Homocysteine is a potent inhibitor of human tumor cell gelatinases

Extracellular matrix-degrading gelatinases are mainly involved in tumor invasion and metastasis. Previous experimental data from our group and others suggested that homocysteine could have a potential modulatory role on the proteolytic balance at the extracellular matrix. Therefore, we studied the effects of homocysteine on extracellular matrix-degrading proteases using model human tumor cell lines and a combination of in vitro fluorogenic assay and zymographic techniques. Homocysteine is shown to be the thiol compound with the most potent inhibitory activity on matrix metalloproteinase 9. Zymographies reveal that matrix metalloproteinase 2 is, at least, as sensitive to inhibition by homocysteine as matrix metalloproteinase 9 is. This study opens new ways to the potential pharmacological use of thiol compounds.     

Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses. Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects. The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation. Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli. The three-dimensional structure of MTHFS has not been solved. The cDNA coding for Mus musculus MTHFS was isolated and expressed in E. coli with a hexa-histidine tag. Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20. Mouse MTHFS has a molecular mass of 23 kDa and is 84% identical in amino acid sequence to the human enzyme. Activity assays confirmed the functionality of the recombinant protein, with K_m=5 μM for (6S)-5-formyltetrahydrofolate and K_m=769 μM for Mg–ATP. This is the first example of a mammalian form of MTHFS expressed in E. coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

Homocysteine inhibits the proliferation and invasive potential of HT-1080 human fibrosarcoma cells

The impairment of homocysteine metabolism has been related to several disorders and diseases. Recently, homocysteine has been shown to inhibit key steps of angiogenesis, including endothelial cell proliferation, invasion, and remodeling of the extracellular matrix. Since these are also key steps in tumor invasion and metastasis, it can be hypothesized that homocysteine can also interfere in these processes. Therefore, we studied the effects of homocysteine on tumor proliferation and invasion, as well as on urokinase, a key extracellular matrix-degrading protease, using a model human tumor cell line. This study demonstrates that, in fact, homocysteine inhibits HT-1080 proliferation and invasion, and is a potent inhibitor of tumor cell urokinase expression.     

早期颈内动脉支架置入对动脉粥样硬化脑梗患者血清Lp-PLA2和Hcy水平的影响

摘要 目的：探讨早期颈内动脉支架置入（carotid artery stenting,CAS）对动脉粥样硬化脑梗死患者血清脂蛋白相关磷脂酶2（lipoprotein-associated phospholipase 2,LP-PLA2）和同型半胱氨酸（Homocysteine,Hcy）水平的影响。方法：选择2018年8月到2021年4月在在西安交通大学第一附属医院诊治的颈动脉重度狭窄性脑梗死患者86例作为研究对象,根据治疗方法将患者分为早期CAS组与对照组各43例,对照组给予药物保守治疗,2周后脑梗死稳定后再给予手术,CAS组在对照组基础上治疗3~5 d后给予颈内动脉支架置入治疗,检测两组患者血清Lp-PLA2和Hcy水平变化情况。结果：CAS组治疗后7 d的总有效率为97.7 %,高于对照组的86.0 %（P<0.05）。CAS组治疗后7 d的高灌注综合征、脑出血、低血压、心动过缓等并发症发生率为4.7 %,对照组无出现高灌注综合征、脑出血、低血压、心动过缓等并发症,对比差异无统计学意义(P>0.05)。两组治疗后7 d的颈内动脉相对脑血流量与脑血容量高于治疗前(P<0.05),CAS组高于对照组(P<0.05)。两组治疗后7 d的血清Lp-PLA2、Hcy含量低于治疗前（P<0.05）,CAS组低于对照组（P<0.05）。结论：早期颈内动脉支架置入在动脉粥样硬化脑梗死患者的应用能抑制Lp-PLA2、Hcy的表达,改善患者的血流动力学变化,从而促进提高治疗效果,在临床上的应用具有很好的安全性。     

血清同型半胱氨酸、胰腺衍生因子、肥胖抑制素与妊娠期糖尿病患者血糖控制情况和妊娠结局的关系分析

摘要 目的：探讨血清同型半胱氨酸（Hcy）、胰腺衍生因子（PANDER）、肥胖抑制素（Obestatin）与妊娠期糖尿病（GDM）患者血糖控制情况和妊娠结局的关系。方法：选择2021年1月至2022年1月于我院就诊的286例GDM患者,根据分娩前糖化血红蛋白（HbA1c）水平分为血糖控制达标组（HbA1c＜7%,173例）和血糖控制不达标组（HbA1c≥7%,113例）,检测并比较两组患者入组时血清Hcy、PANDER、Obestatin、空腹血糖（FPG）、空腹胰岛素（FINS）、胰岛素抵抗（HOMA-IR）水平,采用Pearson相关分析Hcy、PANDER、Obestatin与FPG、FINS、HOMA-IR的相关性。根据妊娠结局分为妊娠结局不良组（90例）和妊娠结局良好组（196例）,采用多因素Logistic回归分析GDM患者妊娠结局的影响因素。结果：血糖控制不达标组血清Hcy、PANDER、FPG、FINS、HOMA-IR水平均高于血糖控制达标组,而Obestatin水平低于血糖控制达标组（P＜0.05）。血清Hcy、PANDER水平与FPG、FINS、HOMA-IR水平均呈正相关,Obestatin水平与FPG、FINS、HOMA-IR水平均呈负相关（P＜0.05）。妊娠结局不良组年龄、入组时体质量指数（BMI）、糖尿病家族史、血糖控制不达标比例以及血清Hcy、PANDER、FPG、FINS、HOMA-IR水平均高于妊娠结局良好组,Obestatin水平则低于妊娠结局良好组（P＜0.05）。血糖控制不达标、血清Hcy、PANDER水平是GDM患者妊娠结局不良的危险因素,而Obestatin水平是保护因素（P＜0.05）。结论：GDM血糖控制不达标患者血清Hcy、PANDER水平增高,Obestatin水平降低,且与胰岛素抵抗和妊娠结局不良有关。     

单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与糖脂代谢紊乱和炎症因子的相关性分析

摘要 目的：研究单纯性肥胖儿童血清同型半胱氨酸（Hcy）、内脂素（visfatin）、上皮型脂肪酸结合蛋白（E-FABP）水平与糖脂代谢紊乱和炎症因子的相关性。方法：将2019年4月～2020年10月于我院就诊的70例单纯性肥胖儿童纳入研究,记作肥胖组。另取同期于我院接受体检的健康儿童70例作为对照组。检测并比较两组血清Hcy、visfatin、E-FABP水平,糖脂代谢紊乱相关指标和炎症因子水平。以Pearson相关性分析明确单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与糖脂代谢紊乱和炎症因子的关系。结果：肥胖组血清Hcy、visfatin、E-FABP水平均高于对照组（均P＜0.05）。肥胖组空腹血糖（FPG）、空腹胰岛素（FINS）水平及胰岛素抵抗指数（HOMA-IR）均高于对照组（均P＜0.05）。肥胖组总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）水平均高于对照组,而高密度脂蛋白胆固醇（HDL-C）水平低于对照组（均P＜0.05）。肥胖组血清白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）及肿瘤坏死因子-α（TNF-α）水平均高于对照组（均P＜0.05）。经Pearson相关性分析可得：单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与FPG、FINS、HOMA-IR、TC、TG、LDL-C、IL-1β、IL-6、TNF-α水平均呈正相关,而与HDL-C水平呈负相关（均P＜0.05）。结论：单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平均异常升高,且与其糖脂代谢紊乱及炎症反应密切相关,值得临床重点关注。     

MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension in Indians

The goals of our present study were to measure plasma homocysteine levels and determine their association with methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms (C677T and A1298C) in essential hypertensive subjects. Plasma total homocysteine and folic acid levels were measured in essential hypertensive patients (n = 153) before and after oral supplementation with either 5 mg folic acid tablet/day or 5 mg placebo/day for 4 weeks and compared with age and sex matched normotensive controls (n = 133). MTHFR gene polymorphisms (C677T and A1298C) were studied by restriction fragment length polymorphism and correlated with plasma homocysteine levels. Homocysteine levels were significantly higher in hypertensive patients as compared to controls and showed a negative correlation with plasma folate levels. Folic acid supplementation (5 mg/day) for 4 weeks resulted in a significant decrease in plasma homocysteine concentrations in these patients. Patients carrying MTHFR 677T allele (OR = 1.90; 95%CI: 1.14–3.19) or MTHFR 1298C (OR = 2.6, 95%CI: 1.55–4.40) allele were at increased risk of hypertension. The frequency of co-occurrence of MTHFR 677 CT/1298 CC genotypes was significantly higher in the patients compared to controls (P < 0.05) and was associated with increased risk of hypertension (OR = 3.54, 95%CI: 0.37–4.30). Subjects with MTHFR 1298 CC genotype had significantly higher homocysteine levels compared to those with MTHFR 1298 AA genotype (P < 0.05). Our results indicate that MTHFR 677T and 1298C alleles and co-occurrence of MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension and MTHFR 1298 CC genotype is associated with higher homocysteine levels in our subjects.     

Effects of atorvastatin therapy on hypercholesterolemic rabbits with respect to oxidative stress, nitric oxide pathway and homocysteine

Hypercholesterolemia is characterized with changes in lipid profile, nitric oxide pathway and oxidative stress markers. This study is designed to evaluate the effects of hypercholesterolemic diet and atorvastatin therapy on oxidative stress, lipid peroxide and thiobarbituric acid reactive substances (TBARS), NO pathway markers, nitric oxide(NO) and asymmetric dimethylarginine (ADMA), homocysteine, and paraoxonase activity (PON1) in rabbits. Twenty rabbits fed with high-cholesterol diet for 8 weeks were randomly divided into 2 groups on the fourth week of the hypercholesterolemic diet. First group was fed with high-cholesterol diet alone, whereas the second group with the same cholesterol diet plus atorvastatin (0.3 mg/kg/day) for 4 weeks. High-cholesterol diet increased total cholesterol, low density lipoprotein (LDL-C), high density lipoprotein (HDL-C), ADMA, TBARS and lipid peroxide levels and reduced PON1 activity and NO levels in rabbits. Four weeks of atorvastatin therapy significantly increased HDL-C, PON1 activity and reduced LDL-C, TBARS and lipid peroxide concentrations. Atorvastatin therapy is beneficial in decreasing oxidative stress related with hypercholesterolemia, mainly affecting lipid profile and PON1 activity.     

同型半胱氨酸与H型高血压左室肥厚的相关性及马来酸依那普利叶酸片的干预效果

目的:探讨同型半胱氨酸(Hcy)与H型高血压左室肥厚的相关性及马来酸依那普利叶酸片的干预效果。方法:选取达州市中心医院于2015年8月-2017年7月收治的450例原发性高血压患者,根据血浆Hcy水平将患者分为Hcy正常组(n=134)和H型高血压组(n=316),比较Hcy正常组、H型高血压组患者超声心动图检测指标的差异,并对Hcy水平与左心室结构改变进行相关性分析。同时将316例H型高血压患者随机分为观察组(n=158)和对照组(n=158),其中对照组患者给予马来酸依那普利片治疗,观察组给予马来酸依那普利叶酸片治疗。两组均连续治疗24个月。分别于治疗前、治疗后6个月、12个月、24个月检测血压、血浆Hcy和左心室质量指数(LVMI)水平,观察脑卒中及药物不良反应发生情况。结果:H型高血压组左室收缩末内径(LVESD)、左室舒张末内径(LVEDD)、左室后壁厚度(LVPWT)、室间隔厚度(IVST)、左心室质量(LVM)、LVMI均较Hcy正常组增大(P0.01)。血浆Hcy与LVPWT、IVST、LVM及LVMI呈正相关(r=0.652、0.526、0.736、0.786,均P0.05);治疗后6个月、12个月、24个月,观察组与对照组的H型高血压患者收缩压(SBP)、舒张压(DBP)水平及观察组Hcy、LVMI均较治疗前降低(P0.05),且观察组SBP、DBP、Hcy及LVMI均低于同时间点的对照组(P0.05)。两组均未见严重药物不良反应发生,观察组脑卒中发生4例(2.53%)较对照组12例(7.59%)明显减少,差异有统计学意义(P0.05)。结论:血浆Hcy水平是影响原发性高血压患者左心室肥厚的危险因素;马来酸依那普利叶酸片干预H型高血压患者后可显著降低血压、血浆Hcy水平,改善患者左心室肥厚程度,降低脑卒中发生率。