Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells:Role of cystathionineγ-lyase/hydrogen sulfide pathway

BACKGROUND Ferroptosis can induce low retention and engraftment after mesenchymal stem cell(MSC)delivery,which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension(PAH)therapy.Interestingly,the cystathionineγ-lyase(CSE)/hydrogen sulfide(H_(2)S)pathway may contribute to mediating ferroptosis.However,the influence of the CSE/H_(2)S pathway on ferroptosis in human umbilical cord MSCs(HUCMSCs)remains unclear.AIM To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H_(2)S pathway-mediated ferroptosis,and to investigate the functions of the CSE/H_(2)S pathway in ferroptosis in HUCMSCs and the underlying mechanisms.METHODS Erastin and ferrostatin-1(Fer-1)were used to induce and inhibit ferroptosis,respectively.HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE.A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions,and pulmonary pressure and vascular remodelling were measured.The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging.Cell viability,iron accumulation,reactive oxygen species production,cystine uptake,and lipid peroxidation in HUCMSCs were tested.Ferroptosis-related proteins and S-sulfhydrated Kelchlike ECH-associating protein 1(Keap1)were detected by western blot analysis.RESULTS In vivo,CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxiainduced PAH.In vitro,CSE overexpression improved H_(2)S production and ferroptosis-related indexes,such as cell viability,iron level,reactive oxygen species production,cystine uptake,lipid peroxidation,mitochondrial membrane density,and ferroptosis-related protein expression,in erastin-treated HUCMSCs.In contrast,in vivo,CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice.In vitro,CSE inhibition decreased H_(2)S levels and restored ferroptosis in Fer-1-treated HUCMSCs.Interestingly,upregulation of the CSE/H_(2)S pathway induced Keap1 S-sulfhydration,which contributed to the inhibition of ferroptosis.CONCLUSION Regulation of the CSE/H_(2)S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH.Moreover,the protective effect of the CSE/H_(2)S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling.The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

Objective To study the relationship between polymorphism of cystathionine beta synthase （CBS） gene and development of congenital heart disease （CHD）. Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method, Results The frequencies of three genotypes （w/w, w/m, and m/m） in control group were 27.2%, 58,4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67,9% in patients and 55.7% in controls CHD parents＇ genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families. Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

Constitutive expression of feedback-insensitive cystathionine γ-synthase increases methionine levels in soybean leaves and seeds

Soybean （Glycine max （L.） Merr.） is a major crop that provides plant-origin protein and oil for humans and livestock. Al- though the soybean vegetative tissues and seeds provide a major source of high-quality protein, they suffer from low con- centration of an essential sulfur-containing amino acid, methionine, which significantly limits their nutritional quality. The level of methionine is mainly controlled by the first unique enzyme of methionine synthesis, cystathione y-synthase （CGS）. Aiming to elevate methionine level in vegetative tissues and seeds, we constitutively over-expressed a feedback-insensitive Arabidopsis CGS （AtD-CGS） in soybean cultivars, Zigongdongdou （ZD） and Jilinxiaoli 1 （JX）. The levels of soluble methionine increased remarkably in leaves of transgenic soybeans compared to wild-type plants （6.6- and 7.3-fold in two transgenic ZD lines, and 3.7-fold in one transgenic JX line）. Furthermore, the total methionine contents were significantly increased in seeds of the transgenic ZD lines （1.5- to 4.8-fold increase） and the transgenic JX lines （1.3- to 2.3-fold increase） than in the wild type. The protein contents of the transgenic soybean seeds were significantly elevated compared to the wild type, suggesting that the scarcity of methionine in soybeans may limit protein accumulation in soybean seeds. The increased protein content did not alter the profile of major storage proteins in the seeds. Generally, this study provides a promising strategy to increase the levels of methionine and protein in soybean through the breeding programs.

The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury

We previously showed that hydrogen sulfide(H2S)has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice.However,the precise mechanism underlying the role of H2S in this situation remains unclear.In this study,we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine,a H2S precursor,attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionineβsynthase(a major H2S synthetase in the brain)in the prefrontal cortex.We also found that an miR-9-5p inhibitor blocked the expression of cystathionineβsynthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia.Furthermore,miR-9-5p overexpression increased cystathionine-β-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury.L-cysteine decreased the expression of CXCL11,an miR-9-5p target gene,in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3,FSTL1,SOCS2 and SOCS5,while treatment with an miR-9-5p inhibitor reversed these changes.These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoringβ-synthase expression,thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.

Relationship between cystathionine γ-lyase gene polymorphism and essential hypertension in Northern Chinese Han population

Background Hydrogen sulfide （H2S） plays an important role in the smooth muscle cell relaxation and thereby participates in the development of hypertension. Cystathionine γ-lyase is the key enzyme in the endogenous production of H2S. Up to now, the reports on the relationship between the polymorphisms of cystathionine γ-lyase gene （CTH） and essential hypertension （EH） are limited. This study was designed to assess their underlying relationship. Methods A total of 503 hypertensive patients and 490 age-, gender- and area-matched normotensive controls were enrolled in this study. Based on the FASTSNP, a web server to identify putative functional single nucleotide polymorphisms （SNPs） of genes, we selected two SNPs, rs482843 and rs1021737, in the CTH gene for genotyping. Genotyping was performed by the polymerase chain reaction and restriction fragment length polymorphism method （PCR-RFLP）. The frequencies of the alleles and genotypes between cases and controls were compared by the chi-square test. The program Haplo.stats was used to investigate the relationship between the haplotypes and EH. Results These two SNPs were in Hardy-Weinberg Equilibrium in both cases and controls. The genotype distribution and allele frequencies of them did not significantly differ between cases and controls （all P〉0.05）. In the stepwise logistic regression analysis we failed to observe their association with hypertension. In addition, none of the four estimated haplotypes or diplotypes significantly increased or decreased the risk of hypertension before or after adjustment for several known risk factors. Conclusions The present study suggests that the SNPs rs482843 and rs1021737 of the CTH gene were not associated with essential hypertension in the Northern Chinese Han population. However, replications in other populations and further functional studies are still necessary to clarify the role of the CTH gene in the pathogenesis of EH.

Localization of cystathionine β synthase in mice ovaries and its expression profile during follicular development

Background In vitro fertilization （IVF） researches have suggested that cystathionine β synthase （CBS） is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development. Methods We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 （C57BL×Balb/c） mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development. Results CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly. Conclusions CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.

Therapeutic importance of hydrogen sulfide in age-associated neurodegenerative diseases

Hydrogen sulfide(H2S)is a gasotransmitter that acts as an antioxidant and exhibits a wide variety of cytoprotective and physiological functions in age-associated diseases.One of the major causes of age-related diseases is oxidative stress.In recent years,the importance of H2S has become clear,although its antioxidant function has not yet been fully explored.The enzymes cystathionineβ-synthase,cystathionineγ-lya-se,and 3-mercaptopyruvate sulfurtransferase are involved in the enzymatic production of H2S.Previously,H2S was considered a neuromodulator,given its role in long-term hippocampal potentiation,but it is now also recognized as an antioxidant in age-related neurodegeneration.Due to aerobic metabolism,the central nervous system is vulnerable to oxidative stress in brain aging,resulting in age-associated degenerative diseases.H2S exerts its antioxidant effect by limiting free radical reactions through the activation of antioxidant enzymes,including superoxide dismutase,catalase,and glutathione peroxidase,which protect against the effects of aging by regulating apoptosis-related genes,including p53,Bax,and Bcl-2.This review explores the implications and mechanisms of H2S as an antioxidant in age-associated neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease,Huntington’s disease,and Down syndrome.

Further evidence of endogenous hydrogen sulphide as a mediator of relaxation in human and rat bladder

We investigated the expression of hydrogen sulphide （H2S） in human and rat lower urinary tract （including bladder, prostate and urethra） tissues, and we sought to determine whether H2S induces relaxation of human and Sprague-Dawley （SD） rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine β-synthase （CBS）, cystathionine y lyase （CSE） and 3-mercaptopyruvate sulphur transferase （MPST, as known as 3-MST） by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine （L-Cys, a substrate of CBS, CSE and MPST） elicited relaxation in a dose-dependent manner on human bladder strips ere-contracted by acetylcholine chloride. This effect could be diminished by the ATe-sensitive potassium ion （KATe） channel blocker glibenclamide （GLB）, the CSE inhibitor DL-propargylglycine （PEG） and the CBS inhibitor hydroxylamine （HA）. H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms （LUTS） or overactive bladder （OAB）.

Changes in Hydrogen Sulfide in Rats with Hepatic Cirrhosis in Different Stages

This study aimed to observe changes in the hydrogen sulfide（H_2S） system in the blood and liver tissue of rats with hepatic cirrhosis at different stages by studying the effect of H_2S on the course of hyperdynamic circulation in rats with hepatic cirrhosis. H_2S concentration in the blood from the portal vein and inferior vena cava of hepatic cirrhosis rat model induced with carbon tetrachloride was detected on the 15 th, 30 th, and 52 nd day. The expression of cystathionine β-synthase（CBS） and cystathionine γ-lyase（CSE） protein, and CBS and CSE mRNA in the liver was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction（RT-PCR）, respectively. The results indicated that H_2S concentration in the blood from the portal vein and inferior vena cava of rats with hepatic cirrhosis was significantly lower than that in the control group. H_2S was gradually decreased with the development of the disease and significantly lower in the blood from portal vein than in the blood of inferior vena cava at the mid-stage and the late stage groups. The expression levels of CBS and CSE protein, and CBS and CSE mR NA in the livers with hepatic cirrhosis at different stages were all higher than those in the control group, and the expression gradually increased with the development of the disease. The expression of CBS was lower than CSE in the same stages. The results indicated that the CSE mRNA was expressed predominantly in the cirrhosis groups as compared with CBS mRNA. Among experimental rats, the H_2S system has an important effect on the occurrence and development of hyperdynamic circulation in rats with hepatic cirrhosis. This finding adds to the literature by demonstrating that H_2S protects vascular remodelling in the liver, and that CSE is indispensable in this process.

同型半胱氨酸与甲基代谢及其胱硫醚酶基因研究进展

同型半胱氨酸(HCY)水平增高是慢性血管疾病的独立危险因子,但HCY的来源不是从食物中吸取,其由蛋氨酸在甲基(CH3)循环代谢的中间产物。甲基化代谢亢进,再甲基化或转硫化代谢酶基因突变或功能障碍等原因均可造成甲基循环代谢失衡,从而导致HCY推积,患者出现高同型半胱氨酸血症(HHCY),动脉硬化等为特征的临床症状和体征。胱硫醚β-合酶(CBS)基因可将HCY转硫化成为胱硫醚,后者再经过胱硫醚-γ-裂解酶(CTH)裂解成丙酮酸、硫酸和水,达到有效降低HCY水平,降低动脉硬化进展,保障机体健康。本文综述近年来HCY代谢通路及其与心血管疾病的关系等的研究许多新进展。

Hyperhomocysteinemia dysregulates plasma levels of polyunsaturated fatty acids-derived eicosanoids

Hyperhomocysteinemia(HHcy)contributes to the incidence of many cardiovascular diseases(CVD).Our group have previously established crucial roles of eicosanoids and homocysteine in the incidence of vascular injury in diabetic retinopathy and renal injury.Using cystathionine-β-synthase heterozygous mice(cβs^(+/-))as a model of HHcy,the current study was designed to determine the impact of homocysteine on circulating levels of lipid mediators derived from polyunsaturated fatty acids(PUFA).Plasma samples were isolated from wild-type(WT)and cβs^(+/-)mice for the assessment of eicosanoids levels using LC/MS.Plasma 12/15-lipoxygenase(12/15-LOX)activity significantly decreased in cβs^(+/-)vs.WT control mice.LOX-derived metabolites from both omega-3 and omega-6 PUFA were also reduced in cβs^(+/-)mice compared to WT control(P<0.05).Contrary to LOX metabolites,cytochrome P450(CYP)metabolites from omega-3 and omega-6 PUFA were significantly elevated in cβs^(+/-)mice compared to WT control.Epoxyeicosatrienoic acids(EETs)are epoxides derived from arachidonic acid(AA)metabolism by CYP with anti-inflammatory properties and are known to limit vascular injury,however their physiological role is limited by their rapid degradation by soluble epoxide hydrolase(sEH)to their corresponding diols(DiHETrEs).In cβs^(+/-)mice,a significant decrease in the plasma EETs bioavailability was obvious as evident by the decrease in EETs/DiHETrEs ratio relative to WT control mice.Cyclooxygenase(COX)metabolites were also significantly decreased in cβs^(+/-)vs.WT control mice.These data suggest that HHcy impacts eicosanoids metabolism through decreasing LOX and COX metabolic activities while increasing CYP metabolic activity.The increase in AA metabolism by CYP was also associated with increase in sEH activity and decrease in EETs bioavailability.Dysregulation of eicosanoids metabolism could be a contributing factor to the incidence and progression of HHcy-induced CVD.

Electrophysiological effects of hydrogen sulfide on human atrial fibers

Background It has been reported that endogenous or exogenous hydrogen sulfide （H2S） exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H2S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H2S on human atrial fibers. Methods Human atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microetectrode technique. Results NariS （H2S donor） （50, 100 and 200 pmol/L） decreased the amplitude of action potential （APA）, maximal rate of depolarization （Vmax）, velocity of diastolic （phase 4） depolarization （VDD） and rate of pacemaker firing （RPF）, and shortened the duration of 90% repolarization （APD90） in a concentration-dependent manner. ATP-sensitive K＋ （KATP） channel blocker glibenclamide （Gli, 20 μmol/L） partially blocked the effects of NariS （100 μmol/L） on human atrial fiber cells. The L-type Ca2＋ channel agonist Bay K8644 （0.5 μmol/L） also partially blocked the effects of NariS （100 μmol/L）. An inhibitor of cystathionine y-lyase （CSE）, DL-propargylglycine （PPG, 200 μmol/L）, increased APA, Vmax, VDD and RPF, and prolonged APDg0. Conclusions H2S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of KATP channels and a concomitant decrease in calcium influx. Endogenous H2S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.