OBJECTIVE: To explore the protective effects and underlying mechanisms of H ₂S against lipid peroxidation-mediated carbonyl stress in the uranium-treated NRK-52E cells. METHODS: Cell viability was evaluated using CCK-8 assay. Apoptosis was measured using flow cytometry. Reagent kits were used to detect carbonyl stress markers malondialdehyde, 4-hydroxynonenal, thiobarbituric acid reactive substances, and protein carbonylation. Aldehyde-protein adduct formation and alcohol dehydrogenase, aldehyde dehydrogenase 2, aldo-keto reductase, nuclear factor E2-related factor 2 (Nrf2), and cystathionine β-synthase (CBS) expression were determined using western blotting or real-time PCR. Sulforaphane (SFP) was used to activate Nrf2. RNA interference was used to inhibit CBS expression. RESULTS: GYY4137 (an H ₂S donor) pretreatment significantly reversed the uranium-induced increase in carbonyl stress markers and aldehyde-protein adducts. GYY4137 effectively restored the uranium-decreased Nrf2 expression, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2, accompanied by a reversal of the uranium-decreased expression of CBS and aldehyde-metabolizing enzymes. The application of CBS siRNA efficiently abrogated the SFP-enhanced effects on the expression of CBS, Nrf2 activation, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2 and concomitantly reversed the SFP-enhanced effects of the uranium-induced mRNA expression of aldehyde-metabolizing enzymes. Simultaneously, CBS siRNA reversed the SFP-mediated alleviation of the uranium-induced increase in reactive aldehyde levels, apoptosis rates, and uranium-induced cell viability. CONCLUSION H ₂S induces Nrf2 activation and nuclear translocation, which modulates the expression of aldehyde-metabolizing enzymes and the CBS/H ₂S axis. Simultaneously, the Nrf2-controlled CBS/H ₂S axis may at least partially promote Nrf2 activation and nuclear translocation. These events form a cycle-regulating mode through which H ₂S attenuates the carbonyl stress-mediated NRK-52E cytotoxicity triggered by uranium.
NF-E2-Related Factor 2/genetics* ; Animals ; Hydrogen Sulfide/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Cell Line ; Uranium/toxicity* ; Antioxidant Response Elements ; Kidney/metabolism* ; Oxidative Stress/drug effects* ; Cell Survival/drug effects* ; Apoptosis/drug effects*

Objective: To investigate the effects of exogenous hydrogen sulfide (H₂S) on pulmonary vascular reactivity induced by endotoxic shock (ES) in rabbits. Methods: In this experiment, the model of endotoxic shock (ES) was induced by injection of lipopolysaccharides (LPS) to New Zealand big eared white rabbit through jugular vein (8 mg/0.8 ml/kg), the intervention was performed by H₂S donor(sodium hydrosulfide, NaHS) which was injected intraperitoneally (28 μmol/kg) 15 min in advance. New Zealand rabbits were randomly divided into 4 groups(n=8):control group, LPS group, LPS+NaHS group and NaHS group. The changes of mean arterial pressure (MAP) and mean pulmonary arterial pressure (MPAP) were detected. The tension of pulmonary artery ring (PARs) was detected byin vitro vascular ring technique. The ultrastructure of pulmonary artery wall and pulmonary artery endothelial cells were observed by light microscope and scanning electron microscope. Results: ①MAP was decreased while MPAP was increased in rabbits after LPS injection, and ES animal model was established successfully. Compared with LPS group, mPAP of rabbit in LPS+NaHS group was decreased significantly (all P＜0.05). ②Compared with normal control group, pulmonary artery of rabbits in LPS group had an increased contractile response to phenylephrine (PE) and a decreased relaxation response to acetylcholine (ACh) (both P＜0.01); Compared with LPS group, pulmonary artery of rabbits in LPS+NaHS group had a decreased contractile response to PE and an increased relaxation response to ACh (both P＜0.05). ③Under light microscope, the structure of vascular endothelial cells was continuous in the normal control group, the elastic fibers were intact in the subcutaneous layer, and the smooth muscle layer was arranged neatly. LPS can shed some of the pulmonary artery endothelial cells, break the subcutaneous elastic fibers, and disorder the smooth muscle layer structure. Compared with LPS group, the injury of pulmonary artery wall in LPS+NaHS group was ameliorated. The morphology of pulmonary artery wall was normal in NaHS group. It is showed that some endothelial cells of pulmonary artery were missing in LPS group by Scanning electron microscopy. The morphology of pulmonary artery endothelial cells in LPS+NaHS group was similar to that in the control group: slightly widened intercellular space was observed, and no cell exfoliation was observed. Conclusion: These results suggest that exogenous H₂S can protect pulmonary artery endothelial cells and regulate the reactivity changes of pulmonary artery during ES, which may be one of the mechanisms reducing PAH in ES rabbits.
Animals ; Endothelial Cells ; Hydrogen Sulfide/pharmacology* ; Hypertension, Pulmonary ; Lipopolysaccharides/adverse effects* ; Pulmonary Artery ; Rabbits ; Shock, Septic

Hydrogen sulfide is one of the most important signal transduction molecules in the body. Its anabolism and catabolism in the gastrointestinal tract(GT) are extremely high, and its role in the physiological and pathological process of the GT is fairly complicated. The study reviewed recent literature on hydrogen sulfide and GT, and proposed that hydrogen sulfide exerted dual modulating effects in the GT; specifically, it promoted the functions of the GT at low concentrations while damaged the GT at high concentrations. Hydrogen sulfide donors or metabolic modifiers exerted their therapeutic effects by restoring the metabolic homeostasis of hydrogen sulfide, and extended their efficacy to other tissues through hydrogen sulfide related gut-axis. Additionally, drugs could deviate hydrogen sulfide metabolism from the normal state due to their instability of structure, local over exposure and/or excessive pharmacological effects, thus inducing toxic and side effects or transforming therapeutic effects into toxic and side effects. This study provided references for the deep research on physiological and pathological mechanisms of hydrogen sulfide and facilitated the development of hydrogen sulfide-related drugs and discovery of their toxicity and efficacy mechanism.
Gastrointestinal Tract ; Hydrogen Sulfide/pharmacology* ; Signal Transduction

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

Objective To explore the effect of hydrogen sulfide on inflammatory factors and energy metabolism of mitochondria after limbs reperfusion injury in rats. Methods Sixty rats were divided into three groups:sham operation group,control group(ischemia-reperfusion injury + saline group),and experimental group(ischemia-reperfusion injury + HS group).Wistar rat models of limb ischemia-reperfusion injury were established.Skeletal muscle samples were collected to determine the levels of necrosis decomposition products [including myoglobin(MB),lipoprotein complex(LPC)and lipid peroxide(LPO)];blood samples were collected to determine the levels of interleukin(IL)-1,IL-6 and tumor necrosis factor-α(TNF-α);mitochondria were extracted for mitochondrial transmembrane potential measurement and ATP content detection.Statistical analysis was made on the test results. Results After ischemia reperfusion injury,the levels of MB,LPO,and LPC in skeletal muscle,liver,lung and renal tissues of the control group were significantly increased(MB:P =0.003,P =0.001,P =0.001,P =0.001;LPO:P =0.001,P =0.001,P =0.001,P =0.002;LPC:P =0.000,P =0.002,P =0.002,P =0.003),and hydrogen sulfide treatment during ischemia reperfusion significantly inhibited the production of MB,LPO,and LPC(MB:P =0.021,P =0.036,P =0.005;LPO:P =0.003,P =0.008,P =0.010,P =0.015;LPC:P =0.002,P =0.026,P =0.007,P =0.006).Ischemia/reperfusion of lower extremity in rats resulted in increased levels of IL-1,IL-6,and TNF-α in the serum of rats,and the levels of IL-1,IL-6,and TNF-increased over time,with statistically significant differences in IL-1,IL-6,and TNF-α among groups at 3 h(IL-1:P =0.019,P =0.011,P =0.009,$P_{12_{h}}$=0.008,and P =0.002;IL-6:P =0.026,P =0.009,P =0.002, $P_{12_{h}}$=0.002,P =0.003;TNF-α:P =0.002,P =0.002,P =0.005,$P_{12_{h}}$=0.002,P =0.003).The levels of IL-1,IL-6,and TNF-α in serum were significantly inhibited during ischemia reperfusion(IL-1:P =0.035,P =0.039,P =0.012,$P_{12_{h}}$=0.005,P =0.006;IL-6:P =0.042,P =0.025,P =0.023,$P_{12_{h}}$=0.006,P =0.005;TNF-α:P =0.005,P =0.003,P =0.022,$P_{12_{h}}$=0.005,P =0.005),and such inhibitory effects became even more obvious over time.After limb ischemia and reperfusion in the control group,the mitochondrial transmembrane potential of skeletal muscle cells significantly decreased compared with that of the sham group(t=6.698;P=0.001).After hydrogen sulfide treatment,the mitochondrial membrane potential energy of the experimental group was significantly higher than that of the control group(t=7.507,P = 0.000).The ATP level in the mitochondria of ischemia reperfusion rats in the control group was significantly lower than that in the sham group(t=7.526,P = 0.000).The content of mitochondrial ATP in the experimental group was significantly higher than that in the control group after hydrogen sulfide treatment(t=8.604,P = 0.000). Conclusions Hydrogen sulfide can alleviate the injury of skeletal muscle and distal organs after limb ischemia-reperfusion and reduce local inflammatory reaction.In addition,it is valuable in alleviating mitochondrial transmembrane potential and energy metabolism disorders during reperfusion injury.
Animals ; Energy Metabolism ; Hydrogen Sulfide ; pharmacology ; Inflammation ; metabolism ; Interleukin-6 ; metabolism ; Mitochondrial Diseases ; pathology ; Rats ; Rats, Wistar ; Reperfusion Injury ; Tumor Necrosis Factor-alpha ; metabolism

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 (H2S) on contraction capacity of diaphragm in type 1 diabetic rats.
 METHODS: Thirty-two male SD rats were randomly divided into a normal group (NC), a diabetic group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8). Intraperitoneal injection of streptozotocin was utilized to establish diabetic rat model. After the modeling, the rats in the DM+NaHS and the NaHS groups were intraperitoneally injected with 28 μmol/kg NaHS solution. 8 weeks later, the diaphragm contractility was assessed by isolated draphragm strips perfusion. The peak twitch tension (Pt), maximum tetanic tension (Po) and maximal rates of contraction/relaxation (±dT/dtmax) were determined. The alterations in diaphragm ultrastructure were observed under electron microscopy. The diaphragm weight/body weight (DW/BW) was measured. The activities of succinic dehydrogenase (SDH), lactate dehydrogenase (LDH) and sarcoplasmic reticulum Ca2+ ATPase (SERCA) were analyzed by spectrophotometric method. The mRNA levels of SERCA and prospholamban (PLB) in diaphragm were detected by RT-PCR.
 RESULTS: Compared with the NC group, there was no significant change in all measured index in the NaHS group (P>0.05), while Pt, Po and ±dT/dtmax were significantly decreased in the DM group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the diaphragm. The DW/BW ratio and the activities of SDH, LDH and SERCA were decreased. The SERCA mRNA was decreased, while PLB mRNA was increased. Compared with the DM group, the diaphragm contractility and ultrastructure damage were improved in the DM+NaHS group. The DW/BW ratio and the activities of SDH, LDH and SERCA were increased. The SERCA mRNA was increased, while PLB mRNA was decreased (all P<0.05).
 CONCLUSION H(2)S can enhance the contraction capacity of diaphragm in type 1 diabetic rats, which is involved in regulating the activities of biological enzymes and the gene expressions of calcium regulatory proteins.
Animals ; Body Weight ; Diabetes Mellitus, Experimental ; physiopathology ; Diaphragm ; drug effects ; ultrastructure ; Hydrogen Sulfide ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Male ; Muscle Contraction ; drug effects ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Succinate Dehydrogenase ; metabolism ; Sulfides ; pharmacology

OBJECTIVE: To investigate the effect of hydrogen sulfide (H2S) on cardiac myosin light chain kinase (MLCK) expression in diabetic rats.
 METHODS: A total of 32 male SD rats were randomly divided into a normal control group (NC group), a diabetic control group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8 in each group). Intraperitoneal injection of streptozotocin was utilized to establish Type 1 diabetic rat model. The diabetic rats in the DM+NaHS and NaHS groups were intraperitoneally injected with 28 μmol/kg NaHS solution. Eight weeks later, the ventricular hemodynamic parameters, the ratio of heart weight/body weight (HW/BW ratio), the levels of lactate dehydrogenase (LDH) and creatine kinase MB isozyme (CK-MB) in serum were determined. The ultrastructures of myocardium were observed under electron microscopy. The expressions of MLCK mRNA and protein level in myocardium were detected by RT-PCR and Western blot, respectively.
 RESULTS: Compared with the NC group, there was no significant difference in the various indexes in the NaHS group (all P>0.05). The function of left ventricular contract and relaxation were decreased obviously in diabetic rats, while the HW/BW ratio was increased (all P<0.01). The levels of LDH and CK-MB were increased (both P<0.01) in serum, while the levels of MLCK mRNA and protein were decreased significantly (both P<0.01) in myocardial tissues. Compared with the DM group, the left ventricular hemodynamic parameters and myocardial ultrastructure damage were improved in the DM+NaHS group, while the HW/BW ratio was decreased (all P<0.05). The levels of LDH and CK-MB were decreased (both P<0.01), while the levels of MLCK mRNA and protein were increased significantly (both P<0.01).
 CONCLUSION H2S can protect myocardium in diabetic rats, which may be associated with upregulation of cardiac MLCK.
Animals ; Cardiotonic Agents ; pharmacology ; Creatine Kinase, MB Form ; blood ; Diabetes Mellitus, Experimental ; drug therapy ; Heart ; drug effects ; Hemodynamics ; Hydrogen Sulfide ; pharmacology ; L-Lactate Dehydrogenase ; blood ; Male ; Myocardium ; ultrastructure ; Myosin-Light-Chain Kinase ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sulfides ; pharmacology ; Ventricular Function, Left ; drug effects

In the present study, a series of novel nitric oxide-hydrogen sulfide releasing derivatives of (S)-3-n-butylphthalide ((S)-NBP) were designed, synthesized, and evaluated as potential antiplatelet agents. Compound NOSH-NBP-5 displayed the strongest activity in inhibiting the arachidonic acid (AA)- and adenosine diphosphate (ADP)-induced platelet aggregation in vitro, with 3.8- and 7.0-fold more effectiveness than (S)-NBP, respectively. Furthermore, NOSH-NBP-5 could release moderate levels of NO and HS, which would be beneficial in improving cardiovascular and cerebral circulation. Moreover, NOSH-NBP-5 could release (S)-NBP when incubated with rat brain homogenate. In conclusion, these findings may provide new insights into the development of novel antiplatelet agents for the treatment of thrombosis-related ischemic stroke.
Animals ; Benzofurans ; chemistry ; Humans ; Hydrogen Sulfide ; chemistry ; Male ; Molecular Structure ; Nitric Oxide ; chemistry ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Thrombosis ; drug therapy ; physiopathology

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*