硫化氢抑制丙酮醛诱导的人皮肤角质形成细胞损伤

目的 观察硫化氢(H2S)对丙酮醛(MGO)诱导的人皮肤角质形成细胞(HaCaT)损伤的影响.方法 HaCaT细胞分为MGO组、对照组和MGO+NSHD(H2S供体)组.MGO组用200、400、600 μmol/LMGO处理HaCaT细胞48 h造成细胞损伤;对照组给予等体积培养基;MGO+NSHD组应用400 μmol/LMGO处理,48 h前用50、100、200μmol/LNSHD预处理1h.通过CCK-8检测各组细胞活性.HaCaT细胞经100 μmol/LNSHD预处理1h,用20 μmol/L的H2S荧光探针WSP-1染色结合荧光照相术检测NSHD预处理1h组和对照组培养基和细胞内的H2S含量.将HaCaT细胞分为MGO组、MGO+NSHD组、NSHD组和对照组.MGO组仅用400 μmol/L MGO处理48 h;MGO+NSHD组在400 μmol/L MGO处理48 h前用100 μmol/L NSHD预处理1 h;NSHD组的细胞仅用100 μmol/L NSHD处理1h;对照组则给予等体积培养基.Hoechst 33258核染色结合荧光照相术检测细胞凋亡.Rh123染色结合荧光照相术检测线粒体膜电位(MMP).结果 HaCaT细胞经200、400和600 μmol/L MGO处理48 h,细胞活性依次为0.325±0.023、0.224±0.009和0.095±0.102,均低于对照组0.415±0.031(F=37.866,P＜0.05),其中400 μmol/LMGO组细胞活性约为对照组的1/2,选用此浓度的MGO作为有效损伤浓度.100 μmol/L NSHD处理1h可使培养基和细胞内的H2S含量较对照组均增加.在400μmol/L MGO处理48 h前,分别用50、100和200 μnol/L NSHD预处理1h,细胞活性依次为0.235±0.028、0.314±0.017、0.346±0.020,均高于单独400μmol/L MGO处理组0.224±0.009(F=61.209,P＜0.05).400 μmol/L MGO处理48 h后细胞凋亡率高于对照组和NSHD组[(32.6±3.5)％比(5.1±1.2)％、(3.4±0.8)％,均P＜0.05],MMP低于对照组和NSHD组(28.5±2.9比46.1±3.8、48.6±4.3,均P＜0.05).MGO+ NSHD组细胞凋亡率(18.3±2.6)％,低于MGO组但高于对照组和NSHD组(均P＜0.05),MMP为38.9±3.2,高于MGO组但低于对照组和NSHD组(均P＜0.05).结论 NSHD能通过释放H2S拮抗MGO诱导的皮肤细胞损伤.     

甲基乙二醛对人血管内皮细胞的毒性作用

目的 探讨甲基乙二醛（methylglyoxal,MGO)对人血管内皮细胞的毒性作用及其机制。方法 体外培养的人脐静脉血管内皮细胞与不同浓度MGO共同孵育后，通过形态学观察和原位末端标记（TUNEL）法检测细胞凋亡，并以荧光Annexin-V及碘化丙碇（PI）染色，经流式细胞仪定量检测凋亡细胞和死亡细胞，细胞内氧化水平以氧化敏感的荧光染料2，7-二氢二氯荧光素（DCFH）染色。用流式细胞仪测定。结果 MGO刺激后，内皮细胞形态和超微结构出现凋亡的特异性变化，并且TUNEL染色阳性，流式细胞仪测定显示凋亡细胞和死亡细胞数量与MGO呈时间和剂量依赖关系；同时，MGO刺激后细胞内氧化水平明显升高，其时效与量效关系与细胞凋亡和死亡的变化相似，抗氧化剂（N-乙酰半胱氨酸和丙丁酚）及羰基化合物清除剂（氨基胍）对MGO引起的细胞氧化应激和细胞毒性具有抑制作用。结论 MGO对人血管内皮细胞具有直接的毒性作用，其机制可能是诱导细胞氧化应激，这些结果提示，活性羰基化合物蓄积可能与慢性肾功能衰竭和糖尿病血管并发症的形成有关。     

Energy metabolism and ageing regulation: metabolically driven deamidation of triosephosphate isomerase may contribute to proteostatic dysfunction

Research carried out up to 3 decades ago by Gracy and co-workers revealed that the activity of the glycolytic enzyme triosephosphate isomerase (TPI), which converts dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), gradually declines whilst carrying out its catalytic function, primarily due to deamidation of certain asparagine residues. It is suggested here that excessive or continuous glycolysis increases TPI deamidation and thereby lowers TPI activity and causes accumulation of its substrate, DHAP, which in turn decomposes into methylglyoxal (MG), a well-recognised reactive bicarbonyl whose actions in cells and tissues, as well as at the whole organism level, mimic much age-relate dysfunction. The proposal helps to explain why suppression of glycolysis by caloric restriction, fasting and increased aerobic activity also suppresses generation of altered proteins which characterise the aged phenotype. It is proposed that these effects on TPI activity, though seemingly neglected in biogerontological contexts, reveal a mechanistic link between energy metabolism and age-related proteostatic dysfunction.     

Method for monitoring of the protein amino group changes during carbonylation

Objectives

Carbonylation of the protein amino, guanidino and thiol groups is one of the important causes of vascular complications in diabetes. We developed a simple spectrophotometric method for monitoring of the changes in the protein amino group contents during carbonylation.

Design and methods

The method is based on the reaction of amino group with p-benzoquinone in the slightly alkaline media. It was applied during carbonylation in vitro with methylglyoxal and in vivo in 13 patients with type 2 diabetes and 20 healthy persons.

Results

The method is simple, fast, precise (RSD in the range of 1.2–1.8%) and accurate (recovery about 100%). The content of amino groups in human serum albumin isolated from diabetics was significantly lower (p < 0.01) in comparison with a control group.

Conclusion

The method developed is suitable for quantification of protein amino groups during in vitro carbonylation as well as for clinical practice.     

Dietary soy isoflavones increase insulin secretion and prevent the development of diabetic cataracts in streptozotocin-induced diabetic rats

Soy isoflavone–containing diets have been reported to be beneficial in diabetes. This present study investigated the hypoglycemic effects of isoflavones in streptozotocin (STZ)-induced diabetes. Diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of 100 mg/kg STZ. Diabetic rats were then randomly divided into 3 groups and received a special diet supplemented with casein (control), low-isoflavone soy (LIS) protein, and high-isoflavone soy protein (HIS) for 8 weeks. Compared with the control or LIS groups, those rats on the HIS diet had significantly increased body weight and serum insulin levels and reduced serum glucose and methylglyoxal levels. Serum glutathione levels were also increased in rats given the HIS diet compared with those in the control or LIS (P < .01). Serum high-density lipoprotein cholesterol level was significantly higher in HIS-fed rats than that of the control or LIS rats (P < .05). More importantly, the death rate and incidence of cataracts in the diabetic rats were markedly decreased in the HIS group. In conclusion, ingestion of high-isoflavone soy protein not only lowers glucose levels but also reduces the incidence of cataracts in diabetic rats. The beneficial effects of soy isoflavones are attributed to increased insulin secretion, a better glycemic control, and antioxidant protection.     

New terpenic and phenolic compounds from Suaeda monoica reverse oxidative and apoptotic damages in human endothelial cells

Elevation in hyperglycemia-associated methylglyoxal level can trigger vascular endothelial cells oxidative stress and apoptosis. The present work assesses the cell proliferative, anti-oxidative and anti-apoptotic potential of Suaeda monoica derived four new terpenes: a norsesquaterpenol (normonisesquaterpenol), a monocyclic triterpenoid (suaedanortriterpene dione), an aromatic monoterpenic ester and a labdane-type norditerpenic xyloside as well as two new phenols: an alkylated β-naphthol and a β-methoxy naphthalene in cultured human umbilical vein endothelial cells (HUVEC). Of these, suaedanortriterpenedione (53.7%), normonisesquaterpenol (51.4%) and norditerpenic xyloside (48%) showed the most promising cell proliferative activities compared to others. Moreover, normonisesquaterpenol, norditerpenic xyloside and suaedanortriterpenedione efficiently reversed the oxidative and apoptotic cell damage via downregulation of capase-3/7 by 44.3%, 42.2% and 39.4%, respectively against dichlorofluorescin, whereas by 46.2%, 43.5% and 42.5%, respectively against methylglyoxal. Aminoguanidine, the reference drug inhibited caspase-3/7 activity by 56.2% and 54.7% through attenuation of dichlorofluorescin and methylglyoxal, respectively. Further in silico molecular docking analysis revealed formation of stable complexes between the tested compounds and caspase-3/7. Conclusively, we for the first time demonstrate the growth stimulatory, anti-oxidative and anti-apoptotic salutations of S. monoica derived novel compounds in human endothelial cells. This warrants their further assessment as vascular cell protective and rejuvenating therapeutics, especially in hyperglycemic conditions.     

Methylglyoxal (MGO) inhibits proliferation and induces cell death of human glioblastoma multiforme T98G and U87MG cells

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM using surgery, chemotherapy and/or radiotherapy are ineffective. Thus new therapeutic strategies to target GBM are urgently needed. The effect of methylglyoxal (MGO) on the cell cycle, cell death and proliferation of human GBM cells was investigated. The T98G and U87MG cell lines were cultured in modified EMEM supplemented with 10% fetal bovine serum and maintained at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were exposed to methylglyoxal (0.025 mM) per 72 h. The influence of MGO on T98G and U87MG cell cycle, proliferation and apoptosis was evaluated as well. Cell cycle phase distribution, proliferation, apoptosis were analyzed by flow cytometry. MGO causes changes in cell cycle and induces accumulation of G1/G0-phase cells and reduced fraction of cells in S and G2/M phases. We have also observed inhibition of cell proliferation and induction of apoptosis in cancer cells. We have also revealed that MGO induces senescence of U87MG but not T98G cells, but further studies are necessary in order to clarify and check mechanism of action of methylglyoxal and it Is a positive phenomenon for the treatment of GBM.     

The hyperglycemic byproduct methylglyoxal impairs anticoagulant activity through covalent adduction of antithrombin III

Introduction

The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition.

Methods

MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition.

Results and conclusions

MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.     

Strategy for identifying unknown hemoglobin adducts using adductome LC-MS/MS data: Identification of adducts corresponding to acrylic acid,glyoxal, methylglyoxal,and 1-octen-3-one

Electrophilic compounds have the ability to form adducts with nucleophilic sites in proteins and DNA in tissues, and thereby constitute risks for toxic effects. Adductomic approaches are developed for systematic screening of adducts to DNA and blood proteins, with the aim to detect unknown internal exposures to electrophiles. In a previous adductomic screening of adducts to N-terminals in hemoglobin, using LC-MS/MS, 19 unknown adducts were detected in addition to seven previously identified adducts. The present paper describes the identification of four of these unknown adducts, as well as the strategy used to identify them. Using LC-MS data from the screening, hypotheses about adduct identities were formulated: probable precursor electrophiles with matching molecular weights were suggested based on the molecular weights of the modifications and the retention times of the analytes, in combination with comparisons of theoretical Log P calculations and databases. Reference adducts were generated by incubation of blood samples with the hypothesized precursor electrophiles. The four identified precursor electrophiles, corresponding to the observed unknown adducts, were glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. Possible origins/exposure sources and toxicological information concerning the electrophilic precursors are discussed. The identified adducts could be explored as possible biomarkers for exposure.