CBS Expression Research Articles

Increased hypothalamic hydrogen sulphide contributes to endotoxin tolerance by down-modulating PGE2 production.

Whereas some patients have important changes in body core temperature (Tb) during systemic inflammation, others maintain a normal Tb, which is intrinsically associated to immune paralysis. One classical model to study immune paralysis is the use of repeated administration of lipopolysaccharide (LPS), the so-called endotoxin tolerance. However, the neuroimmune mechanisms of endotoxin tolerance remain poorly understood. Hydrogen sulphide (H2 S) is a gaseous neuromodulator produced in the brain by the enzyme cystathionine β-synthase (CBS). The present study assessed whether endotoxin tolerance is modulated by hypothalamic H2 S. Rats with central cannulas (drug microinjection) and intraperitoneal datalogger (temperature record) received a low-dose of lipopolysaccharide (LPS; 100µgkg-1 ) daily for four consecutive days. Hypothalamic CBS expression and H2 S production rate were assessed, together with febrigenic signalling. Tolerant rats received an inhibitor of H2 S synthesis (AOA, 100pmol1µL-1 icv) or its vehicle in the last day. Antero-ventral preoptic area of the hypothalamus (AVPO) H2 S production rate and CBS expression were increased in endotoxin-tolerant rats. Additionally, hypothalamic H2 S inhibition reversed endotoxin tolerance reestablishing fever, AVPO and plasma PGE2 levels without altering the absent plasma cytokines surges. Endotoxin tolerance is not simply a reflection of peripheral reduced cytokines release but actually results from a complex set of mechanisms acting at multiple levels. Hypothalamic H2 S production modulates most of these mechanisms.

Exogenous Hydrogen Sulfide Offers Neuroprotection on Intracerebral Hemorrhage Injury Through Modulating Endogenous H2S Metabolism in Mice.

Hydrogen sulfide (H2S), an important endogenous signaling molecule, has a significant neuroprotective role in the central nervous system. In this study, we examined the protective effects of exogenous H2S against intracerebral hemorrhage (ICH), as well as its underlying mechanisms. We investigated the effects of exogenous H2S on ICH using Western blotting, injury volume, measurement of brain edema, propidium iodide (PI) staining, and behavior assessment, respectively. We found that endogenous H2S production was downregulated in the brain after ICH, which is caused by the decrease in cystathionine β-synthase (CBS) as the predominant cerebral H2S-generating enzyme in the brain. Treatment with sodium hydrosulfide (NaHS; an H2S producer) could restore the H2S production and the expression of CBS. NaHS could also attenuate brain edema, injury volume, and neurological deficits in the Morris water maze test after ICH. Western blotting results indicated that H2S pretreatment reversed the increase in caspase 3 cleavage and the decrease in Bcl-2, suppressed the activation of autophagy marker (LC3II and Beclin-1), and maintained the p62 level in injured striatum post-ICH. However, H2S could not restore brain CBS expression and H2S content, reduce brain edema, and improve motor performance and memory function after ICH through modulating autophagy and apoptosis when pretreated with the CBS inhibitor aminooxyacetic acid (AOAA). We also found that AOAA reduced the endogenous H2S production through inhibiting the enzyme activity of CBS rather than modulating the expression of CBS protein level. These present results indicate that H2S may possess potential therapeutic value in the treatment of brain injury after ICH, and the protective effect of exogenous H2S against ICH may be not a direct action but an indirect effect through inducing endogenous H2S metabolism responses.

Dietary Egg Protein Prevents Hyperhomocysteinemia via Upregulation of Hepatic Betaine-Homocysteine S-Methyltransferase Activity in Folate-Restricted Rats

Hyperhomocysteinemia is associated with increased cardiovascular disease risk. Whole eggs contain several nutrients known to affect homocysteine regulation, including sulfur amino acids, choline, and B vitamins. The aim of this study was to determine the effect of whole eggs and egg components (i.e., egg protein and choline) with respect to 1) homocysteine balance and 2) the hepatic expression and activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine β-synthase (CBS) in a folate-restricted (FR) rat model of hyperhomocysteinemia. Male Sprague Dawley rats (n=48; 6 wk of age) were randomly assigned to a casein-based diet (C; n=12), a casein-based diet supplemented with choline (C+Cho; 1.3%, wt:wt; n=12), an egg protein-based diet (EP; n=12), or a whole egg-based diet (WE; n=12). At week 2, half of the rats in each of the 4 dietary groups were provided an FR (0 g folic acid/kg) diet and half continued on the folate-sufficient (FS; 0.2 g folic acid/kg) diet for an additional 6 wk. All diets contained 20% (wt:wt) total protein. Serum homocysteine was measured by HPLC and BHMT and CBS expression and activity were evaluated using real-time quantitative polymerase chain reaction, Western blot, and enzyme activity. A 2-factor ANOVA was used for statistical comparisons. Rats fed FR-C exhibited a 53% increase in circulating homocysteine concentrations compared with rats fed FS-C (P<0.001). In contrast, serum homocysteine did not differ between rats fed FS-C and FR-EP (P=0.078). Hepatic BHMT activity was increased by 45% and 40% by the EP (P<0.001) and WE (P=0.002) diets compared with the C diets, respectively. Dietary intervention with egg protein prevented elevated circulating homocysteine concentrations in a rat model of hyperhomocysteinemia, due in part to upregulation of hepatic BHMT. These data may support the inclusion of egg protein for dietary recommendations targeting hyperhomocysteinemia prevention.

Anomalous Kv 7 channel activity in human malignant hyperthermia syndrome unmasks a key role for H2 S and persulfidation in skeletal muscle.

Human malignant hyperthermia (MH) syndrome is induced by volatile anaesthetics and involves increased levels of cystathionine β-synthase (CBS)-derived H2 S within skeletal muscle. This increase contributes to skeletal muscle hypercontractility. Kv 7 channels, expressed in skeletal muscle, may be a molecular target for H2 S. Here, we have investigated the role of Kv 7 channels in MH. Skeletal muscle biopsies were obtained from MH-susceptible (MHS) and MH-negative (MHN) patients. Immunohistochemistry, RT-PCR, Western blot, and in vitro contracture test (IVCT) were carried out. Development and characterization of primary human skeletal muscle cells (PHSKMC) and evaluation of cell membrane potential were also performed. The persulfidation state of Kv 7 channels and polysulfide levels were measured. Kv 7 channels were similarly expressed in MHN and MHS biopsies. The IVCT revealed an anomalous contractility of MHS biopsies following exposure to the Kv 7 channel opener retigabine. Incubation of negative biopsies with NaHS, prior to retigabine addition, led to an MHS-like positive response. MHS-derived PHSKMC challenged with retigabine showed a paradoxical depolarizing effect, compared with the canonical hyperpolarizing effect. CBS expression and activity were increased in MHS biopsies, resulting in a major polysulfide bioavailability. Persulfidation of Kv 7.4 channels was significantly higher in MHS than in MHN biopsies. In skeletal muscle of MHS patients, CBS-derived H2 S induced persulfidation of Kv 7 channels. This post-translational modification switches the hyperpolarizing activity into depolarizing. This mechanism can contribute to the pathological skeletal muscle hypercontractility typical of MH syndrome. This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Treatment of Cystathionine β-Synthase Deficiency in Mice Using a Minicircle-Based Naked DNA Vector.

Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 μM before injection to 176 μM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.

Reverse Erythroblastosis Virus α Antagonism Promotes Homocysteine Catabolism and Ammonia Clearance.

Metabolic homeostasis of amino acids is essential for human health. Here, we aimed to investigate a potential role for the clock component reverse erythroblastosis virus α (Rev-erbα) in circadian regulation of amino acid metabolism. RNA-seq with Rev-erbα-/- mice showed expression changes in genes involved in amino acid metabolism, particularly, the urea cycle and methionine metabolism. Rev-erbα ablation increased hepatic mRNA, protein, and enzymatic activity of betaine homocysteine methyltransferase (Bhmt), cystathionine β-synthase (Cbs), and cystathionine γ-lyase (Cth) and decreased the levels of plasma and liver homocysteine in mice. Cell-based assays confirmed negative regulation of these three genes by Rev-erbα. Combined luciferase reporter, mobility-shift, and chromatin immunoprecipitation assays identified Rev-erbα as a transcriptional repressor of Bhmt, Cbs, and Cth. Rev-erbα ablation or antagonism alleviated chemical-induced hyperhomocysteinemia in mice. This was accompanied by elevated expressions of Bhmt, Cbs, and Cth. Moreover, Rev-erbα ablation or antagonism promoted urea production and ammonia clearance. Of urea cycle-related genes, arginase 1 (Arg1), ornithine transcarbamylase (Otc), and carbamoyl-phosphate synthase 1 (Cps1) expressions were up-regulated in Rev-erbα-/- mice. Negative regulation of these urea cycle genes by Rev-erbα was validated using cell-based experiments. Mechanistic studies revealed that Rev-erbα inhibited CCAAT-enhancer-binding protein α transactivation to repress the transcription of Arg1, Cps1, and Otc. Conclusion: Rev-erbα antagonism alleviates hyperhomocysteinemia and promotes ammonia clearance. Targeting Rev-erbα represents an approach for the management of homocysteine- and ammonia-related diseases.

Sustained‐release of hydrogen sulfide by GYY4137 protected diabetic‐induced kidney injury

Hydrogen sulfide (H2S) has been recognized as the third gaseous signaling molecule after nitric oxide and carbon monoxide. It plays an important role in regulation of physiological and pathophysiological functions in many systems including the renal system. It has been reported that sustained‐release of H2S by GYY4137 suppresses diabetes‐accelerated atherosclerosis and has protective effects on diabetic nephropathy. However, whether the increase of H2S by GYY4137 can also protect diabetic kidney disease (DKD) ‐ induce proteinuria and reduce the damages in podocytes is unknown. The diabetes mellitus mouse model was used for this study. The CD‐1 mice were intraperitoneally (ip) injected with streptozotocin (STZ) 50mg/kg for 4 days and then diabetic mice were fed with high fat diet for 12 weeks. Under this condition, the DKD was development as indicated by proteinuria and damages in podocytes in these mice. The experiments were performed in four groups, control group (without STZ), DKD miceand GYY4137 treated groups with (5mg/kg) and (20mg/kg) respectively. GYY4137 was given by daily ip injection for 12 weeks (during this period animals feeding with high fat diet). Urinary albumin concentrations, kidney morphology changes and podocytes foot process was examined by light and electron microscopes. The level of apoptosis was examined by TUNEL staining. These changes were analyzed and compared among groups. In addition, the levels of H2S in the plasma (in both human and mice) and in kidneys (mice) and the expression levels of the H2S producing enzymes, Cystyl sulfide synthetase (CBS), cystyl sulfide beta lyase (CSE) and the 3‐thiol pyruvate transferase (3‐MST) in the kidney were detected by Western blotting (WB) and immunohistochemical methods. Experimental results showed that the plasma H2S was lower in DKD patient than healthy controls (17.8 ± 2.2 vs 24.8 ± 3.6 mmol/l, P&lt;0.01), and also in DKD mice compared with the control mice (18.7 ± 4.1 vs 40.7 ± 4.5 mmol/l, P&lt;0.01). The H2S level was also lower in DKD mouse kidney compare with the control kidney (52.1 ± 7.2 vs 81.6 ± 14.1 nmol/g, P&lt;0.01). WB showed the expression of CBS was reduced, but not CSE and MST in DKD kidney compare with the control. Administrations of GYY4137 significantly reduced urine albumin in DKD mice compare to the untreated group. The 24‐hour urine albumin was 46.6 ± 47.2 μg in treated and 144.6 ± 61μg in untreated group respectively (P&lt;0.05). Although the blood glucose levels were the same in both treated and untreated groups (30 ± 4.2 vs. 31.8 ± 2.1 mmol/l, P&gt;0.05), GYY4137 reduced both the mesangial hyperplasia and fusion of foot process in DKD kidney, indicating less damage of mesangia cells and the podocytes. GYY4137 also reduced DKD increased apoptosis in the kidney, detected by TUNEL staining in renal tissue. Our study shown that sustained‐release of H2S by GYY4137 decreases the urinary albumin and reduces kidney tissue apoptosis, mesangial proliferation and foot process infusion in DKD mice, thus indicating that H2S has significant protective effects on DKD.Support or Funding InformationNIH/NIDDK RO1 DK099284 and P01 DK 17433This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alterations in folate-dependent one-carbon metabolism as colon cell transition from normal to cancerous

Folate-dependent one-carbon cycle metabolism (FOCM) plays a critical role in maintaining genomic stability through regulating DNA biosynthesis, repair and methylation. Folate metabolites as well as other metabolites in the FOCM are hypothesized to be altered when cells transition from normal to cancerous state. Using cells at different stages in their development into colorectal cancer, the FOCM metabolites were profiled as an effort to phenotype the cells, and the metabolite levels were compared to the expressions of related genes. Here, we investigate whether there is a correlation between the metabolite levels, DNA methylation levels and the expression of the related genes that drive the levels of these metabolites. Using CRL1459, APC10.1, HCT116 and Caco-2, we show for the first time that FOCM metabolites correlate with the gene expression patterns. These differences follow a trend that may facilitate distinguishing colon cells at the different stages as they transition into cancerous state. The folate distribution and methionine levels were found to be key in determining the staging of the colon cells in CRC development. Also, expression of CBS, MTRR and MAT genes may facilitate distinguishing between untransformed and transformed colon cells.

Cystathionine β-Synthase in the Brain of the Trout Oncorhynchus mykiss after Unilateral Eye Damage and in Conditions of in vitro Cultivation

Expression of cystathionine β-synthase (CBS) in the brain of adult trout under normal conditions and 1 week after an eye injury was assessed using Western blot analysis. The study of CBS distribution in the brain of intact trout and after a mechanical eye injury in the telencephalon, tectum, cerebellum, and brainstem was carried out by the method of immunoperoxidase labeling on free-floating sections. The results of the study showed an increase in CBS expression in different brain divisions after an eye injury. In the visual projection center of the brain (tectum), radial glia cells expressing CBS were revealed after the injury. The emergence of CBS+ heterogeneous radial glia in the trout tectum after eye injury indicates the ability of the reactive neural stem cells (NSC) to synthesize hydrogen sulfide. CBS+ fibers and cells were found in the proliferative zones of cerebellum (valvula) and telencephalon (ventral region). In the intact trout tegmentum, CBS expression was observed in large neurons of the dorsal region and in the proliferative zones. After the eye injury, reactive neurogenic niches appeared in the brainstem, and no CBS-immunopositivity was detected in the periventricular zone. The increased expression of H2S-producing CBS enzyme after a traumatic impact can probably be explained by the neuroprotective functions of hydrogen sulfide, which are implemented in the matrix zones of the brain associated with reparative neurogenesis. A cultivation of trout brain cells showed the formation of neurosphere-like complexes, a part of which exhibited CBS-immunopositivity in the monolayer.

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats.

Thyroid hormones have a role in the regulation of hydrogen sulfide (H2 S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H2 S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l-thyroxine (12 mg/L) to drinking water for a period of 21 days. H2 S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H2 S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H2 S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H2 S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H2 S-producing enzymes in the rat.

Ovine uterine artery hydrogen sulfide biosynthesis in vivo: effects of ovarian cycle and pregnancy†.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term≈145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P>>>NP follicular>luteal UAendo and UAvsm (P<0.001). H2S production was greater in P>>>NP UAendo and UAvsm (P <0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Decreased Expression of Cystathionine β-Synthase Exacerbates Intestinal Barrier Injury in Ulcerative Colitis.

Endogenous H2S regulates multiple physiological and pathological processes in colon epithelial tissues. The current study investigated the role of cystathionine β-synthase [CBS], a major producer of H2S in colon epithelial cells, in the pathogenesis of ulcerative colitis [UC]-related intestinal barrier injury. The expression and DNA methylation level of CBS were investigated in inflamed and non-inflamed colon tissues collected from UC patients, and the effect of decreased CBS levels on Caco-2 monolayer barrier injury and altered status of tight junctions elicited by tumour necrosis factor/interferon [TNF/IFN] was determined. The expression of CBS and the methylation level of the CBS promoter were assessed in non-inflamed and inflamed colon epithelial tissue samples collected from UC patients. Barrier function, status of tight junction proteins and activation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway were further investigated in Caco-2 monolayers. Decreased expression of CBS and elevated methylation levels of the CBS promoter were observed in inflamed sites compared with in non-inflamed sites in the colon epithelial samples from UC patients. In Caco-2 monolayers, decreased expression of CBS exacerbated TNF/IFN-induced barrier injury and altered localization of tight junction proteins. Decreased expression of CBS predisposed Caco-2 monolayers to injury elicited by TNF/IFN via augmentation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway. Decreased expression of CBS propagates the pathogenesis of UC by exacerbating inflammation-induced intestinal barrier injury. Elevated methylation of the CBS promoter might be one of the mechanisms underlying the decreased expression of CBS in inflamed sites of colon epithelial tissues from UC patients.

Cystathionine β-synthase Induces Multidrug Resistance and Metastasis in Hepatocellular Carcinoma.

This study aims to analyze whether Cystathionine β-synthase (CBS) plays roles in hepatocellular carcinoma (HCC) drug resistance. MTS assay was used to detect the effect of chemotherapeutic drugs doxorubicin (DOX) and sunitinib on HCC cell viability and cell growth. Intracellular doxorubicin accumulation assay was performed to evaluate the sensitivity of DOX and sunitinib in HCC cells and the function of multidrug resistance-associated protein Pglycoprotein (P-gp). Quantification of H2S production was performed using the methylene blue method. Production of intracellular ROS was quantified using the DCFHDA assay. The scratch wound and transwell assays were used to determine the cell migration and invasion. Expression of proteins was tested by western blot analysis. HepG2 cells with high CBS expression were less sensitive to DOX and sunitinib and knockdown of CBS significantly elevated the sensitivity to DOX and sunitinib in HepG2 cells. In contrast, CBS overexpression increased the resistance of DOX and sunitinib in BEL-7404 cells. Moreover, the overexpression of CBS caused the up-regulation of the expression level of P-gp and the decrease of DOX accumulation in BEL-7404 cells. In further mechanism research, we found that STAT3/Akt/Bcl-2 pathway activation, reactive oxygen species (ROS) inhibition as well as enhancement of the metastatic ability of hepatoma cells were responsible for the HCC drug resistance. CBS overexpression conferred HCC cell resistance.

Inhibited Endogenous H2S Generation and Excessive Autophagy in Hippocampus Contribute to Sleep Deprivation-Induced Cognitive Impairment.

Background and Aim: Sleep deprivation (SD) causes deficit of cognition, but the mechanisms remain to be fully established. Hydrogen sulfide (H2S) plays an important role in the formation of cognition, while excessive and prolonged autophagy in hippocampus triggers cognitive disorder. In this work, we proposed that disturbances in hippocampal endogenous H2S generation and autophagy might be involved in SD-induced cognitive impairment.Methods: After treatment of adult male wistar rats with 72-h SD, the Y-maze test, object location test (OLT), novel object recognition test (NORT) and the Morris water maze (MWM) test were performed to determine the cognitive function. The autophagosome formation was observed with electron microscope. Generation of endogenous H2S in the hippocampus of rats was detected using unisense H2S microsensor method. The expressions of cystathionine-β-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), beclin-1, light chain LC3 II/LC3 I, and p62 in the hippocampus were assessed by western blotting.Results: The Y-maze, OLT, NORT, and MWM test demonstrated that SD-exposed rats exhibited cognitive dysfunction. SD triggered the elevation of hippocampal autophagy as evidenced by enhancement of autophagosome, up-regulations of beclin-1 and LC3 II/LC3 I, and down-regulation of p62. Meanwhile, the generation of endogenous H2S and the expressions of CBS and 3-MST (H2S producing enzyme) in the hippocampus of SD-treated rats were reduced.Conclusion: These results suggested that inhibition of endogenous H2S generation and excessiveness of autophagy in hippocampus are involved in SD-induced cognitive impairment.

Hyperhomocysteinemia induced endothelial progenitor cells dysfunction through hyper-methylation of CBS promoter

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are the key players in angiogenesis and vascular function. Cystathionine-β-synthase (CBS), an H2S-generating enzyme in methionine metabolism, regulates the function of these EPCs. This study aims to examine whether CBS hyper-methylation contributes to the bone marrow endothelial progenitor cell (BM-EPCs) function and subsequent bone blood flow in mice fed with a high methionine diet (HMD). Bone marrow (BM) cells were collected from HMD and control mice, differentiated into BM-EPCs, and were characterized by acLDL-DiI labeling. CBS mRNA expression was analyzed by real-time PCR, and the global methylation status and methylation of the CBS promoter were detected by nuclear 5-mC assay and methylation-specific PCR (qMSP) respectively. The result reveals that CBS promoter in BM-EPCs from HMD mice was hyper-methylated and the methylation level was, indeed, negatively correlated with CBS mRNA and angiogenic function of BM-EPCs. In addition, global methylation (5-mC) and DNA methyltransferase-1 (DNMT1) expression were increased in HMD condition. In vitro study also shows that HMD induced hyperhomocysteinemia (HHcy) impaired both adhesion and angiogenesis properties of BM-EPCs, accompanied by higher methylation level of CBS promoter that compared to control. Furthermore, bone blood flow was found to be decreased in HMD mice as compared to wild-type mice. To dissect the epigenetic mechanism, we also administrated DNMT inhibitor, 5-azacytidine (5-Aza) to HMD mice. The administration of 5-Aza in HMD mice restored the CBS expression, EPC mediated angiogenesis and blood flow by reducing abnormal DNA hyper-methylation. In conclusion, HHcy dismantles BM-EPCs function and bone blood flow through the hyper-methylation of the CBS promoter in HMD fed mice.

Comparative localization of cystathionine beta synthases and cystathionine gamma lyase in canine, non-human primate and human retina

Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H2S) and glutathione (GSH) may play an important role in ameliorating disease progression. H2S production is driven by cystathionine-γ-lyase (CSE) and cystathionine-β–synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H2S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primate (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and Mϋller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser capture microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H2S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.

MiR-191 secreted by platelet-derived microvesicles induced apoptosis of renal tubular epithelial cells and participated in renal ischemia-reperfusion injury via inhibiting CBS

ABSTRACT In this study, we aimed to reveal the role of miR-191 in apoptosis of renal tubular epithelial cells and in the involvement of renal ischemia-reperfusion injury. Renal transplantation rat model was established. miR-191 and Cystathionine-β-synthase (CBS) were measured by qRT-PCR and Western blot. The regulation of miR-191 on CBS was detected by luciferase reporter assay. We found miR-191 expression in platelets and platelet microvesicles (P-MVs) of patients and model rats was significantly upregulated than that of health and normal rats. Also, mRNA and protein levels of CBS in renal tissues of patients were significantly downregulated than that of health and normal rats. We also found that P-MVs could transfer miR-191 to HK-2 cells. Luciferase reporter assay showed that CBS was a direct target of miR-191. In addition, we proved that P-MVs-secreted miR-191 inhibited CBS expression in HK-2 cells, and P-MVs-secreted miR-191 promoted HK-2 cell apoptosis via CBS. Finally, we verified the trends of CBS expressions, HK-2 cell apoptosis and apoptosis-related proteins in vivo were similar as the trends in vitro. Therefore, CBS was a direct target of miR-191, and miR-191 could transfer to HK-2 cells via P-MVs to decrease the expression of CBS, thus to promote cell apoptosis and renal IR injury.

Possible involvement of peripheral TRP channels in the hydrogen sulfide-induced hyperalgesia in diabetic rats

BackgroundPeripheral diabetic neuropathy can be painful and its symptoms include hyperalgesia, allodynia and spontaneous pain. Hydrogen sulfide (H2S) is involved in diabetes-induced hyperalgesia and allodynia. However, the molecular target through which H2S induces hyperalgesia in diabetic animals is unclear. The aim of this study was to determine the possible involvement of transient receptor potential (TRP) channels in H2S-induced hyperalgesia in diabetic rats.ResultsStreptozotocin (STZ) injection produced hyperglycemia in rats. Intraplantar injection of NaHS (an exogenous donor of H2S, 3–100 µg/paw) induced hyperalgesia, in a time-dependent manner, in formalin-treated diabetic rats. NaHS-induced hyperalgesia was partially prevented by local intraplantar injection of capsazepine (0.3–3 µg/paw), HC-030031 (100–316 µg/paw) and SKF-96365 (10–30 µg/paw) blockers, at 21 days post-STZ injection. At the doses used, these blockers did not modify formalin-induced nociception. Moreover, capsazepine (0.3–30 µg/paw), HC-030031 (100–1000 µg/paw) and SKF-96365 (10–100 µg/paw) reduced formalin-induced nociception in diabetic rats. Contralateral injection of the highest doses used did not modify formalin-induced flinching behavior. Hyperglycemia, at 21 days, also increased protein expression of cystathionine-β-synthase enzyme (CBS) and TRPC6, but not TRPA1 nor TRPV1, channels in dorsal root ganglia (DRG). Repeated injection of NaHS enhanced CBS and TRPC6 expression, but hydroxylamine (HA) prevented the STZ-induced increase of CBS protein. In addition, daily administration of SKF-96365 diminished TRPC6 protein expression, whereas NaHS partially prevented the decrease of SKF-96365-induced TRPC6 expression. Concordantly, daily intraplantar injection of NaHS enhanced, and HA prevented STZ-induced intraepidermal fiber loss, respectively. CBS was expressed in small- and medium-sized cells of DRG and co-localized with TRPV1, TRPA1 and TRPC6 in IB4-positive neurons.ConclusionsOur data suggest that H2S leads to hyperalgesia in diabetic rats through activation of TRPV1, TRPA1 and TRPC channels and, subsequent intraepidermal fibers loss. CBS enzyme inhibitors or TRP-channel blockers could be useful for treatment of painful diabetic neuropathy.

The Neuro Engraftment and Neuroregenerative effects of Hydrogen Sulphide Donor, Intracerebral MSCs, Ginko Biloba and Kefir in Attenuating Neuropathological hallmarks of Lipopolysaccharide induced Alzheimer’s disease Rat models

Background: Memory disorders have been characterized by being a devastating long term incurable diseases with a huge social impact in addition to a diminished efficient available medical treatments. Deep Brain stimulation via using neuroprotective inducers for treatment of brain structure degenerative diseases such as Alzheimer’s disease (AD) can be considered as being a promising successful therapy due to its various targets and underlying mechanisms for improving brain dysfunction. Objectives: The main aim of this study is to suggest therapeutic protocol having the potentials for restoring normal neurons diverse population and modifying neuropathological deposited hallmarks including both positive and negative lesions. Materials and Methods: Rats were divided into nine groups: (G1) control ;(G2) rats received LPS as a method of inducing nongenetically manipulated AD;(G3)AD rats received NaHS;(G4) AD rats received MSCs intracerebrally;(G5) AD rats received MSCs+NaHS;(G6)AD rats received kefir+GB;(G7)AD rats received MSCs+kefir+GB;(G8)AD rats received NaHS+kefir+GB; (G9) AD rats received MSCs+NaHS+kefir+GB. Results: AD induction resulted in down-regulation of CBS expression and GSH brain tissue level accompanied with overexpression in amyloid-? protein, MAPK, tau protein, ACAT expression and MDA brain tissue level in addition to elevated caspase-3 serum level. Conclusion: The implantation of amyloid reliving therapy that do have a wide clinical impact if initiated at benign plaques stage before irreversible brain damage occurs. The following effects have been observed following the administration of suggested medical protocol where a decrease in AD pathological deposited hallmarks has been observed with maintaining inflammatory brain factors by functioning as a potent neuroregenerative.

Optic Nerve Crush Induces Changes of Hydrogen Sulfide Synthases Expression in the Rat Retina.

To investigate the expression changes of the hydrogen sulfide synthases cystathionine γ-lyase （CSE）, cystathionine β-synthase （CBS）, and 3-mercaptopyruvate sulfurtransferase （3-MST）, after optic nerve crush （ONC） in rat the retina. The rat model of ONC was established. Rats were divided into normal control, ONC, and sham control groups. Histopathologic changes in retina, the number of retinal ganglion cells （RGC） and retinal thickness of inner part （RTIP） were measured. The changes of CSE, CBS and 3-MST mRNA expression were detected with quantitative real-time PCR. The retinal histostructure was normal in normal controls and with minor changes in sham controls, respectively. Compared with sham group, significant retina damages were found in the ONC group： a time-dependent reduction of RGC number and RTIP. Expressions of CSE, CBS and 3-MST mRNA in rat retina were detected in normal control. Compared with normal controls, the expressions of CSE, CBS and 3-MST mRNA did not show any significant changes in the sham controls. Compared with sham controls, CBS mRNA expressions showed a time-dependent increase at 3 d, 7 d and 14 d after crush in the ONC group; CSE mRNA expressions increased to the peak at 3 d and then slightly reduced at 14 d after crush; 3-MST mRNA expressions showed the trend of increase at 3 d and 7 d and then enhanced remarkably at 14 d after crush. Hydrogen sulfide synthases CSE, CBS and 3-MST expressions were up-regulated in rat retina following ONC, which may cause an increase in local endogenous hydrogen sulfide production in the retina and a compensatory protective effect.