Exogenous methionine contributes to reversing the resistance of Streptococcus suis to macrolides.

Streptococcus suis (S. suis) has been increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Multidrug-resistant Streptococcus suis is becoming increasingly prevalent, and novel strategies to treat bacterial infections caused by these organisms are desperately needed. In the present study, an untargeted metabolomics analysis showed that the significant decrease in methionine content and the methionine biosynthetic pathway were significantly affected by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis in drug-resistant S. suis. The addition of L-methionine restored the bactericidal activity of macrolides, doxycycline, and ciprofloxacin on S. suis in vivo and in vitro. Further studies showed that the exogenous addition of methionine affects methionine metabolism by reducing S-adenosylmethionine synthetase activity and the contents of S-adenosylmethionine, S-adenosyl homocysteine, and S-ribose homocysteine. Methionine can decrease the total methylation level and methylesterase activity in multidrug resistant S. suis. The drug transport proteins and efflux pump genes were significantly downregulated in S. suis by exogenous L-methionine. Moreover, the exogenous addition of methionine can reduce the survival of S. suis by affecting oxidative stress and metal starvation in bacteria. Thus, L-methionine may influence the development of resistance in S. suis through methyl metabolism and metal starvation. This study provides a new perspective on the mitigation of drug resistance in S. suis.IMPORTANCEBacterial antibiotic resistance has become a severe threat to human and animal health. Increasing the efficacy of existing antibiotics is a promising strategy against antibiotic resistance. Here, we report that L-methionine enhances the efficacy of macrolides, doxycycline, and ciprofloxacin antibiotics in killing Streptococcus suis, including multidrug-resistant pathogens. We investigated the mechanism of action of exogenous methionine supplementation in restoring macrolides in Streptococcus suis and the role of the methionine cycle pathway on methylation levels, efflux pump genes, oxidative stress, and metal starvation in Streptococcus suis. It provides a theoretical basis for the rational use of macrolides in clinical practice and also identifies a possible target for restoring drug resistance in Streptococcus suis.

Methionine sources at different dietary levels alters the growth and expression of genes related to homocysteine remethylation in the jejunum of broilers.

Sulfur amino acids are essential for the proper development of broilers and are required throughout the bird's life to perform important physiological functions. Studies that seek to understand the actions of sulfur amino acids in the body of birds are essential. The present study evaluated the influence of sulfur amino acid supplementation using DL-Methionine (DL-Met) and DL-Methionine hydroxy analogue (DL-HMTBA), on the performance and expression of genes related to methionine metabolism, in the jejunum of broilers. Four hundred and fifty male broilers (Cobb-700 slow feathering) were distributed in a completely randomized design, in a factorial scheme (2x3), with two sources of methionine (DL-Met and DL-HMTBA) and three levels of methionine (deficiency, requirement and excess). The mRNA expression of the MAT1, MTR, BHMT, MTRR, CBG and GSS genes, and performance data such as feed intake, weight gain, and feed conversion were evaluated. DL-HMTBA increased the expression of BHMT (p = 0.0072) and MTRR (p = 0.0003) in the jejunum of the birds. Methionine deficiency increased the expression of BHMT (p = 0.0805) and MTRR (p = 0.0018). Higher expression of GSS was observed in birds that were supplemented with DL-HMTBA (p = 0.0672). Analyzing our results, it is preferable to supplement sulfur amino acids with DL-Met at the requirement level. Birds fed with DL-HMTBA showed worse weight gain (p = 0.0117) and higher feed conversion (p = 0.0170); methionine deficiency resulted in higher feed intake (p = 0.0214), lower weight gain (p<0.0001) and consequently higher feed conversion (p<0.0001). Based on the information found in this work, it is recommended to supplement sulfur amino acids with DL-Met at the level of compliance with the requirement.

Hypermethylation of the CTRP9 promoter region promotes Hcy induced VSMC lipid deposition and foam cell formation via negatively regulating ER stress.

To provide a theoretical basis for the prevention and treatment of atherosclerosis (As), the current study aimed to investigate the mechanism underlying the effect of homocysteine (Hcy) on inducing the lipid deposition and foam cell formation of the vascular smooth muscle cell (VSMC) via C1q/Tumor necrosis factor-related protein9 (CTRP9) promoter region Hypermethylation negative regulating endoplasmic reticulum stress (ERs). Therefore, apolipoprotein E deficient (ApoE-/-) mice were randomly divided into the control [ApoE-/- + normal diet (NC)] and high methionine [ApoE-/- + (normal diet supplemented with 1.7% methionine (HMD)] groups (n = 6 mice/group). Following feeding for 15 weeks, the serum levels of Homocysteine (Hcy), total cholesterol (TC), and triglyceride (TG) were measured using an automatic biochemical analyzer. HE and oil red O staining were performed on the aorta roots to observe the pathological changes. Additionally, immunofluorescence staining was performed to detect the protein expression levels of CTRP9, glucose-regulated protein 78 kD (GRP78), phosphorylated protein kinase RNA-like ER kinase (p-PERK), activating transcription factor 6a (ATF6a), phosphorylated inositol-requiring enzyme-1α (p-IRE1α), sterol regulatory element binding proteins-1c (SREBP1c) and sterol regulatory element binding proteins-2 (SREBP2) in VSMC derived from murine aortic roots. In vitro, VSMC was stimulated with 100 µmol/l Hcy. After transfection of plasmids with overexpression and interference of CTRP9, ERs agonist (TM) and inhibitor (4-PBA) were given to stimulate VSMC cells. HE staining and oil red O staining were used to observe the effect of Hcy stimulation on lipid deposition in VSMC. Additionally, The mRNA and protein expression levels of CTRP9, GRP78, PERK, ATF6a, IRE1α, SREBP1c, and SREBP2 in VSMC were detected by RT-qPCR and western blot analysis, respectively. Finally, The methylation modification of the CTRP9 promoter region has been studied. The NCBI database was used to search the promoter region of the CTRP9 gene, and CpG Island was used to predict the methylation site. After Hcy stimulation of VSMC, overexpression of DNMT1, and intervention with 5-Azc, assess the methylation level of the CTRP9 promoter through bisulfite sequencing PCR (BSP). The results showed that the serum levels of Hcy, TC, and TG in the ApoE-/- + HMD group were significantly increased compared with the ApoE-/- + NC group. In addition, HE staining and oil red O staining showed obvious AS plaque formation in the vessel wall, and a large amount of fat deposition in VSMC, thus indicating that the hyperhomocysteinemia As an animal model was successfully established. Furthermore, CTRP9 were downregulated, while GRP78, p-PERK, ATF6a, p-IRE1α, SREBP1c, SREBP2 was upregulated in aortic VSMC in the ApoE-/- + HMD group. Consistent with the in vivo results, Hcy can inhibit the expression of CTRP9 in VSMC and induce ERs and lipid deposition in VSMC. Meanwhile, the increased expression of CTRP9 can reduce ERs and protect the lipid deposition in Hcy induced VSMC. Furthermore, ERs can promote Hcy induced VSMC lipid deposition, inhibition of ERs can reduce Hcy induced VSMC lipid deposition, and CTRP9 may play a protective role in Hcy induced VSMC lipid deposition and foam cell transformation through negative regulation of ERs. In addition, The CTRP9 promoter in the Hcy group showed hypermethylation. At the same time as Hcy intervention, overexpression of DNMT1 increases the methylation level of the CTRP9 promoter, while 5-Azc can reduce the methylation level of the CTRP9 promoter. Finally, Hcy can up-regulate the expression of DNMT1 and down-regulate the expression of CTRP9. After overexpression of DNMT1, the expression of CTRP9 is further decreased. After 5-Azc inhibition of DNMT1, the expression of DNMT1 decreases, while the expression of CTRP9 increases. It is suggested that the molecular mechanism of Hcy inhibiting the expression of CTRP9 is related to the hypermethylation of the CTRP9 promoter induced by Hcy and regulated by DNMT1. 5-Azc can inhibit the expression of DNMT1 and reverse the regulatory effect of DNMT1 on CTRP9. Overall, the results of the present study suggested that Hcy induces DNA hypermethylation in the CTRP9 promoter region by up-regulating DNMT1 expression, and negatively regulates ERs mediated VSMC lipid deposition and foam cell formation. CTRP9 may potentially be a therapeutic target in the treatment of hyperhomocysteinemia and As.

Shedding Light on Lysosomal Malondialdehyde Affecting Vitamin B12 Transport during Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) is often accompanied by upregulation of homocysteine (Hcy). Excessive Hcy damages cerebral vascular endothelial cells and neurons, inducing neurotoxicity and even neurodegeneration. Normally, supplementation of vitamin B12 is an ideal intervention to reduce Hcy. However, vitamin B12 therapy is clinically inefficacious for CIRI. Considering oxidative stress is closely related to CIRI, the lysosome is the pivotal site for vitamin B12 transport. Lysosomal oxidative stress might hinder the transport of vitamin B12. Whether lysosomal malondialdehyde (lysosomal MDA), as the authoritative biomarker of lysosomal oxidative stress, interferes with the transport of vitamin B12 has not been elucidated. This is ascribed to the absence of effective methods for real-time and in situ measurement of lysosomal MDA within living brains. Herein, a fluorescence imaging agent, Lyso-MCBH, was constructed to specifically monitor lysosomal MDA by entering the brain and targeting the lysosome. Erupting the lysosomal MDA level in living brains of mice under CIRI was first observed using Lyso-MCBH. Excessive lysosomal MDA was found to affect the efficacy of vitamin B12 by blocking the transport of vitamin B12 from the lysosome to the cytoplasm. More importantly, the expression and function of the vitamin B12 transporter LMBD1 were proved to be associated with excessive lysosomal MDA. Altogether, the revealing of the lysosomal MDA-LMBD1 axis provides a cogent interpretation of the inefficacy of vitamin B12 in CIRI, which could be a prospective therapeutic target.

Proximal tubular Bmal1 protects against chronic kidney injury and renal fibrosis by maintaining of cellular metabolic homeostasis.

Recent studies suggest that deletion of the core clock gene Bmal1 in the kidney has a significant influence on renal physiological functions. However, the role of renal Bmal1 in chronic kidney disease (CKD) remains poorly understood. Here by generating mice lacking Bmal1 in proximal tubule (Bmal1flox/flox-KAP-Cre+, ptKO) and inducing CKD with the adenine diet model, we found that lack of Bmal1 in proximal tubule did not alter renal water and electrolyte homeostasis. However, adenine-induced renal injury indexes, including blood urea nitrogen, serum creatinine, and proteinuria, were markedly augmented in the ptKO mice. The ptKO kidneys also developed aggravated tubulointerstitial fibrosis and epithelial-mesenchymal transformation. Mechanistically, RNAseq analysis revealed significant downregulation of the expression of genes related to energy and substance metabolism, in particular fatty acid oxidation and glutathione/homocysteine metabolism, in the ptKO kidneys. Consistently, the renal contents of ATP and glutathione were markedly reduced in the ptKO mice, suggesting the disruption of cellular metabolic homeostasis. Moreover, we demonstrated that Bmal1 can activate the transcription of cystathionine ß-synthase (CBS), a key enzyme for homocysteine metabolism and glutathione biosynthesis, through direct recruitment to the E-box motifs of its promoter. Supporting the in vivo findings, knockdown of Bmal1 in cultured proximal tubular cells inhibited CBS expression and amplified albumin-induced cell injury and fibrogenesis, while glutathione supplementation remarkably reversed these changes. Taken together, we concluded that deletion of Bmal1 in proximal tubule may aggravate chronic kidney injury and exacerbate renal fibrosis, the mechanism is related to suppressing CBS transcription and disturbing glutathione related metabolic homeostasis. These findings suggest a protective role of Bmal1 in chronic tubular injury and offer a novel target for treating CKD.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

Hyperhomocysteinemia Promotes Cardiac Hypertrophy in Hypertension.

Hyperhomocysteinemia (HHcy) is positively linked with several cardiovascular diseases; however, its role and underlying mechanisms in pathological cardiac hypertrophy are still unclear. Here, we focused on the effects and underlying mechanisms of HHcy in hypertensive cardiac hypertrophy, one of the most common and typical types of pathological cardiac hypertrophy. By a retrospective analysis of the association between HHcy and cardiac hypertrophy in a hypertensive cohort, we found that the prevalence of HHcy was higher in patients with hypertrophy and significantly associated with the presence of cardiac hypertrophy after adjusting for other conventional risk factors. In mice, HHcy induced by a methionine (2% wt/wt) diet feeding significantly promoted cardiac hypertrophy as well as cardiac inflammation and fibrosis induced by 3-week angiotensin ІІ (AngІІ) infusion (1000 ng/kg/min), while folic acid (0.006% wt/wt) supplement corrected HHcy and attenuated AngII-stimulated cardiac phenotypes. Mechanistic studies further showed that homocysteine (Hcy) exacerbated AngII-stimulated expression of Calcineurin and nuclear factor of activated T cells (NFAT), which could be attenuated by folic acid both in mice and in neonatal rat cardiomyocytes. Moreover, treatment with cyclosporin A, an inhibitor of Calcineurin, blocked Hcy-stimulated Calcineurin-NFAT signaling and hypertrophy in neonatal rat cardiomyocytes. In conclusion, our study indicates that HHcy promotes cardiac hypertrophy in hypertension, and Calcineurin-NFAT pathway might be involved in the pro-hypertrophic effect of Hcy.

The effects of vitamin B12 supplementation on metabolic profile of patients with non-alcoholic fatty liver disease: a randomized controlled trial.

The present study is the first effort to evaluate the effects of vitamin B12 supplementation on the serum level of liver enzymes, homocysteine, grade of hepatic steatosis, and metabolic profiles in patients with non-alcoholic fatty liver disease (NAFLD). Forty patients with NAFLD were enrolled in a double-blind placebo-controlled trial to receive either one oral tablet of vitamin B12 (1000 µg cyanocobalamin) or a placebo per day for 12 weeks. We investigated serum levels of homocysteine, aminotransferases, fasting blood glucose (FBG), lipids, malondialdehyde (MDA), and homeostasis model assessment of insulin resistance (HOMA-IR). The grade of liver steatosis and fibrosis was measured by real-time 2-dimensional shear wave elastography. Vitamin B12 supplementation significantly decreased serum levels of homocysteine compared to placebo (medians: - 2.1 vs. - 0.003 µmol/l; P = 0.038). Although serum alanine transaminase (ALT) in the vitamin B12 group decreased significantly, this change did not reach a significant level compared to the placebo group (medians: - 7.0 vs. 0.0 IU/l; P > 0.05). Despite the significant within-group decrease in FBG, MDA, and liver steatosis in the vitamin B12 group, between-group comparisons did not reveal any significant difference. Vitamin B12 supplementation might decrease serum levels of homocysteine in patients with NAFLD. The fasting blood glucose and serum levels of MDA were significantly improved in the trial group who received vitamin B12. However, these changes did not reach a significant level compared to the placebo group. In this respect, further studies with larger sample sizes, different doses, and types of vitamin B12 will reveal additional evidence.Trial Registration: At  http://irct.ir/  as IRCT20120718010333N5 on December 25, 2019.

Tetrahydroxystilbene glucoside attenuated homocysteine-upregulated endothelin receptors in vascular smooth muscle cells via the ERK1 /2 /NF-κB signaling pathway.

2,3,5,4'-Tetrahydrostilbene-2-o-ß-d-glucoside (TSG) is the main active component of Polygonum multiflorum Thunb. It has effects on hypertension. However, the mechanism is unclear. Current research is devoted to exploring the mechanism of TSG improving HHcy-induced hypertension. The mice received a subcutaneous injection of Hcy in the presence or absence of TSG for 4 weeks. Blood pressure (BP) was measured using a noninvasive tail-cuff plethysmography method. Levels of plasma Hcy and endothelin-1 were measured using ELISA. Rat SMA without endothelium was cultured in a serum-free medium in the presence or absence of TSG with or without Hcy. The contractile response to sarafotoxin 6c or endothein-1 was studied using a sensitive myography. The levels of protein were detected using Western blotting. The results showed that TSG lowered HHcy-elevated BP and decreased levels of plasma Hcy and endothelin-1 in mice. Furthermore, the results showed that TSG inhibited Hcy-upregulated ET receptor expression and ET receptor-mediated contractile responses as well as the levels of p-ERK1/2 and p-p65 in SMA. In vivo results further validate the in vitro results. In conclusion, TSG can decrease the levels of plasma Hcy and ET-1 and downregulate Hcy-upregulated ET receptors in VSMCs by inhibiting the ERK1/2 /NF-κB/ETB2 pathway to lower the BP.

Homocysteine-Induced Disturbances in DNA Methylation Contribute to Development of Stress-Associated Cognitive Decline in Rats.

Chronic stress is generally accepted as the main risk factor in the development of cognitive decline; however, the underlying mechanisms remain unclear. Previous data have demonstrated that the levels of homocysteine (Hcy) are significantly elevated in the plasma of stressed animals, which suggests that Hcy is associated with stress and cognitive decline. To test this hypothesis, we analyzed the cognitive function, plasma concentrations of Hcy, and brain-derived neurotropic factor (BDNF) levels in rats undergoing chronic unpredicted mild stress (CUMS). The results showed that decreased cognitive behavioral performance and decreased BDNF transcription and protein expression were correlated with hyperhomocysteinemia (HHcy) levels in stressed rats. Diet-induced HHcy mimicked the cognitive decline and BDNF downregulation in the same manner as CUMS, while Hcy reduction (by means of vitamin B complex supplements) alleviated the cognitive deficits and BDNF reduction in CUMS rats. Furthermore, we also found that both stress and HHcy disturbed the DNA methylation process in the brain and induced DNA hypermethylation in the BDNF promoter. In contrast, control of Hcy blocked BDNF promoter methylation and upregulated BDNF levels in the brain. These results imply the possibility of a causal role of Hcy in stress-induced cognitive decline. We also used ten-eleven translocation (TET1), an enzyme that induces DNA demethylation, to verify the involvement of Hcy and DNA methylation in the regulation of BDNF expression and the development of stress-related cognitive decline. The data showed that TET1-expressing viral injection into the hippocampus inhibited BDNF promoter methylation and significantly mitigated the cognitive decline in HHcy rats. Taken together, novel evidence from the present study suggests that Hcy is likely involved in chronic stress-induced BDNF reduction and related cognitive deficits. In addition, the negative side-effects of HHcy may be associated with Hcy-induced DNA hypermethylation in the BDNF promoter. The results also suggest the possibility of Hcy as a target for therapy and the potential value of vitamin B intake in preventing stress-induced cognitive decline.

Clinical Study of Clopidogrel Combined with Huoxue Tongluo Prescription in Improving Transient Ischemic Attack and the Effect on MMP-9, Hcy, and CRP.

Background: This study aimed to explore the clinical study of clopidogrel combined with Huoxue Tongluo prescription in improving transient ischemic attack (TIA) and the effect on MMP-9, Hcy, and CRP. Methods: A total of 84 patients with TIA admitted to our hospital from December 2019 to February 2021 were selected. The patients were divided into the control group (42 cases: not treated with Huoxue Tongluo prescription) and study group (42 cases: treatment with Huoxue Tongluo prescription). The clinical efficacy, adverse reactions, the levels of blood pressure and lipid, blood rheology and cerebral hemodynamics, neurological function-related indicators, MMP-9, Hcy, and CRP of the two groups were compared. Results: The total effective rate in the study group was higher than the control group. Compared with before treatment, the levels of SBP and DBP in both groups decreased memorably after treatment, and those in the study group decreased more particularly than the control group. The levels of LDL, HDL, TC, and TG in the study group were significantly better than those in the control group. The plasma viscosity, whole blood high shear viscosity, whole blood low shear viscosity, and hematocrit of patients in the study group were lower than those in the control group, and the maximum blood flow velocity, minimum blood flow velocity, average blood flow velocity, and average blood flow were higher than those in the control group. The levels of NSE, MBP, and S100ß in the study group were more memorably lower than those in the control group. After treatment, the levels of MMP-9, Hcy, and CRP in the study group were significantly lower than those in the control group. There was no obvious difference in the incidence of adverse reactions between the study group and control group. Conclusion: Clopidogrel combined with Huoxue Tongluo prescription can significantly improve the therapeutic effect and reduce the levels of MMP-9, Hcy, and CRP in patients with TIA.

Omega 3 fatty acids - Potential modulators for oxidative stress and inflammation in the management of sickle cell disease.

OBJECTIVE: Sickle cell disease is characterized by clinical complications resulting in vaso-occlusive crisis with prominent attributes of oxidative stress, inflammation, and pain. Inflammation is an integral part of this disease which further exacerbates the pain during a crisis. Omega-3 fatty acids are known to possess anti-inflammatory and anti-aggregatory properties and assist in diminishing the slow physiological inactivation. METHODS: A pilot nutritional interventional study was conducted wherein forty-three children with sickle cell disease aged 5-16 years were supplemented with omega-3 fatty acids for a period of six months. Analysis of oxidative stress, as well as inflammatory parameters, was done pre and post-supplementation. RESULTS: Increased free oxygen radical transference values depicting free radical generation is enhanced in these patients along with a reduced antioxidant defense, as seen by decreased free oxygen radical defense values. Supplementation with omega-3 fatty acids for a period of six months significantly reduced the inflammatory marker homocysteine in all patients, whereas high sensitive C reactive protein was significantly reduced only in females of the age group 11-16years. Simultaneously a significant reduction in oxidative stress parameters with a concomitant increase of antioxidant defense was observed in all patients. CONCLUSION: The authors' findings suggest the regulatory effects of omega-3 fatty acids as cellular activators in alleviating the complications due to sickle cell disease. Omega-3 fatty acids hold promise as future therapeutic candidates in patients with sickle cell disease.

Investigating the causality of metabolites involved in one-carbon metabolism with the risk and age at onset of Parkinson's disease: A two-sample mendelian randomization study.

With the aging population and increasing life expectancy, Parkinson's disease (PD), a neurological disorder rapidly increasing in morbidity and mortality, is causing a huge burden on society and the economy. Several studies have suggested that one-carbon metabolites, including homocysteine, vitamin B6, vitamin B12 and folate acid, are associated with PD risk. However, the results remain inconsistent and controversial. Thus, we performed a two-sample Mendelian randomization (MR) study to detect the causality between one-carbon metabolites and PD susceptibility as well as age at PD onset. We collected several genetic variants as instrumental variables from large genome-wide association studies of one-carbon metabolites (homocysteine: N = 14, vitamin B6: N = 1, vitamin B12: N = 10, folate acid: N = 2). We then conducted MR analyses using the inverse variance-weighted (IVW) approach and additional MR-Egger regression, weighted median and MR-pleiotropy residual sum and outlier (MR-PRESSO) methods to further test causality. The results showed no causal association between circulating homocysteine levels and PD risk (p = 0.868) or age at PD onset (p = 0.222) with the IVW method. Meanwhile, similar results were obtained by three complementary analyses. In addition, we did not observe any evidence that the circulating levels of vitamin B6, vitamin B12 and folate acid affected the risk of PD or age at onset of PD. Our findings implied that lowering homocysteine levels through vitamin B6, vitamin B12 or folate acid supplementation may not be clinically helpful in preventing PD or delaying the age at PD onset.

Effect of N, N-Dimethylglycine on Homocysteine Metabolism in Rats Fed Folate-Sufficient and Folate-Deficient Diets.

OBJECTIVE: This study aimed to investigate the effects of N,N-dimethylglycine (DMG) on the concentration and metabolism of plasma homocysteine (pHcy) in folate-sufficient and folate-deficient rats. METHODS: In this study, 0.1% DMG was supplemented in 20% casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system. RESULTS: Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 µmol/L to 28.49 ± 0.50 µmol/L; P < 0.05). When supplemented with DMG, pHcy concentration was significantly decreased (12.23 ± 0.18 µmol/L) in rats fed 20C diet but significantly increased (31.56 ± 0.59 µmol/L) in rats fed 20CFD. The hepatic methionine synthase activity in the 20CFD group was significantly lower than that in the 20C group; enzyme activity was unaffected by DMG supplementation regardless of folate sufficiency. The activity of hepatic cystathionine ß-synthase (CBS) in the 20CFD group was decreased but not in the 20C group; DMG supplementation enhanced hepatic CBS activity in both groups, in which the effect was significant in the 20C group but not in the other group. CONCLUSION: DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.

Changes in Biothiol Levels Are Closely Associated with Alzheimer's Disease.

BACKGROUND: Serum homocysteine (Hcy) level is considered to be an important biomarker for Alzheimer's disease (AD); however, the status of Hcy in brain tissue, and the association between brain and serum levels of Hcy in AD patients remain unclear. OBJECTIVE: We aimed to examine whether the changes of three thiols are consistent in serum of AD patients and the brain of APP/PS1 mice, and to verify the effectiveness of Hcy as a biomarker for early AD detection. METHODS: The levels of Hcy, cysteine (Cys), and glutathione (GSH) in Aß1-42-treated PC12 cells, the brain and hippocampus of APP/PS1 mouse, and the serum of AD patients were evaluated using ethyl (E)-3-(9-chloro-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f] pyrido [3,2,1 -ij] quinolin-10-yl)-2-cyanoacrylate (Probe 1) and ELISA assay or LC-MS. RESULTS: Measurement by Probe 1 revealed a significant increase in Hcy level, and a decrease in Cys and GSH levels in Aß1-42-treated PC12 cells and the serum of AD patients. The hippocampus and whole brain of APP/PS1 mice also showed a significant increase in Hcy level alongside the accumulation of age-related AD symptoms. The upregulation of Hcy and the downregulation of Cys and GSH were reversed in the Aß1-42-treated PC12 cells and the brain of APP/PS1 mice when supplemented with VB6. CONCLUSION: Changes in Hcy, Cys, and GSH levels in the brain of APP/PS1 mice and Aß1-42-treated PC12 cells were observed in situ with a new fluorescent probe, which were consistent with the abnormal changes in Hcy, Cys, and GSH levels in the serum of AD patients. VB6 supplementation was successful in ameliorating abnormal increases in Hcy levels.

A Novel Review of Homocysteine and Pregnancy Complications.

Homocysteine (Hct) is a substance produced in the metabolism of methionine. It is an essential type of amino acid gained from the daily diet. Methylenetetrahydrofolate reductase (MTHFR) gene mutation is related to elevated total homocysteine (tHct) expressions, in particular, among women with low folate intake. Hyperhomocysteinemia (HHct) is caused by numerous factors, such as genetic defects, lack of folic acid, vitamin B6 and B12 deficiency, hypothyroidism, drugs, aging, and renal dysfunction. Increased Hct in peripheral blood may lead to vascular illnesses, coronary artery dysfunction, atherosclerotic changes, and embolic diseases. Compared to nonpregnant women, the Hct level is lower in normal pregnancies. Recent studies have reported that HHct was associated with numerous pregnancy complications, including recurrent pregnancy loss (RPL), preeclampsia (PE), preterm delivery, placental abruption, fetal growth restriction (FGR), and gestational diabetes mellitus (GDM). Besides, it was discovered that neonatal birth weight and maternal Hct levels were negatively correlated. However, a number of these findings lack consistency. In this review, we summarized the metabolic process of Hct in the human body, the levels of Hct in different stages of normal pregnancy reported in previous studies, and the relationship between Hct and pregnancy complications. The work done is helpful for obstetricians to improve the likelihood of a positive outcome during pregnancy complications. Reducing the Hct level with a high dosage of folic acid supplements during the next pregnancy could be helpful for females who have suffered pregnancy complications due to HHct.

The Effect of Omega-3 Fatty Acids on Thromboxane, Brain-Derived Neurotrophic Factor, Homocysteine, and Vitamin D in Depressive Children and Adolescents: Randomized Controlled Trial.

In the DEPOXIN project, we have found that a high ratio of omega-6/omega-3 fatty acids (FA) is associated with worsening of depressive symptoms in children and adolescents with depressive disorder (DD) and that the 12-week omega-3 FA supplementation modulates DD symptoms. Here we present our results of the secondary outcomes: the levels of thromboxane (TXB), brain-derived neurotrophic factor (BDNF), homocysteine (HCy) and vitamin D. Fifty-eight patients were randomized into two arms. One group received a fish oil emulsion enriched with omega-3 FA, and the other received a sunflower oil emulsion containing omega-6 FA, for 12 weeks. Depressive symptoms were evaluated, using the Child's Depressive Inventory (CDI). The patients with DD had elevated TXB levels and decreased vitamin D levels, as compared to healthy controls. Both CDI and omega-6/omega-3 ratio correlated positively with TXB and negatively with BDNF at baseline. Compared to the omega-6 FA group, the supplementation with omega-3 FA for 12 weeks significantly reduced plasma TXB (p = 0.024) and increased BDNF (p = 0.011) levels. No changes in HCy and vitamin D were observed. Our results demonstrate the possible role of TXB and BDNF in the pathophysiology of DD and the benefits of omega-3 FA supplementation. The study was registered with the ISRCTN registry (ISRCTN81655012).

Cardiovascular manifestations of intermediate and major hyperhomocysteinemia due to vitamin B12 and folate deficiency and/or inherited disorders of one-carbon metabolism: a 3.5-year retrospective cross-sectional study of consecutive patients.

BACKGROUND: The association of moderate hyperhomocysteinemia (HHcy) (15-30 µmol/L) with cardiovascular diseases (CVD) has been challenged by the lack of benefit of vitamin supplementation to lowering homocysteine. Consequently, the results of interventional studies have confused the debate regarding the management of patients with intermediate/severe HHcy. OBJECTIVE: We sought to evaluate the association of intermediate (30-100 µmol/L) and severe (>100 µmol/L) HHcy related to vitamin deficiencies and/or inherited disorders with CVD outcomes. METHODS: We performed a retrospective cross-sectional study on consecutive patients who underwent a homocysteine assay in a French University Regional Hospital Center. Patients with CVD outcomes were assessed for vitamin B12, folate, Hcy, methylmalonic acid, and next-generation clinical exome sequencing. RESULTS: We evaluated 165 patients hospitalized for thromboembolic and other cardiovascular (CV) manifestations among 1006 patients consecutively recruited. Among them, 84% (138/165) had Hcy >30 µmol/L, 27% Hcy >50 µmol/L (44/165) and 3% Hcy >100 µmol/L (5/165). HHcy was related to vitamin B12 and/or folate deficiency in 55% (87/165), mutations in one or more genes of one-carbon and/or vitamin B12 metabolisms in 11% (19/165), and severe renal failure in 15% (21/141) of the studied patients. HHcy was the single vascular risk retrieved in almost 9% (15/165) of patients. Sixty % (101/165) of patients received a supplementation to treat HHcy, with a significant decrease in median Hcy from 41 to 17 µmol/L (IQR: 33.6-60.4 compared with 12.1-28). No recurrence of thromboembolic manifestations was observed after supplementation and antithrombotic treatment of patients who had HHcy as a single risk, after ∼4 y of follow-up. CONCLUSION: The high frequency of intermediate/severe HHcy differs from the frequent moderate HHcy reported in previous observational studies of patients with pre-existing CVD. Our study points out the importance of diagnosing and treating nutritional deficiencies and inherited disorders to reverse intermediate/severe HHcy associated with CVD outcomes.

Physcion, a tetra-substituted 9,10-anthraquinone, prevents homocysteine-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways.

BACKGROUND: Homocysteine (Hcy) induced vascular endothelial dysfunction is known to be closely associated with oxidative stress and impaired NO system. 1,8-Dihydroxy-3-methoxy-6-methylanthracene-9,10-dione (physcion) has been known to has antioxidative and anti-inflammatory properties. PURPOSE: The purpose of the present study was to define the protective effect of physcion on Hcy-induced endothelial dysfunction and its mechanisms involved. STUDY DESIGN AND METHODS: Hyperhomocysteinemia (HHcy) rat model was induced by feeding 3% methionine. A rat thoracic aortic ring model was used to investigate the effects of physcion on Hcy-induced impairment of endothelium-dependent relaxation. Two doses, low (L, 30 mg/kg/day) and high (H, 50 mg/kg/day) of physcion were used in the present study. To construct Hcy-injured human umbilical vein endothelial cells (HUVECs) model, the cells treated with 3 mM Hcy. The effects of physcion on Hcy-induced HUVECs cytotoxicity and apoptosis were studied using MTT and flow cytometry. Confocal analysis was used to determine the levels of intracellular Ca2+. The levels of protein expression of the apoptosis-related markers Bcl-2, Bax, caspase-9/3, and Akt and endothelial nitric oxide synthase (eNOS) were evaluated by western blot. RESULTS: In the HHcy rat model, plasma levels of Hcy and malondialdehyde (MDA) were elevated (20.45 ± 2.42 vs. 4.67 ± 1.94 µM, 9.42 ± 0.48 vs. 3.47 ± 0.59 nM, p < 0.001 for both), whereas superoxide dismutase (SOD) and nitric oxide (NO) levels were decreased (77.11 ± 4.78 vs. 115.02 ± 5.63 U/ml, 44.51 ± 4.45 vs. 64.18 ± 5.34 µM, p < 0.001 and p < 0.01, respectively). However, treatment with physcion significantly reversed these changes (11.82 ± 2.02 vs. 20.45 ± 2.42 µM, 5.97 ± 0.72 vs. 9.42 ± 0.48 nM, 108.75 ± 5.65 vs. 77.11 ± 4.78 U/ml, 58.14 ± 6.02 vs. 44.51 ± 4.45 µM, p < 0.01 for all). Physcion also prevented Hcy-induced impairment of endothelium-dependent relaxation in HHcy rats (1.56 ± 0.06 vs. 15.44 ± 2.53 nM EC50 for ACh vasorelaxation, p < 0.05 vs. HHcy). In Hcy-injured HUVECs, physcion inhibited the impaired viability, apoptosis and reactive oxygen species. Hcy treatment significantly increased the protein phosphorylation levels of p38 (2.26 ± 0.20 vs. 1.00 ± 0.12, p <0.01), ERK (2.11 ± 0.21 vs. 1.00 ± 0.11, p <0.01) and JNK. Moreover, physcion reversed the Hcy-induced apoptosis related parameter changes such as decreased mitochondrial membrane potential (MMP) and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9/3 in HUVECs. Furthermore, the downregulation of Ca2+, Akt, eNOS and NO caused by Hcy were recovered with physcion treatment in HUVECs. CONCLUSION: Physcion prevents Hcy-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways. This study provides the first evidence that physcion might be a candidate agent for the prevention of cardiovascular disease induced by Hcy.

Effect of / 生物医学与环境科学（英文）

Objective@#This study aimed to investigate the effects of @*Methods@#In this study, 0.1% DMG was supplemented in 20% casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system.@*Results@#Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 μmol/L to 28.49 ± 0.50 μmol/L; @*Conclusion@#DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.