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1.
Electron. j. biotechnol ; 50: 23-28, Mar. 2021. graf, ilus
Article in English | LILACS | ID: biblio-1292311

ABSTRACT

BACKGROUND: H2S is proved to be functioning as a signaling molecule in an array of physiological processes in the plant and animal kingdom. However, the H2S synthesis pathway and the responses to cold conditions remain unclear in postharvest mushroom. RESULTS: The biosynthesis of H2S in the Agaricus bisporus mushroom tissues exhibited an increasing tendency during postharvest storage and was significantly triggered by cold treatment. The cystathionine clyase (AbCSE) and cystathionine b-synthase (AbCBS) genes were cloned and proved responsible for H2S biosynthesis. Furthermore, transcriptional and posttranscriptional regulation of AbCSE and AbCBS were crucial for the enzyme activities and subsequent H2S levels. However, the AbMST was not involved in this process. Moreover, the AbCSE and AbCBS genes displayed low identity to the characterized genes, but typical catalytic domains, activity sites, subunit interface sites, and cofactor binding sites were conserved in the respective protein sequences, as revealed by molecular modeling and docking study. The potential transcription factors responsible for the H2S biosynthesis in cold conditions were also provided. CONCLUSIONS: The H2S biosynthetic pathway in postharvest mushroom was unique and distinct to that of other horticultural products.


Subject(s)
Agaricus/chemistry , Cystathionine beta-Synthase/metabolism , Cystathionine gamma-Lyase/metabolism , Hydrogen Sulfide/chemical synthesis , Agricultural Cultivation , Agaricus campestris , Cold Temperature , Food Storage
2.
Acta Physiologica Sinica ; (6): 148-156, 2020.
Article in Chinese | WPRIM | ID: wpr-827073

ABSTRACT

The adrenal gland is an important endocrine organ of human body. CYP11B1 gene was specifically expressed in the zona fasciculata in adrenal cortex. In order to better study the function of genes specifically expressed in the zona fasciculata in adrenal cortex, the mice with Cre recombinase specifically expressed in the zona fasciculata in adrenal cortex were constructed. It was then confirmed that CYP11B1 was specifically expressed in adrenal glands. Then, using CRISPR/Cas9 technique, CYP11B1-2A-GfpCre recombinant vector was constructed and subsequently injected into the fertilized eggs of mice. It was confirmed that the Cre gene was mainly expressed in the zona fasciculata in adrenal cortex of CYP11B1Cre mice by using mTmG and LacZ staining. The CYP11B1Cre mice were then mated with cystathionine γ-lyase (CTH) mice, thereby generating CTH/CYP11B1Cre mice. It was also confirmed that CTH gene in the zona fasciculata in adrenal cortex was specifically knocked out in these mice. These results suggest that transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex were constructed successfully. This animal model can be a powerful tool for the study of the function of genes expressed in the zona fasciculata in adrenal cortex.


Subject(s)
Adrenal Cortex , Animals , CRISPR-Cas Systems , Cystathionine gamma-Lyase , Genetics , Integrases , Genetics , Metabolism , Mice , Mice, Transgenic , Zona Fasciculata
3.
Acta Physiologica Sinica ; (6): 299-307, 2020.
Article in Chinese | WPRIM | ID: wpr-827057

ABSTRACT

The purpose of the present study is to investigate the effect of L-cysteine on colonic motility and the underlying mechanism. Immunohistochemical staining and Western blot were used to detect the localization of the HS-generating enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Organ bath system was used to observe the muscle contractile activities. Whole-cell patch-clamp technique was applied to record ionic channels currents in colonic smooth muscle cells. The results showed that both CBS and CSE were localized in mucosa, longitudinal and circular muscle and enteric neurons. L-cysteine had a dual effect on colonic contraction, and the excitatory effect was blocked by pretreatment with CBS inhibitor aminooxyacetate acid (AOAA) and CSE inhibitor propargylglycine (PAG); L-cysteine concentration-dependently inhibited L-type calcium channel current (I) without changing the characteristic of L-type calcium channel (P < 0.01); In contrast, the exogenous HS donor NaHS increased I at concentration of 100 μmol/L, but inhibited I and modified the channel characteristics at concentration of 300 μmol/L (P < 0.05); Furthermore, L-cysteine had no effect on large conductance calcium channel current (I), but NaHS significantly inhibited I (P < 0.05). These results suggest that L-cysteine has a potential dual effect on colonic smooth muscle and the inhibitory effect might be directly mediated by L-type calcium channel while the excitatory effect might be mediated by endogenous HS.


Subject(s)
Cystathionine beta-Synthase , Cystathionine gamma-Lyase , Cysteine , Pharmacology , Hydrogen Sulfide , Muscle, Smooth
4.
Article in Chinese | WPRIM | ID: wpr-776550

ABSTRACT

OBJECTIVE@#To investigate the hypothesis that hydrogen could ameliorate cecal ligation and puncture (CLP)-induced lung injury of rats by inhibiting cystathionine-gamma-lyase/hydrogen sulfide (CSE/HS) system.@*METHODS@#A total number of 24 healthy male SD rats weighting 250~300 g were randomly divided into four groups (n=6 in each group): sham operation group(sham group), hydrogen-rich saline control group(H group), CLP group and hydrogen-rich saline treatment group(CLP+H group). The rats were treated with hydrogen-rich saline or saline 10 min before CLP or sham operation. At 8 h of sham or CLP operation, lung samples were obtained to detect the changes of the CSE/HS system using biochemical and RT-PCR methods. In order to further confirm the role of HS during hydrogen improve the lung injury of CLP rats, we also observed the effect of hydrogen-rich saline on the lung injury induced by HS donor-sodium sodium hydrosulfide (NaHS). Thirty-two healthy male SD rats (250~300 g) were randomly divided into four groups (n=8 in each group): control group, HS group, HS+H group and H group. Saline(10 mg/kg) or NaHS(HS donor, 56 μmol/kg) was injected intraperitoneally (10 mg/kg) respectively into rats in the control rats or HS group. For rats in the HS+H and H group, hydrogen-rich saline (10 mg/kg) was injected 10 min before saline or NaHS administration. Eight hours after the LPS saline or NaHS administration, lung coefficient, MDA content, and MPO activity were detected. The contents of TNF-α, IL-6 and IL-10 in lung tissue were measured, and the morphological changes of lung tissue were also observed.@*RESULTS@#CSE/HS system up-regulating were observed in animals exposed to CLP. Hydrogen-rich saline treatment significantly inhibited CSE/HS system as indicated by significantly reduced HS production in lung, along with a decreased CSE activity and CSE mRNA expression (all P<0.05). Importantly, the results showed that lung injury and lung tissue inflammation were observed in animals exposed to NaHS. Hydrogen-rich saline treatment significantly attenuated lung injury as indicated by significantly improved histological changes in lung, significantly reduced index of quantitative assessment (IQA), MDA content and lung coefficient (all P<0.05). MPO activity in lung tissue was significantly reduced along with decreased productions of TNF-α and IL-6, and an increased production of IL-10 in the presence of hydrogen (all P<0.05), demonstrating antioxidant and anti-inflammatory effect of hydrogen in NaHS-induced ALI.@*CONCLUSION@#These results indicate that hydrogen-rich saline peritoneal injection improves the lung injury induced by CLP operation. The therapeutic effects of hydrogen-rich saline may be related to suppressing the production of HS.


Subject(s)
Animals , Cecum , General Surgery , Cystathionine gamma-Lyase , Metabolism , Cytokines , Metabolism , Hydrogen , Pharmacology , Hydrogen Sulfide , Metabolism , Ligation , Lung Injury , Therapeutics , Male , Punctures , Random Allocation , Rats , Rats, Sprague-Dawley , Saline Solution , Pharmacology
5.
Article in Chinese | WPRIM | ID: wpr-773808

ABSTRACT

OBJECTIVE@#To investigate the effects of hydrogen sulfide (HS) on the negatively regulation of cardiomyocyte hypertrophy and the relationship between the effect of HS with miRNA-133a-mediated Ca/calcineurin/NFATc4 signal pathway.@*METHODS@#Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expression of brain natriuretic peptide(BNP), β-myosin heavy chain(β-MHC), cystathionase (CSE), miRNA-133a, calcineurin (CaN) were detected by qRT-PCR. The protein expressions of CaN、nuclear factors of activated T cells (NFATc4) were detected by Western blot. The concentration of HS in the cardiomyocyte was detected by Elisa. The concentration of intracellular calcium was measured by calcium imaging using confocal microscope. The nuclear translocation of NFATc4 was checked by immuno-fluorescence cell staining technique.@*RESULTS@#①The level of system of CSE/HS and expression of miRNA-133a were significantly reduced in cardiomyocyte hypertrophy. Pretreatment with NaHS increased the concentration of HS and the expression of miRNA-133a mRNA in cardiomyocytes, and suppressed cardiomyocyte hypertrophy. ②The concentration of intracellular calcium, the expression of CaN and nulear protein NFATc4 were significantly increased, and the nuclear translocation of NFATc4 were obviously enhanced in cardiomyocyte hypertrophy. NaHS pretreatment markedly inhibited these effects of ISO induced cardiomyocyte hypertrophy. ③Application of antagomir-133a reversed the inhibitory effects of NaHS on cardiomyocyte hypertrophy, and increased the influx of intracellular calcium, and elevated the expression of CaN and nuclear protein NFATc4, and enhanced the nuclear translocation of NFATc4.@*CONCLUSIONS@#HS can negatively regulate cardiomyocyte hypertrophy. The effects might be associated with HS increasing expression of miRNA-133a and inhibiting inactivation of Ca/calcineurin/NFATc4 signal pathway.


Subject(s)
Animals , Calcineurin , Metabolism , Cardiomegaly , Metabolism , Cells, Cultured , Cystathionine gamma-Lyase , Metabolism , Hydrogen Sulfide , Metabolism , MicroRNAs , Metabolism , Myocytes, Cardiac , Metabolism , Myosin Heavy Chains , Metabolism , NFATC Transcription Factors , Metabolism , Natriuretic Peptide, Brain , Metabolism , Nerve Tissue Proteins , Metabolism , Rats , Signal Transduction
6.
Braz. arch. biol. technol ; 61: e18160308, 2018. graf
Article in English | LILACS | ID: biblio-974118

ABSTRACT

ABSTRACT Background and aim: It is well established that the rate of gastric lesions increases in diabetic rats. Recently, the protective effect of hydrogen sulfide (H2S) in gastric mucosa has been proven. This study aimed to determine the release of H2S and mRNA expression of cystathionine gamma lyase (CSE) in gastric mucosa in alloxan-diabetic rats in response to distention-induced gastric acid secretion. Twenty-four rats were randomly assigned to 4 groups (6 in each). They were the normal-control, distention-control, diabetic-control, and distention-diabetic groups. Under anesthesia, animals underwent a tracheotomy and midline laparotomy. To washout the gastric contents, a catheter was inserted in the stomach through the duodenum. To determine the effect of distention-induced gastric acid secretion on H2S release and mRNA expression of CSE, the stomachs were distended by normal saline. At the end of experiments, animals were sacrificed and the gastric mucosa was collected to determine H2S concentration and to quantify mRNA expression of CSE by quantitative real-time PCR. Mucosal release of H2S and mRNA expression of CSE significantly increased in response to stimulated gastric acid secretion in normal rats (P<0.01), while the increases in diabetic rats were not significant. Basal release of H2S and mRNA expression of CSE in gastric mucosa were significantly in diabetic rats lower than normal rats. On the basis of the results, we conclude that the decreased release of H2S in response to basal and stimulated gastric acid output in alloxan-diabetic rats compared to normal rats is largely due to downregulation of mRNA expression of CSE.


Subject(s)
Animals , Rats , Cystathionine gamma-Lyase , Gastric Acid , Hydrogen Sulfide , Alloxan
7.
Acta Physiologica Sinica ; (6): 65-73, 2015.
Article in Chinese | WPRIM | ID: wpr-255971

ABSTRACT

The present study was designed to investigate the potential role of endogenous hydrogen sulfide (H2S) in chronic stress-induced colonic hypermotility. Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. The total number of fecal pellets was counted at the end of each 1 h of WAS or SWAS session. Organ bath recordings were used to test the colonic motility. H₂S production of colon was determined, and immunohistochemistry and Western blot were performed on rat colonic samples to detect the distribution and expression of H₂S-producing enzymes. The results showed that i) repeated WAS increased the number of fecal pellets per hour and the area under the curve (AUC) of the spontaneous contractions of colonic strips (P < 0.05), ii) repeated WAS decreased the endogenous production of H₂S and the expression of H₂S-producing enzymes in the colon devoid of mucosa and submucosa (P < 0.001), iii) cystathionine-γ-lyase (CSE) was strongly expressed in the cytosols of the circular and longitudinal smooth muscle cells and the nucleus of the myenteric plexus neurons, iv) cystathionine-β-synthase (CBS) was primarily localized in the cytosols of myenteric plexus neurons and weakly localized in the epithelial cells and v) inhibitors of H₂S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats (P < 0.001). In conclusion, the results suggest that the colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H2S.


Subject(s)
Animals , Colon , Cystathionine beta-Synthase , Metabolism , Cystathionine gamma-Lyase , Metabolism , Gastrointestinal Motility , Hydrogen Sulfide , Metabolism , Male , Muscle Contraction , Myocytes, Smooth Muscle , Metabolism , Neurons , Metabolism , Rats , Rats, Wistar , Stress, Physiological
8.
Article in Chinese | WPRIM | ID: wpr-260347

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of intestinal resection on hydrogen sulfide (H2S) biosynthesis and interstitial cells of Cajal(ICC) in mice.</p><p><b>METHODS</b>After intestinal resection mouse model was established, the activity of MPO in the proximal anastomosis intestinal tissue were detected. Sensitive sulphur electrode assay was applied to measure the H2S level. RT-PCR technique was employed to investigate the mRNA expression of the endogenous H2S biosynthesis enzymes, cystathionine-b-synthase (CBS) and cystathionine-c-lyase (CSE). Immunofluorescence staining was used to detect the expression of c-kit in order to calculate the area of ICC.</p><p><b>RESULTS</b>The mRNA expression of CSE was detected in the small intestine tissue of mice, while no CBS mRNA was found. The mRNA expression of CSE in proximal anastomotic stoma increased in time-dependent manner in the model group. CSE mRNA expression began to increase 1 hour after operation, reached the peak at 6th hour, then decreased gradually, and was similar to the control group at postoperative 24th hour. Compared to the model group, in the intestinal tissues of proximal 3 cm to anastomotic stoma, the mRNA expression of CSE (1.16 ± 0.18 vs. 1.63 ± 0.13, P<0.05), the activity of MPO [(0.54 ± 0.07) U/g vs. (0.83 ± 0.09) U/g, P<0.05], the H2S level [(36.1 ± 6.1) nmol/mg vs. (5.3 ± 5.6) nmol/mg, P<0.05] were significantly reduced in the PPG group. Meanwhile, average percentage of positive ICC area in the PPG groups was significantly higher [(2.26 ± 0.19)% vs. (1.65 ± 0.24)%, P<0.05].</p><p><b>CONCLUSIONS</b>Inflammatory reaction in muscular layer induced by intestinal resection up-regulates the mRNA expression of CSE proximal to anastomotic stoma, generates excess H2S to damage ICC leading to intestinal motor dysfunction. Preoperative inhibition of endogenous H2S generation may protect the ICC.</p>


Subject(s)
Animals , Cystathionine gamma-Lyase , Disease Models, Animal , Hydrogen Sulfide , Inflammation , Interstitial Cells of Cajal , Intestines , Mice , Proto-Oncogene Proteins c-kit , RNA, Messenger
9.
National Journal of Andrology ; (12): 299-303, 2014.
Article in Chinese | WPRIM | ID: wpr-309718

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the impact of hyperglycemia on the hydrogen sulfide (H2S) signaling pathway in rat penile tissue and its relationship with erectile function.</p><p><b>METHODS</b>Twenty healthy male Sprague Dawley (SD) rats aged 8 weeks were randomly divided into groups A (4-week healthy control), B (4-week diabetes mellitus model), C (6-week healthy control) and D (6-week diabetes mellitus model). The rats in groups B and D were injected intraperitoneally with streptozotocin at 50 mg/kg to induce diabetes mellitus, while those in groups A and C with the same volume of normal saline. The animals were killed at 4 (groups A and B) and 6 weeks (groups C and D) after treatment for measurement of the maximal intracavernous pressure/mean arterial blood pressure (ICP(max)/MAP) by electrostimulation, determination of the H2S concentration in the plasma and penile tissue, and detection of the expressions of cystathionine-beta-synthetase (CBS) and cystathionine-gamma-lyase (CSE) in the penile corpus cavernosum by immunohisto- chemistry and Western blot.</p><p><b>RESULTS</b>With electrostimulation of the pelvic ganglia at 5V and 7 V, ICP(max)/MAP was significantly reduced in groups B (0.19 +/- 0.03 and 0.29 +/- 0.04) and D (0.14 +/- 0.04 and 0.25 +/- 0.04) as compared with A (0.46 +/- 0.07 and 0.68 +/- 0.09) and C (0.43 +/- 0.07 and 0.65 +/- 0.16) (P < 0.05). No statistically significant differences were found in the level of serum testosterone either between groups A and B ([469.19 +/- 126.46] ng/dl vs [359.08 +/- 60.06] ng/dl, P > 0.05) or between C and D ([470.44 +/- 209.28] ng/dl vs [297.01 +/- 96.58] ng/dl, P > 0.05). Groups B and D showed remarkable reduction in the H2S concentration (P < 0.05) and the expressions of CBS and CSE (P < 0.05) in comparison with A and C, and the CBS and CSE expressions were even more significantly decreased in D than in B (P < 0.05).</p><p><b>CONCLUSION</b>The reduced concentration of H2S and decreased expressions of CBS and CSE in the penile corpus cavernosum of the diabetic rats suggested that the H2S signaling pathway might be involved in hyperglycemia-induced erectile dysfunction.</p>


Subject(s)
Animals , Blood Pressure , Physiology , Cystathionine gamma-Lyase , Metabolism , Diabetes Mellitus, Experimental , Metabolism , Electric Stimulation , Methods , Erectile Dysfunction , Humans , Hydrogen Sulfide , Metabolism , Hyperglycemia , Metabolism , Lyases , Metabolism , Male , Penis , Physiology , Random Allocation , Rats , Rats, Sprague-Dawley , Signal Transduction , Testosterone , Metabolism
10.
National Journal of Andrology ; (12): 605-612, 2014.
Article in Chinese | WPRIM | ID: wpr-309667

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of endogenous hydrogen sulfide (H2S) in erectile dysfunction (ED) induced by androgen deficiency.</p><p><b>METHODS</b>We randomly divided 30 eight-week-old healthy male SD rats into six groups: 2-week control (A), 4-week control (B), 2-week castration (C), 4-week castration (D), 2-week castration + androgen replacement (E), and 4-week castration + androgen replacement (F), those in groups E and F subcutaneously injected with testosterone propionate (TP) at the physiological dose of 3 mg/kg per day after castration, while those in the other groups with isodose oil instead. At 2 and 4 weeks after operation, we determined the level of serum testosterone (T) , intracavernous pressure (ICP) , mean carotid arterial pressure (MAP) of the rats, measured the concentration of H2S in the plasma and corpus cavernosum tissue, and detected the expressions of cystathionine-P3-synthase (CBS) and cystathionine-gamma-lyase (CSE) by immunohistochemistry and Western blot.</p><p><b>RESULTS</b>The serum T level was significantly lower in group C ([0.63 +/- 0.15] nmol/L) than in A ( [ 16.55 +/- 4.17] nmol/L) and E ( [ 18.99 +/- 4.62] nmol/L) (P <0.05), as well as in group D ([0.70 +/-0.22] nmol/L) than in B ([15.44 +/-5.18] nmol/L) and F ([20.99 +/-6.41] nmol/L) (P <0. 05) , and so were ICP/MAP after 5 and 7 V electrical stimulation of the pelvic ganglia (P <0. 05) , H2 S concentration (P <0.05), and the expressions of CBS and CSE (P <0.05). The expressions of CBS and CSE proteins were also significantly decreased in group C as compared with D (P <0.05).</p><p><b>CONCLUSION</b>The reduced expressions of CBS and CSE may inhibit the H2 S signaling pathway, which might be one of the mechanisms underlying androgen deficiency-induced ED in rats.</p>


Subject(s)
Androgens , Animals , Cystathionine beta-Synthase , Metabolism , Cystathionine gamma-Lyase , Metabolism , Erectile Dysfunction , Metabolism , Hydrogen Sulfide , Metabolism , Male , Orchiectomy , Penis , Metabolism , Rats , Rats, Sprague-Dawley
11.
Chinese Medical Journal ; (24): 3695-3699, 2014.
Article in English | WPRIM | ID: wpr-240701

ABSTRACT

<p><b>BACKGROUND</b>Acute lymphoblastic leukemia (ALL) and chemotherapy can cause immune imbalance, and gaseous molecule hydrogen sulfide (H2S) can participate in the process of immune response. This study aimed to investigate the immune regulation of H2S in pediatric ALL.</p><p><b>METHODS</b>Children (n = 78) with ALL admitted during 2010-2013 were included in this study. Two blood samples were collected in period of before chemotherapy, bone marrow remission and two days after chemotherapy, respectively. Serum contents of H2S and cytokines, including interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10) and macrophage inflammatory protein-1α (MIP-1α), were detected using ELISA method. Stepwise regression was used to analyze the correlation between H2S and cytokines. Furthermore, human Jurkat cells were cultured in vitro, and nucleoprotein of Jurkat cells and peripheral blood mononuclear cells (PBMCs) were collected, contents of cystathionine γ-lyase (CSE) and certain cytokines were measured by Western blotting.</p><p><b>RESULTS</b>Serum concentrations of H2S, IL-1β, IL-6, IL-10 and MIP-1a in children with ALL were increased significantly (P < 0.01), while concentrations of IL-2, TNF-α, IFN-γ and IL-4 decreased obviously (P < 0.01). In patients after chemotherapy, concentrations of H2S and IL-10 were decreased significantly (P < 0.05), but IL-4 and IFN-γ concentrations increased markedly (P < 0.05). At remission stage, H2S, IL-1β, IL-4, IL-6, IL-10 and MIP-1α concentrations were further decreased markedly (P < 0.05), but concentrations of IL-2, TNF-α and IFN-γ increased again (P < 0.05). Protein contents of CSE, IL-10, IL-4 and IL-2 of PBMCs also increased markedly in children with ALL. Moreover, changes of CSE protein contents of PBMCs were consistent with serum H2S contents, and there were significant correlation between H2S and certain cytokines based on stepwise regression analysis. Furthermore, compared with those of PBMCs group, in vitro study indicated that Jurkat cells of H2S group expressed IFN-γ, IL-10, IL-4 and IL-2 protein increased obviously (P < 0.05), while IL-4, IL-2 and CSE expression of PPG group decreased markedly (P < 0.05).</p><p><b>CONCLUSION</b>Gaseous molecule H2S might participate in the process of immune regulation in pediatric ALL through modulating transcription and expression of cytokines.</p>


Subject(s)
Child , Child, Preschool , Cystathionine gamma-Lyase , Blood , Female , Humans , Hydrogen Sulfide , Blood , Interferon-gamma , Blood , Interleukin-10 , Blood , Interleukin-1beta , Blood , Interleukin-2 , Blood , Interleukin-4 , Blood , Interleukin-6 , Blood , Leukocytes, Mononuclear , Metabolism , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Blood , Tumor Necrosis Factor-alpha , Blood
12.
Article in Chinese | WPRIM | ID: wpr-267952

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expressions of cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) in the corpus cavernosum of spontaneous hypertensive rats (SHR) and their relationship with erectile dysfunction.</p><p><b>METHODS</b>This study included 10 male SHRs and 10 healthy male Wistar-Kyoto (WKY) rats as controls, all aged 12 weeks. We applied a series of electric stimuli to the major pelvic ganglions of the rats, observed changes in the ratio of intracavernosal to mean arterial blood pressure (ICP/MAP), measured the levels of serum testosterone (T) and endogenous H2S, and determined the expressions of CSE and CBS in the corpus cavernosum by Western blot and immunohistochemistry.</p><p><b>RESULTS</b>No obvious difference was found in the serum T level between the two groups. Compared with the WKY rats, the SHRs showed significant reduction in the ICP/MAP ratio, the contents of plasma H2S ([21.92 +/- 2.75] micromol/L vs [10.49 +/- 1.35] micromol/L, P < 0.05) and endogenous corpus cavernosal H2S ([87.67 +/- 2.12] nmol/mg prot vs [52.60 +/- 3.44] nmol/mg prot, P < 0.05), the level of endogenous H2S synthesis ([4.35 +/- 0.32] nmol/mg per min vs [1.14 +/- 0.07] nmol/mg per min, P < 0.05) and the expressions of CBS and CSE (P < 0.05). Immunohistochemistry showed that CSE and CBS were distributed mainly in the smooth muscle cells and vascular endothelial cells of the corpus cavernosum. The ICP/MAP ratio was highly positively correlated with the expressions of CSE (r = 0.977, P < 0.05) and CBS (r = 0.955, P < 0.05) in the corpus cavernosal tissue.</p><p><b>CONCLUSION</b>Hypertension inhibits endogenous H2S synthesis by suppressing the expressions of CSE and CBS in the corpus cavernosum, which might be related with hypertension-induced reduction of erectile function.</p>


Subject(s)
Animals , Cystathionine beta-Synthase , Metabolism , Cystathionine gamma-Lyase , Metabolism , Gene Expression Regulation , Hydrogen Sulfide , Blood , Hypertension , Metabolism , Male , Penis , Rats , Rats, Inbred SHR , Rats, Inbred WKY
13.
Braz. j. med. biol. res ; 46(8): 708-714, ago. 2013. tab, graf
Article in English | LILACS | ID: lil-684527

ABSTRACT

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg, ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.


Subject(s)
Animals , Female , Rats , Alendronate/antagonists & inhibitors , Gastric Mucosa/drug effects , Hydrogen Sulfide/pharmacology , Indicators and Reagents/pharmacology , Organothiophosphorus Compounds/pharmacology , Stomach Diseases/chemically induced , Analysis of Variance , Cystathionine gamma-Lyase/analysis , Diagnosis, Computer-Assisted , Diazoxide/administration & dosage , Gastric Mucosa/pathology , Glutathione/analysis , Glyburide/administration & dosage , Interleukin-1beta/analysis , KATP Channels/pharmacology , Malondialdehyde/analysis , Peroxidase/analysis , Peroxidase/metabolism , Rats, Wistar , Stomach Diseases/enzymology , Stomach Diseases/pathology , Tumor Necrosis Factor-alpha/analysis
14.
Arq. gastroenterol ; 50(2): 157-160, abr. 2013. tab, graf
Article in English | LILACS | ID: lil-679152

ABSTRACT

Context Hydrogen sulphide (H2S) has been proved to be a neuromodulator and contributes to the maintenance of gastric mucosal integrity in damage caused by anti-inflammatory nonsteroidal drugs. Previously, we demonstrated that H2S synthesis is essential to gastric protection against ethanol. Objective To better understanding the role of H2S and the detailed localization of its production in both normal and injured stomach due to ethanol injection, we studied the expression of cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) isoforms in gastric mucosa of mice treated with saline or 50% ethanol. Methods Mice were treated by gavage with saline or 50% ethanol (0.5 mL/25 g). After 1 hour, mice were sacrificed, and gastric tissue was evaluated by histological and immunohistochemical analysis specific for CSE and CBS. Results We have demonstrated a non-specific expression of CBS in the normal gastric mucosa and expression of CSE occurring mainly in the parietal cells of the animals treated with ethanol. Conclusion Thus, we demonstrated that the expression of CBS appears to be constitutive and diffuse across the gastric epithelium, while the expression of CSE appears to be induced in parietal cells by damage agents such as ethanol. .


Contexto O sulfeto de hidrogênio (H2S) tem sido mostrado como um neuromodulador e contribuidor para a manutenção da integridade da mucosa gástrica na lesão causada por drogas antiinflamatórias não esteroidais. Previamente, demonstramos que a síntese de H2S é essencial para a proteção da mucosa gástrica contra a administração de etanol. Objetivo Para compreender o papel do H2S e a localização detalhada de sua produção no estômago normal e após lesão induzida pela administração de etanol, estudou-se a expressão das isoformas cistationina-γ-liase (CSE) e cistationina-β-sintetase (CBS) na mucosa gástrica de camundongos tratados com salina ou etanol 50%. Métodos Os camundongos foram tratados por gavagem com salina ou etanol 50% (0,5 mL/25 g). Após 1 hora, os camundongos foram sacrificados e os tecidos gástricos foram avaliados por análise histológica e imunoistoquímica específica para CBS e CSE. Resultados Foi demonstrado expressão não específica de CBS na mucosa gástrica normal e expressão de CSE ocorrendo principalmente nas células parietais dos animais tratados com etanol. Conclusão Assim, demonstramos que a expressão de CBS parece ser constitutiva e difusa através do epitélio gástrico, enquanto a expressão de CSE parece ser induzida nas células parietais por agentes lesivos como o etanol. .


Subject(s)
Animals , Mice , Cystathionine beta-Synthase/metabolism , Cystathionine gamma-Lyase/metabolism , Gastric Mucosa/enzymology , Hydrogen Sulfide/metabolism , Disease Models, Animal , Ethanol/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/pathology , Immunohistochemistry
15.
Article in Chinese | WPRIM | ID: wpr-345684

ABSTRACT

<p><b>OBJECTIVE</b>To observe the level in plasma hydrogen sulfide (H2S) and the expression of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) (two key synthetases for endogenous H2S generation in the kidney) in obstructed kidney tissue among rats with tubulointerstitial fibrosis (TIF) induced by unilateral ureteral obstruction (UUO), and to explore the role of H2S in TIF.</p><p><b>METHODS</b>Ninety-six male Sprague-Dawley rats were randomly divided into sham-operated, model, low-dose NaHS and high-dose NaHS groups (n=24 each). TIF was induced by UUO in the model, low-dose NaHS and high-dose NaHS groups. The low-dose and high-dose NaHS groups were intraperitoneally injected with NaHS (1.4 and 7.0 μmol/kg respectively) twice daily immediately after operation, and the sham-operated and model groups were intraperitoneally injected with an identical volume of normal saline. In each group, 8 rats were randomly selected and sacrificed at 7, 14 or 21 days after operation. Plasma H2S concentration was measured by deproteinization. The obstructed kidney tissue was subjected to hematoxylin and eosin staining and Masson staining, and the renal tubulointerstitial injury was evaluated under a microscope. mRNA and protein expression of CBS and CSE in the obstructed kidney tissue was measured by RT-PCR and immunohistochemistry respectively.</p><p><b>RESULTS</b>The degree of UUO-induced renal tubulointerstitial injury was negatively correlated with plasma H2S concentration in (r=-0.891, P<0.01). With H2S supplementation, renal tubulointerstitial injury was reduced (P<0.01), the expression of mRNA and protein of CBS and CSE in the kidney tissue and plasma H2S level were upregulated (P<0.01), and the degree of TIF was reduced (P<0.01). There were no significant differences in plasma H2S level and mRNA and protein expression of CBS and CSE between the low-dose and high-dose NaHS groups (P>0.05).</p><p><b>CONCLUSIONS</b>H2S is involved in the development of UUO-induced TIF, and the CBS/H2S and CSE/H2S systems play key roles in this process. H2S supplementation can delay the progression of TIF.</p>


Subject(s)
Animals , Cystathionine beta-Synthase , Genetics , Cystathionine gamma-Lyase , Genetics , Dietary Supplements , Fibrosis , Hydrogen Sulfide , Blood , Kidney Tubules , Pathology , Male , RNA, Messenger , Rats , Rats, Sprague-Dawley , Ureteral Obstruction , Blood , Pathology
16.
National Journal of Andrology ; (12): 599-603, 2013.
Article in Chinese | WPRIM | ID: wpr-350853

ABSTRACT

<p><b>OBJECTIVE</b>To study the expressions of cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) in the corpus cavernosum smooth muscle of castrated rats and their roles in erectile dysfunction after castration.</p><p><b>METHODS</b>We randomly assigned 40 eight-week-old male SD rats to groups A (2-week sham-operation), B (4-week sham-operation), C (2-week castration) and D (4-week castration). We determined the level of serum testosterone (T) and the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats after operation using immunohistochemistry and RT-PCR.</p><p><b>RESULTS</b>The T level was significantly decreased in groups C ([11.85 +/- 6.73] nmol/L) and D ([1.96 +/- 1.23] nmol/L) as compared with A ([89.65 +/- 17.13] nmol/L) and B ([106.75 +/- 19.68] nmol/L) (P < 0.05). CBS and CSE were expressed in all groups of rats, but the relative expressions of CBS and CSE mRNA were significantly lower in groups C (0.93 +/- 0.14 and 0.87 +/- 0.20) and D (0.79 +/- 0.17 and 0.71 +/- 0.12) than in A (2.13 +/- 0.65 and 1.93 +/- 0.15) and B (2.07 +/- 0.53 and 1.89 +/- 0.45) (P < 0. 05), so were the optical density values (IA) of the CBS and CSE proteins, 130.35 +/- 23.56 and 93.56 +/- 36.64 in group C and 80.29 +/- 29.65 and 58.56 +/- 19.95 in group D, as compared with 310.57 +/- 130.56 and 269.56 +/- 116.76 in group A and 349.68 +/-112.35 and 298.35 +/- 100.76 in group B (P < 0.05). The androgen level was positively correlated with the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats.</p><p><b>CONCLUSION</b>Androgen regulates erectile function via the expressions of CBS and CSE.</p>


Subject(s)
Animals , Cystathionine beta-Synthase , Metabolism , Cystathionine gamma-Lyase , Metabolism , Male , Muscle, Smooth , Orchiectomy , Penis , Rats , Rats, Sprague-Dawley , Testosterone , Blood
17.
Acta Pharmaceutica Sinica ; (12): 1233-1240, 2013.
Article in English | WPRIM | ID: wpr-259489

ABSTRACT

This study is to investigate the role of endogenous CSE/H2S in regulating apoptosis of HepG2 cells. MTT and Trypan blue assay were performed to determine the effect of CSE inhibitor PAG and CSE siRNA on proliferation of HepG2. Production of H2S from HepG2 cells was assessed spectrophotometrically using N, N-dimethyl-p-phenylenediamine-dihydrochloride. Cells apoptosis was detected by means of double staining of Hoechst 33342 and PI with Array Scan V(TI)HCS600 High-Contents. Dihydroethidine (DHE) and 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine intracellular superoxide anion and ROS level. Reduced glutathione (GSH) was determined by OxiSelect Total Glutathione Assay Kit. Recombinant plasmid pcDNA 3.1/myc-His(-)-CSE was constructed and transfected into 293T cells to rescue the ROS and GSH level to further investigate the effect of CSE/H2S on ROS and GSH. Western blotting was performed to test the effect of CSE siRNA on expression of activated caspase 3 and p-AKT and Nrf2 protein. The results showed that PAG and CSE siRNA could significantly decrease the production of H2S in HepG2 cells and inhibit the proliferation of HepG2 cells at a dose-dependent and time-dependent manner, respectively. PAG and CSE siRNA could promote the cell apoptosis of HepG2 cells. Moreover, PAG and CSE siRNA induced increased ROS generation and depletion of the critical antioxidant GSH and recombinant plasmid pcDNA 3.1/myc-His(-)-CSE rescued the level of ROS and GSH. Meanwhile, CSE siRNA increased the expression of activated caspase 3, but CSE siRNA did not affect the expression of p-AKT and Nrf2. These results suggested that the CSE/H2S pathway was involved in suppression of HepG2 cell growth and promoted apoptosis of HepG2 cells in an oxidative stress-dependent manner.


Subject(s)
Alkynes , Pharmacology , Apoptosis , Caspase 3 , Metabolism , Cell Proliferation , Cystathionine gamma-Lyase , Genetics , Metabolism , Glutathione , Metabolism , Glycine , Pharmacology , HEK293 Cells , Hep G2 Cells , Humans , Hydrogen Sulfide , Metabolism , NF-E2-Related Factor 2 , Metabolism , Plasmids , Proto-Oncogene Proteins c-akt , Metabolism , RNA, Small Interfering , Genetics , Reactive Oxygen Species , Metabolism , Recombinant Proteins , Genetics , Metabolism , Signal Transduction , Transfection
18.
National Journal of Andrology ; (12): 823-826, 2012.
Article in Chinese | WPRIM | ID: wpr-286416

ABSTRACT

Hydrogen sulfide (H2S) is the third type of active endogenous gaseous signal molecule following nitric oxide (NO) and carbon monoxide (CO). In mammalians, H2S is mainly synthesized by two proteases, cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). H2S plays an essential function of physiological regulation in vivo, and promotes penile erection by acting on the ATP-sensitive potassium channels to relax the vascular smooth muscle as well as by the synergistic effect with testosterone and NO to relax the corpus cavernosum smooth muscle (CCSM). At present, the selective phosphodiesterase type 5 (PDE5) inhibitor is mainly used for the treatment of erectile dysfunction (ED), but some ED patients fail to respond. Therefore, further studies on the mechanism of H2S regulating penile erection may provide a new way for the management of erectile dysfunction.


Subject(s)
Cystathionine beta-Synthase , Metabolism , Cystathionine gamma-Lyase , Metabolism , Humans , Hydrogen Sulfide , Male , Penile Erection
19.
Chinese Journal of Pediatrics ; (12): 136-140, 2012.
Article in Chinese | WPRIM | ID: wpr-356322

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the impact of sodium nitroprusside (a nitric oxide donor) in the ductus arteriosus in preterm rabbits on hydrogen sulfide (H(2)S)-cystathionine-γ-lyase (CSE) system.</p><p><b>METHODS</b>For 16 Japanese white rabbits pregnant for 21 days were randomly divided into four groups, each of the following groups had 4 rabbits: control group, intraperitoneal injection of sodium nitroprusside 1 mg/kg, 2.5 mg/kg, and 5.0 mg/kg groups. The rabbits in control group had a peritoneal puncture with a simple hollow needle, and those in the other groups were given corresponding dose of intraperitoneal injection of sodium nitroprusside at gestational age 23 and 25 days, respectively. At gestational age 26 days the fetuses of the pregnant rabbits were removed surgically, and 28 fetal rabbits were obtained from the control group, 27 from the sodium nitroprusside small dose group, 29 from the medium dose group, and 26 from the large dose group. The fetal heart blood sample of 1 ml was taken from each fetus, and immediately after sampling the arterial ductal tissues were dissected. Fetal rabbit plasma proteins hydrogen sulfide content was determined by using de-protein method, and real time quantitative RT-PCR was used for determination of arterial tissue CSE gene and western-blotting was used for measuring protein expression of CSE.</p><p><b>RESULTS</b>In control group hydrogen sulfide content of fetal rabbits plasma (55.68 ± 6.57) µmol/L and arterial tissue CSE mRNA expression was 1.07 ± 4.12; the parameters in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (60.02 ± 6.09) µmol/L and 3.46 ± 0.18; in intraperitoneal injection of sodium nitroprusside group 2.5 mg/kg, were (64.71 ± 7.12) µmol/L and 10.95 ± 0.22; and in intraperitoneal injection of sodium nitroprusside group 1 mg/kg were (70.63 ± 8.07) µmol/L and 19.56 ± 0.17. Comparison between small dose group and control group, medium dose group and small dose group, high dose group and medium dose group showed that the above data were significantly different P < 0.05, with the injection of sodium nitroprusside CSE protein expression increased gradually with increasing doses.</p><p><b>CONCLUSION</b>Sodium nitroprusside showed an enhancing effect on preterm CSE-H(2)S system in rabbit ductus arteriosus in a certain range of concentration in a dose-dependent manner.</p>


Subject(s)
Animals , Cystathionine gamma-Lyase , Blood , Ductus Arteriosus , Metabolism , Female , Hydrogen Sulfide , Blood , Nitric Oxide , Blood , Nitroprusside , Pharmacology , Pregnancy , Rabbits
20.
Chinese Journal of Pediatrics ; (12): 890-894, 2011.
Article in Chinese | WPRIM | ID: wpr-356348

ABSTRACT

<p><b>OBJECTIVE</b>To explore the impact of sulfur dioxide (SO(2)) on hydrogen sulfide (H(2)S)/cystathionine-γ-lyase (CSE) and H(2)S/mercaptopyruvate sulfurtransferase (MPST) pathways in the pathogenesis of hypoxic pulmonary hypertension.</p><p><b>METHODS</b>Thirty-two male Wistar rats were randomly divided into four groups: control group (n = 8), hypoxic group (n = 8), hypoxic + SO(2) group (n = 8) and hypoxic + hydroxamate (HDX) group (n = 8). After 21 days of experiment, the concentration and production of H(2)S in lung tissues were measured respectively for each rat. The protein expression of CSE and MPST in intima and media of small pulmonary arteries in rats was detected with immunohistochemical method.</p><p><b>RESULTS</b>Compared with control group, the mean pulmonary artery pressure (mPAP) in rats of hypoxic group was increased significantly [(33.38 ± 6.32) mm Hg vs. (16.74 ± 3.81) mm Hg, P < 0.01]. Compared with hypoxic group, the mPAP in rats of hypoxic + SO(2) group was decreased significantly [(29.65 ± 2.53) mm Hg vs. (33.38 ± 6.32) mm Hg, P < 0.01]. However, compared with hypoxic group, the mPAP in rats of hypoxic + HDX group was increased significantly [(39.44 ± 6.26) mm Hg vs. (33.38 ± 6.32) mm Hg, P < 0.01]. Compared with control group, the concentration [(2.02 ± 0.43) µmol/g vs. (3.11 ± 0.42) µmol/g, P < 0.01] and production [(19.64 ± 3.48) nmol/(g·min)vs. (28.20 ± 5.95) nmol/(g·min), P < 0.05] of H(2)S were decreased significantly in rats of hypoxic group, respectively. When treated with SO(2), hypoxic rats showed an increased concentration [(2.73 ± 0.20) µmol/g vs. (2.02 ± 0.43) µmol/g, P < 0.01] and production [(26.24 ± 1.92) nmol/(g·min) vs. (19.64 ± 3.48) nmol/(g·min), P < 0.01] of H(2)S in lung tissue compared with those without receiving SO(2) treatment. When treated with HDX, hypoxic rats showed a significant decrease in concentration [(1.64 ± 0.23) µmol/g vs. (2.02 ± 0.43) µmol/g, P < 0.05] and production [(13.94 ± 3.63) nmol/(g·min) vs. (19.64 ± 3.48) nmol/(g·min), P < 0.05] of H(2)S in lung tissue compared with those without receiving HDX treatment. As for the expression of CSE in small pulmonary arteries (SPAs), compared with control group, the expression of CSE in intima [(0.31 ± 0.02) vs. (0.36 ± 0.01), P < 0.01] and media [(0.27 ± 0.01) vs. (0.30 ± 0.01), P < 0.01] in rats of hypoxic group was decreased significantly. While compared with hypoxic group, the expression of CSE in intima [(0.35 ± 0.02) vs. (0.31 ± 0.02), P < 0.01] in SPAs of hypoxic + SO(2) group was increased significantly. With HDX treatment, the expression of CSE in intima [(0.26 ± 0.01) vs. (0.31 ± 0.02), P < 0.01] in SPAs of hypoxic group was lower than that without HDX treatment. As for the expression of MPST in SPAs, compared with hypoxic group, the expression of MPST in media [(0.32 ± 0.02) vs. (0.29 ± 0.01), P < 0.01] in SPAs of hypoxic + SO(2) group was increased significantly.</p><p><b>CONCLUSION</b>SO(2) might upregulate H(2)S/CSE and H(2)S/MPST pathways in pulmonary arteries of hypoxic rats.</p>


Subject(s)
Animals , Cystathionine gamma-Lyase , Metabolism , Hydrogen Sulfide , Metabolism , Hypertension, Pulmonary , Hypoxia , Metabolism , Male , Pulmonary Artery , Metabolism , Rats , Rats, Wistar , Sulfur Dioxide , Pharmacology , Sulfurtransferases , Metabolism
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