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.
Cystathionine beta-Synthase ; Cystathionine gamma-Lyase ; Cysteine ; pharmacology ; Hydrogen Sulfide ; Muscle, Smooth

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.
Adrenal Cortex ; enzymology ; Animals ; CRISPR-Cas Systems ; Cystathionine gamma-Lyase ; genetics ; Integrases ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Zona Fasciculata ; enzymology

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.
Animals ; Cecum ; surgery ; Cystathionine gamma-Lyase ; metabolism ; Cytokines ; metabolism ; Hydrogen ; pharmacology ; Hydrogen Sulfide ; metabolism ; Ligation ; Lung Injury ; therapy ; Male ; Punctures ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saline Solution ; pharmacology

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.
Animals ; Calcineurin ; metabolism ; Cardiomegaly ; chemically induced ; 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

OBJECTIVE: To explore the role of hydrogen sulfide (H2S) in acute liver injury induced by crushing hind limbs of rats. METHODS: The rats were randomly divided into the following groups: control, crushing, H2S donor sodium hydrosulfide (NaHS) + crushing, H2S inhibitor propargylglycine (PAG) + crushing group. The acute liver injury model was established by 'crushing the hind limbs of rats with standard weight. Rats were sacrificed at 30 min and 120 min after the crush. The activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by colorimetric method, and the content of H2S in plasma and the contents of malondialdehyde (MDA), protein carbonyl, glutathione (GSH) in the liver and the activity of H2S generating enzyme (cystathionine y-lyase, CSE) were determined by chemical method. The expression of CSE mRNA in liver was detected by RT-PCR. RESULTS: For crush injury group, the levels of AST and ALT in serum, MDA and protein carbonyl in liver increased. The levels of GSH, CSE, CSE mRNA in liver and H2S in serum decreased. The administration of NaHS before limbs crush could attenuate the changes of liver injury, but the pre-treatment with PAG could exacerbate the changes. CONCLUSION The decrease of H2S production could involve in mediating the acute liver injury induced by traumatic stress in rats.
Alanine Transaminase/blood* ; Alkynes/pharmacology* ; Animals ; Aspartate Aminotransferases/blood* ; Cystathionine gamma-Lyase/metabolism* ; Glutathione/metabolism* ; Glycine/pharmacology* ; Hydrogen Sulfide/pharmacology* ; Liver/injuries* ; Malondialdehyde/metabolism* ; Protein Carbonylation ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sulfides/pharmacology*

OBJECTIVETo investigate the effect of intestinal resection on hydrogen sulfide (H2S) biosynthesis and interstitial cells of Cajal(ICC) in mice.

METHODSAfter 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.

RESULTSThe 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].

CONCLUSIONSInflammatory 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.

Animals ; Cystathionine gamma-Lyase ; Disease Models, Animal ; Hydrogen Sulfide ; Inflammation ; Interstitial Cells of Cajal ; Intestines ; Mice ; Proto-Oncogene Proteins c-kit ; RNA, Messenger

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-&gamma;-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.
Animals ; Colon ; physiopathology ; 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

OBJECTIVETo investigate the role of endogenous hydrogen sulfide (H2S) in erectile dysfunction (ED) induced by androgen deficiency.

METHODSWe 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.

RESULTSThe 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).

CONCLUSIONThe 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.

Androgens ; deficiency ; Animals ; Cystathionine beta-Synthase ; metabolism ; Cystathionine gamma-Lyase ; metabolism ; Erectile Dysfunction ; metabolism ; Hydrogen Sulfide ; metabolism ; Male ; Orchiectomy ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the impact of hyperglycemia on the hydrogen sulfide (H2S) signaling pathway in rat penile tissue and its relationship with erectile function.

METHODSTwenty 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.

RESULTSWith 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).

CONCLUSIONThe 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.

Animals ; Blood Pressure ; physiology ; Cystathionine gamma-Lyase ; metabolism ; Diabetes Mellitus, Experimental ; chemically induced ; metabolism ; Electric Stimulation ; methods ; Erectile Dysfunction ; etiology ; Humans ; Hydrogen Sulfide ; metabolism ; Hyperglycemia ; metabolism ; Lyases ; metabolism ; Male ; Penis ; enzymology ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Testosterone ; metabolism

OBJECTIVETo 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.

METHODSThis 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.

RESULTSNo 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.

CONCLUSIONHypertension 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.

Animals ; Cystathionine beta-Synthase ; metabolism ; Cystathionine gamma-Lyase ; metabolism ; Gene Expression Regulation ; Hydrogen Sulfide ; blood ; Hypertension ; metabolism ; Male ; Penis ; enzymology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY