal squamous cell carcinoma through metabolic reprogramming and promoting the Warburg effect

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor with poor prognosis. And different individuals respond to the same drug differently. Increasing evidence has confirmed that metabolism reprogramming was involved in the drug sensitivity of tumor cells. However, the potential molecular mechanism of 5-fluorouracil (5-FU) sensitivity remains to be elucidated in ESCC cells. In this study, we found that the 5-FU sensitivity of TE1 cells was lower than that of EC1 and Eca109 cells. Gas chromatography-mass spectrometry analysis results showed that nicotinate and nicotinamide metabolism and tricarboxylic acid cycle were significantly different in these three cell lines. Nicotinamide N-methyltransferase (NNMT), a key enzyme of nicotinate and nicotinamide metabolism, was significantly higher expressed in TE1 cells than that in EC1 and Eca109 cells. Therefore, the function of NNMT on 5-FU sensitivity was analyzed in vitro and in vivo. NNMT downregulation significantly increased 5-FU sensitivity in TE1 cells. Meanwhile, the glucose consumption and lactate production were decreased, and the expression of glycolysis-related enzymes hexokinase 2, lactate dehydrogenase A, and phosphoglycerate mutase 1 were downregulated in NNMT knockdown TE1 cells. Besides, overexpression of NNMT in EC1 and Eca109 cells caused the opposite effects. Moreover, when glycolysis was inhibited by 2-deoxyglucose, the roles of NNMT on 5-FU sensitivity was weakened. In vivo experiments showed that NNMT knockdown significantly increased the sensitivity of xenografts to 5-FU and suppressed the Warburg effect. Overall, these results demonstrated that NNMT decreases 5-FU sensitivity in human ESCC cells through promoting the Warburg effect, suggesting that NNMT may contribute to predict the treatment effects of the clinical chemotherapy in ESCC.     

Adipokines in dental pulp: Physiological,pathological, and potential therapeutic roles

BackgroundHundreds of adipokines have been identified, and their extensive range of endocrine functions—regulating distant organs such as oral tissues—and local autocrine/paracrine roles have been studied. In dentistry, however, adipokines are poorly known proteins in the dental pulp; few of them have been studied despite their large number. This study reviews recent advances in the investigation of dental-pulp adipokines, with an emphasis on their roles in inflammatory processes and their potential therapeutic applications.HighlightsThe most recently identified adipokines in dental pulp include leptin, adiponectin, resistin, ghrelin, oncostatin, chemerin, and visfatin. They have numerous physiological and pathological functions in the pulp tissue: they are closely related to pulp inflammatory mechanisms and actively participate in cell differentiation, mineralization, angiogenesis, and immune-system modulation.ConclusionAdipokines have potential clinical applications in regenerative endodontics and as biomarkers or targets for the pharmacological management of inflammatory and degenerative processes in dental pulp. A promising direction for the development of new therapies may be the use of agonists/antagonists to modulate the expression of the most studied adipokines.     

Combined genotypes of ACE and NADPH oxidase p22phox associated with somatic mutation of mtDNA and carotid intima-media thickness in Japanese patients with type 2 diabetes mellitus

Background: We previously reported that the carotid artery intima-media thickness (IMT) increased with age and that patients with type 2 diabetes mellitus (DM) had a significantly larger IMT than did age-matched nondiabetic subjects with normal glucose tolerance. Although the exact mechanism behind the increase in IMT in diabetic patients has not been determined, data obtained from in vivo and in vitro studies suggest that hyperglycemia-induced oxidative stress may lead to atherogenesis.Objective: The aim of this single-center study was to determine whether long-term oxidative stress and the carotid IMT are influenced by differences of the angiotensin-converting enzyme insertion/deletion (ACE I/D) and NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase p22phox C242T genotypes.Methods: Eligible subjects were Japanese patients with type 2 DM. Polymorphism of the ACE I/D and p22phox gene was investigated using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively. The rate of an acquired mutation of mitochondrial DNA—that is, A-to-G substitution at position 3243 (mtDNA A3243G)—was determined by real-time PCR. As a marker of early atherosclerosis, the carotid artery IMT was measured using high-resolution B-mode ultrasonography.Results: A total of 262 Japanese patients (173 men, 89 women; mean [SEM] age, 58 [0.6] years [range, 18-80 years]) were recruited and enrolled for study. An ACED-positive (DD or DI) and p22phox 242T-negative genotype (CC) was associated with a significantly higher mtDNA A3243G mutation rate than the other 3 possible genotypes (0.0219% [0.0028%] vs 0.0097% [0.0012%]; P < 0.05). This genotype also had higher maximum and mean IMT values than the other genotypes (1.13 [0.048] mm vs 0.99 [0.03] mm and 1.01 [0.036] mm vs 0.92 [0.023] mm; P < 0.05). These parameters were similar among the other 3 genotypes.Conclusion: In this study, the ACED-positive and p22phox 242T-negative genotype showed higher rates of somatic mtDNA mutation (mtDNA A3243G) and higher carotid mean and maximum IMT levels.     

基于ND2基因序列的燕窝DNA条形码鉴别

目的:利用DNA条形码鉴定技术,快速、准确地鉴别不同种类及产地的燕窝,为该药材的质量评价提供参考。方法:提取不同产地及品种燕窝的DNA,对烟酰胺腺嘌呤二核苷酸(NADH)脱氧酶亚单位2(ND2)部分序列进行扩增和测序,利用软件MEGA6.0进行序列比对,分析变异位点,计算Kimura-2参数遗传距离,构建邻接(neighbor-joining,NJ)系统发育树。结果:NJ系统发育树显示31个官燕窝样品均与爪哇金丝燕Aerodramus fuciphagus聚为1支;1个毛燕窝样品与大金丝燕A.maximus聚为1支。但官燕窝中经细胞色素b(Cytb)鉴定生物基原为爪哇金丝燕亚种(淡腰金丝燕A.fuciphagus germani)的8个样品单独聚为1支,支持率82%,并在该段ND2序列表现为2个位点的差异,表明该段ND2序列可鉴别爪哇金丝燕的亚种。结论:ND2条形码可用于快速、准确地鉴别燕窝的生物基原。     

苯肾上腺素对大鼠成骨细胞放射损伤的保护作用

目的:探讨苯肾上腺素对放射损伤大鼠成骨细胞的细胞形态以及烟酰胺磷酸核糖转移酶(Nampt)mRNA表达水平的影响,阐明苯肾上腺素对其可能的保护作用机制。方法:组织块法培养大鼠颅骨成骨细胞,随机分为空白对照组、苯肾上腺素组、单纯照射组和苯肾上腺素+照射组。苯肾上腺素组在照射前0.5 h以100 mmol·L^-1苯肾上腺素孵育成骨细胞,照射组给予成骨细胞8 Gy X射线照射。照射后8、16、32和64 h在倒置显微镜下观察并分别收集细胞,提取细胞总RNA,应用RT-PCR法检测成骨细胞Nampt mRNA表达水平。结果:倒置显微镜下,与空白对照组比较,苯肾上腺素组成骨细胞形态、数量未见明显改变;单纯照射组细胞形态发生明显皱缩,尤以8 h明显,细胞数量在8、16、32和64 h时分别是对照组的0.82、0.37、0.24和0.21倍;苯肾上腺素+照射组在照射8和16 h后细胞皱缩,32 h后其形态恢复,在8、16、32和64 h后其数量分别为空白对照组的0.91、0.83、0.72和0.75倍,是单纯照射组的1.09、2.24、3.00和3.60倍。RT-PCR法检测,在照射8、16、32和64 h后,单纯照射组、苯肾上腺素+照射组Nampt mRNA表达水平较空白组明显减少(P<0.01),苯肾上腺素+照射组较单纯照射组明显增加(P<0.01)。结论:苯肾上腺素可减轻电离辐射导致的大鼠成骨细胞萎缩,并上调Nampt mRNA表达水平,其对成骨细胞放射损伤的保护作用可能与上调Nampt mRNA表达水平有关。     

虾青素对过氧化氢诱导胎盘滋养细胞HTR-8/SVneo的影响

目的观察虾青素通过烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶/活性氧(ROS)信号通路对过氧化氢(H2O2)诱导人胎盘滋养细胞HTR-8/SVneo的影响。方法实验分为空白组、模型组和实验组,每组8个复孔;实验组HTR-8/SVneo细胞预先以10 nmol·L^-1虾青素处理24 h,之后实验组和模型组细胞均以250μmol·L-1H2O2作用24 h;空白组未进行任何药物干预。以噻唑蓝(MTT)法检测各组HTR-8/SVneo细胞增殖情况,以DCF-DA荧光染色检测各组HTR-8/SVneo细胞内ROS水平,检测各组HTR-8/SVneo细胞培养上清中乳酸(LDH)及氧化应激指标含量,以蛋白质印迹法检测各组细胞NADPH氧化酶4(NOX4)、p22phox蛋白表达情况。结果干预后24 h,空白组、模型组及实验组HTR-8/SVneo细胞内ROS荧光强度值分别为2.76±0.43,34.15±2.34,15.61±1.85,LDH分别为(756.24±31.05),(1785.46±34.69),(1235.26±26.75)U·L^-1,超氧化物歧化酶(SOD)分别为(23.56±2.24),(10.04±2.02),(15.16±3.08)U·mg^-1,丙二醛(MDA)分别为(0.46±0.14),(0.96±0.21),(0.68±0.13)U·mg^-1,Nox4蛋白相对表达量分别为0.32±0.04,0.89±0.06,0.64±0.03,p22phox蛋白相对表达量分别为0.15±0.03,0.75±0.04,0.49±0.02,模型组分别与空白组和实验组比较,差异均有统计学意义(均P<0.05)。结论虾青素对H2O2诱导人胎盘滋养细胞HTR-8/SVneo氧化应激损伤具有保护作用,可能与干扰NADPH氧化酶/ROS信号通路活性有关。     

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice

Nicotinamide adenine dinucleotide (NAD⁺) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD⁺ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD⁺ results in impaired mitochondrial and cellular functions. Administration of NAD⁺ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD⁺ degradation and cellular death. In this study, we identified a novel link between NAD⁺ metabolism and mitochondrial dynamics. A single dose (62.5 mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD⁺ pools for up to 24 hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD⁺ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.     

还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶在脂多糖诱导的小鼠急性心肌损伤组织中的表达及意义

目的探讨还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的非吞噬细胞氧化酶(Nox)家族在脂多糖(LPS)诱导的小鼠急性心肌损伤组织中的表达及意义。方法选取8~10周龄无特定病原体C57/BL6雄性小鼠30只,随机分为对照组和LPS组,每组15只。LPS组小鼠通过腹腔注射大剂量LPS(10 mg/kg)诱导急性心肌损伤模型,对照组腹腔注射等量生理盐水,观察6 h后取材,逆转录-聚合酶链反应(RT-PCR)检测Nox、B细胞淋巴瘤/白血病蛋白-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天门冬氨酸特异性蛋白酶3(Caspase 3)基因表达,Western blotting检测Nox 2、Nox 4、Bax、Bcl-2和Caspase 3蛋白表达,免疫组织化学法检测4羟基壬烯醛(4-HNE)表达,末端脱氧核糖核酸转移酶(Td T)介导的脱氧尿苷三磷酸(dUTP)切口末端标记技术(TUNEL)法检测心肌组织细胞凋亡,比较两组结果差异。使用SPSS 13.0统计软件对数据进行分析,两组比较采用t检验。结果相比对照组,LPS组小鼠心肌Nox 2、Nox 4、Bax基因和蛋白以及Caspase 3蛋白表达水平明显升高,Bcl-2基因和蛋白表达水平明显降低,差异具有统计学意义(P0.05)。免疫组织化学检测结果表明相比对照组,LPS组心肌组织4-HNE表达明显增加,差异具有统计学意义(P0.05)。TUNEL检测结果表明相比对照组,LPS组心肌组织心肌凋亡细胞明显增加,差异具有统计学意义(P0.05)。结论 NADPH氧化酶通过调控氧化应激和细胞凋亡参与急性心肌损伤的发生发展。